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1

Anthrax toxin-induced rupture of artificial lipid bilayer membranes.  

PubMed

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. PMID:23947891

Nablo, Brian J; Panchal, Rekha G; Bavari, Sina; Nguyen, Tam L; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E; Robertson, Joseph W F; Balijepalli, Arvind; Halverson, Kelly M; Kasianowicz, John J

2013-08-14

2

Anthrax toxin-induced rupture of artificial lipid bilayer membranes  

NASA Astrophysics Data System (ADS)

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

2013-08-01

3

Human genetic variation altering anthrax toxin sensitivity  

PubMed Central

The outcome of exposure to infectious microbes or their toxins is influenced by both microbial and host genes. Some host genes encode defense mechanisms, whereas others assist pathogen functions. Genomic analyses have associated host gene mutations with altered infectious disease susceptibility, but evidence for causality is limited. Here we demonstrate that human genetic variation affecting capillary morphogenesis gene 2 (CMG2), which encodes a host membrane protein exploited by anthrax toxin as a principal receptor, dramatically alters toxin sensitivity. Lymphoblastoid cells derived from a HapMap Project cohort of 234 persons of African, European, or Asian ancestry differed in sensitivity mediated by the protective antigen (PA) moiety of anthrax toxin by more than four orders of magnitude, with 99% of the cohort showing a 250-fold range of sensitivity. We find that relative sensitivity is an inherited trait that correlates strongly with CMG2 mRNA abundance in cells of each ethnic/geographical group and in the combined population pool (P = 4 × 10?11). The extent of CMG2 expression in transfected murine macrophages and human lymphoblastoid cells affected anthrax toxin binding, internalization, and sensitivity. A CMG2 single-nucleotide polymorphism (SNP) occurring frequently in African and European populations independently altered toxin uptake, but was not statistically associated with altered sensitivity in HapMap cell populations. Our results reveal extensive human diversity in cell lethality dependent on PA-mediated toxin binding and uptake, and identify individual differences in CMG2 expression level as a determinant of this diversity. Testing of genomically characterized human cell populations may offer a broadly useful strategy for elucidating effects of genetic variation on infectious disease susceptibility.

Martchenko, Mikhail; Candille, Sophie I.; Tang, Hua; Cohen, Stanley N.

2012-01-01

4

Ratcheting up protein translocation with anthrax toxin  

PubMed Central

Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradient-driven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heat-engine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation.

Feld, Geoffrey K; Brown, Michael J; Krantz, Bryan A

2012-01-01

5

Ratcheting up protein translocation with anthrax toxin.  

PubMed

Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradient-driven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heat-engine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation. PMID:22374876

Feld, Geoffrey K; Brown, Michael J; Krantz, Bryan A

2012-03-30

6

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1.  

PubMed

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-? responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection. PMID:23607659

Ingram, R J; Harris, A; Ascough, S; Metan, G; Doganay, M; Ballie, L; Williamson, E D; Dyson, H; Robinson, J H; Sriskandan, S; Altmann, D M

2013-07-01

7

Anthrax Toxin Entry into Polarized Epithelial Cells  

PubMed Central

We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl? secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl? secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease.

Beauregard, Kathryn E.; Wimer-Mackin, Susan; Collier, R. John; Lencer, Wayne I.

1999-01-01

8

Cellular and Systemic Effects of Anthrax Lethal Toxin and Edema Toxin  

PubMed Central

Anthrax lethal toxin (LT) and edema toxin (ET) are the major virulence factors of anthrax and can replicate the lethality and symptoms associated with the disease. This review provides an overview of our current understanding of anthrax toxin effects in animal models and the cytotoxicity (necrosis and apoptosis) induced by LT in different cells. A brief reexamination of early historic findings on toxin in vivo effects in the context of our current knowledge is also presented.

Moayeri, Mahtab; Leppla, Stephen H.

2009-01-01

9

Changing the Receptor Specificity of Anthrax Toxin  

PubMed Central

ABSTRACT The actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.

Mechaly, Adva; McCluskey, Andrew J.; Collier, R. John

2012-01-01

10

Anthrax lethal toxin induces endothelial barrier dysfunction.  

PubMed

Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection. PMID:15920171

Warfel, Jason M; Steele, Amber D; D'Agnillo, Felice

2005-06-01

11

Anthrax Edema Toxin Impairs Clearance in Mice  

PubMed Central

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.

Sastalla, Inka; Tang, Shixing; Crown, Devorah; Liu, Shihui; Eckhaus, Michael A.; Hewlett, Indira K.; Leppla, Stephen H.

2012-01-01

12

Anthrax toxins and the host: a story of intimacy.  

PubMed

Although the dramatic events of the year 2001 have revitalized the interest in anthrax, research on Bacillus anthracis and its major virulence factors is one of the oldest theme in microbiology and started with the early works of Robert Koch and Louis Pasteur. The anthrax toxins are central to anthrax pathogenesis. They were discovered in the mid-1950s and since then there has been an enormous amount of work to elucidate both the molecular and physiopathological details of their mode of action. In this review, after a brief introduction of B. anthracis, we will focus on the latest findings that concern two aspects of anthrax toxin research: the environmental signals and the molecular mechanisms that regulate toxin synthesis, and the mechanisms of intoxication. We hope to convince the reader that the anthrax toxins are highly specialized determinants of B. anthracis pathogenicity: their synthesis is integrated within a global virulence programme and they target key eukaryotic cell proteins. We conclude with a consideration of the therapeutic perspectives arising from our current knowledge of how the toxins work. PMID:12542467

Mock, Michèle; Mignot, Tâm

2003-01-01

13

The unfolding story of anthrax toxin translocation  

PubMed Central

Summary The essential cellular functions of secretion and protein degradation require a molecular machine to unfold and translocate proteins either across a membrane or into a proteolytic complex. Protein translocation is also critical for microbial pathogenesis, namely bacteria can use translocase channels to deliver toxic proteins into a target cell. Anthrax toxin (Atx), a key virulence factor secreted by Bacillus anthracis, provides a robust biophysical model to characterize transmembrane protein translocation. Atx is comprised of three proteins: the translocase component, protective antigen (PA); and two enzyme components, lethal factor (LF) and edema factor (EF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and EF. These complexes are endocytosed into mammalian host cells, where PA forms a protein-conducting translocase channel. The proton motive force unfolds and translocates LF and EF through the channel. Recent structure and function studies have shown that LF unfolds during translocation in a force-dependent manner via a series of metastable intermediates. Polypeptide-binding clamps located throughout the PA channel catalyze substrate unfolding and translocation by stabilizing unfolding intermediates through the formation of a series of interactions with various chemical groups and ?-helical structure presented by the unfolding polypeptide during translocation.

Thoren, Katie L.; Krantz, Bryan A.

2011-01-01

14

The Receptors that Mediate the Direct Lethality of Anthrax Toxin  

PubMed Central

Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin ?1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 ?g PA + 2 ?g FP59 being sufficient to kill a mouse. While TEM8?/? and wild type control mice succumbed to a 5 ?g PA + 5 ?g FP59 challenge, CMG2?/? mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin ?1, CMG2?/?/TEM8?/? mice were challenged with as many as five doses of 50 ?g PA + 50 ?g FP59. Strikingly, the CMG2?/?/TEM8?/? mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.

Liu, Shihui; Zhang, Yi; Hoover, Benjamin; Leppla, Stephen H.

2012-01-01

15

Structure-based Design of a Heptavalent Anthrax Toxin Inhibitor  

PubMed Central

The design of polyvalent molecules, consisting of multiple copies of a biospecific ligand attached to a suitable scaffold, represents a promising approach to inhibit pathogens and oligomeric microbial toxins. Despite the increasing interest in structure-based drug design, few polyvalent inhibitors based on this approach have shown efficacy in vivo. Here we demonstrate the structure-based design of potent biospecific heptavalent inhibitors of anthrax lethal toxin. Specifically, we illustrate the ability to design potent polyvalent ligands by matching the pattern of binding sites on the biological target. We used a combination of experimental studies based on mutagenesis and computational docking studies to identify the binding site for an inhibitory peptide on the heptameric subunit of anthrax toxin. We developed an approach based on copper-catalyzed azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a ?-cyclodextrin core via a polyethylene glycol linker of an appropriate length. The resulting heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors show considerable promise as anthrax anti-toxins.

Joshi, Amit; Kate, Sandesh; Poon, Vincent; Mondal, Dhananjoy; Boggara, Mohan B.; Saraph, Arundhati; Martin, Jacob T.; McAlpine, Ryan; Day, Ryan; Garcia, Angel E.; Mogridge, Jeremy; Kane, Ravi S.

2011-01-01

16

NANO APTASENSOR FOR PROTECTIVE ANTIGEN TOXIN OF ANTHRAX  

PubMed Central

We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (Kd) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1nM. The sensitivity was 0.11 per nM and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.

Cella, Lakshmi N.; Sanchez, Pablo; Zhong, Wenwan; Myung, Nosang V.; Chen, Wilfred; Mulchandani, Ashok

2010-01-01

17

Identification of the cellular receptor for anthrax toxin  

NASA Astrophysics Data System (ADS)

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.

Bradley, Kenneth A.; Mogridge, Jeremy; Mourez, Michael; Collier, R. John; Young, John A. T.

2001-11-01

18

Fluorescence resonance energy transfer studies on anthrax lethal toxin  

Microsoft Academic Search

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA63, a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for

John C. Croney; Kristina M. Cunningham; R. John Collier; David M. Jameson

2003-01-01

19

Crystallographic studies of the Anthrax lethal toxin. Annual report  

SciTech Connect

The lethal form of Anthrax results from the inhalation of anthrax spores. Death is primarily due to the effects of the lethal toxin (Protective Antigen (PA) + Lethal Factor) from the causative agent, Bacillus anthracis. All the Anthrax vaccines currently in use or under development contain or produce PA, the major antigenic component of anthrax toxin, and there is a clear need for an improved vaccine for human use. In the previous report we described the first atomic resolution structure of PA, revealing that the molecule is composed largely of beta-sheets organized into four domains. This information can be used in the design. of recombinant PA vaccines. In this report we describe additional features of the full-length PA molecule derived from further crystallographic refinement and careful examination of the structure. We compare two crystal forms of PA grown at different pH values and discuss the functional implications. A complete definition of the function of each domain must await the crystal structure of the PA63 heptamer. We have grown crystals of the heptamer under both detergent and detergent-free conditions, and made substantial progress towards the crystal structure. The mechanism of anthrax intoxication in the light of our results is reviewed.

Frederick, C.A.

1996-07-01

20

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis.  

PubMed

The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and ?1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization. PMID:24085852

Jeong, Sun-Young; Martchenko, Mikhail; Cohen, Stanley N

2013-10-01

21

Search for Cyclodextrin-Based Inhibitors of Anthrax Toxins: Synthesis, Structural Features, and Relative Activities  

Microsoft Academic Search

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of -cyclodex- trin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new -cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-

Vladimir A. Karginov; Ekaterina M. Nestorovich; Adiamseged Yohannes; Tanisha M. Robinson; Nour Eddine Fahmi; Frank Schmidtmann; Sidney M. Hecht; Sergey M. Bezrukov

2006-01-01

22

Anthrax Toxins Induce Shock in Rats by Depressed Cardiac Ventricular Function  

Microsoft Academic Search

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts

Linley E. Watson; Shu-Ru Kuo; Khurshed Katki; Tongyun Dang; Seong Kyu Park; David E. Dostal; Wei-Jen Tang; Stephen H. Leppla; Arthur E. Frankel; Adam Ratner

2007-01-01

23

Effects of dynamin inactivation on pathways of anthrax toxin uptake.  

PubMed

Internalization and traffic to acidic endosomes of anthrax lethal factor (LF) and protective antigen (PA), bound to the anthrax toxin receptor (ATR), is required for LF translocation into the cytosol, where it can elicit its toxic effects. Dynamin is required for clathrin-mediated endocytosis, and long-term disruption of dynamin function blocks internalization of PA. We have used LFn-DTA, a surrogate of LF consisting of the N-terminal domain of LF fused to the catalytic subunit of diphtheria toxin, to differentiate the effects of acute and long-term block of dynamin function on LFn-DTA toxicity. Both forms of interference reduce LFn-DTA toxicity only partially, consistent with alternative routes for LFn-DTA endocytosis. In contrast, a long-term block of dynamin activity results in a further interference with LFn-DTA toxicity that is consistent with an altered endosomal environment, probably an increase in endosomal pH. PMID:15511085

Boll, Werner; Ehrlich, Marcelo; Collier, R John; Kirchhausen, Tomas

2004-07-01

24

Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin  

Microsoft Academic Search

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that

Anshu Agrawal; Jai Lingappa; Stephen H. Leppla; Sudhanshu Agrawal; Abdul Jabbar; Conrad Quinn; Bali Pulendran

2003-01-01

25

Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax ? †  

PubMed Central

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ?50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Wycoff, Keith L.; Belle, Archana; Deppe, Dorothee; Schaefer, Leah; Maclean, James M.; Haase, Simone; Trilling, Anke K.; Liu, Shihui; Leppla, Stephen H.; Geren, Isin N.; Pawlik, Jennifer; Peterson, Johnny W.

2011-01-01

26

Pathogenesis of the Lethal Effect of Anthrax Toxin in the Rat. Ii. Morphologic Studies.  

National Technical Information Service (NTIS)

Intravenous injections of anthrax toxin consistently produce pulmonary edema, cyanosis, and death in Fischer 344 rats. A light and electron microscopic study of the lungs of rats sacrificed serially after injections of toxin revealed diffuse pulmonary ede...

F. A. Beall F. G. Dalldorf

1965-01-01

27

Multicomponent anthrax toxin display and delivery using bacteriophage T4.  

PubMed

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases. PMID:17069938

Shivachandra, Sathish B; Li, Qin; Peachman, Kristina K; Matyas, Gary R; Leppla, Stephen H; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

2006-10-17

28

Chloroquine prevents T lymphocyte suppression induced by anthrax lethal toxin.  

PubMed

Lysosomal processing of lethal toxin (LTX) is a key event in the pathogenesis of anthrax. This study investigated the ability of chloroquine (CQ) to interfere with this processing and thereby to reduce suppression of T lymphocytes. T lymphocytes isolated from blood were activated, by cross-linking of CD3, in both the absence and presence of LTX and CQ and then were assayed by flow cytometry and immunoblotting. LTX was found to disrupt intracellular signaling, and it down-regulated T lymphocyte function. CQ significantly reduced the harmful effects of LTX and protected the activation and cytokine production of T lymphocytes. This effect may indicate a promising strategy in the treatment of anthrax. PMID:16960789

Hirsh, Mark I; Cohen, Victoria

2006-08-29

29

Nasal immunization with a dual antigen anthrax vaccine induced strong mucosal and systemic immune responses against toxins and bacilli  

Microsoft Academic Search

Anthrax-vaccine-adsorbed (AVA), the only anthrax vaccine licensed in the U.S., suffers from many major drawbacks. Therefore, there is a need to develop new generation anthrax vaccines that can be easily administered and induce strong immune responses not only against the anthrax toxins, but also against the toxin-producing vegetative anthrax bacilli. In the present study, we evaluated the feasibility of inducing

Brian R. Sloat; Zhengrong Cui

2006-01-01

30

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-beta-Lactamase Fusion Protein  

Microsoft Academic Search

BackgroundTo initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.Methodology\\/Principal FindingsTo measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin

Haijing Hu; Stephen H. Leppla; Adam J. Ratner

2009-01-01

31

Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor  

Microsoft Academic Search

Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host. The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene. The ATR\\/TEM8-PA interaction is mediated by the receptor's extracellular domain

Heather M. Scobie; G. Jonah A. Rainey; Kenneth A. Bradley; John A. T. Young

2003-01-01

32

Anthrax lethal toxin induces cell death-independent permeability in zebrafish vasculature  

PubMed Central

Vascular dysfunction has been reported in human cases of anthrax, in mammalian models of Bacillus anthracis, and in animals injected with anthrax toxin proteins. To examine anthrax lethal toxin effects on intact blood vessels, we developed a zebrafish model that permits in vivo imaging and evaluation of vasculature and cardiovascular function. Vascular defects monitored in hundreds of embryos enabled us to define four stages of phenotypic progression leading to circulatory dysfunction. We demonstrated increased endothelial permeability as an early consequence of toxin action by tracking the extravasation of fluorescent microspheres in toxin-injected embryos. Lethal toxin did not induce a significant amount of cell death in embryonic tissues or blood vessels, as shown by staining with acridine orange, and endothelial cells in lethal toxin-injected embryos continued to divide at the normal rate. Vascular permeability is strongly affected by the VEGF/vascular permeability factor (VPF) signaling pathway, and we were able to attenuate anthrax lethal toxin effects with chemical inhibitors of VEGFR function. Our study demonstrates the importance of vascular permeability in anthrax lethal toxin action and the need for further investigation of the cardiovascular component of human anthrax disease.

Bolcome, Robert E.; Sullivan, Sarah E.; Zeller, Rene; Barker, Adam P.; Collier, R. John; Chan, Joanne

2008-01-01

33

Anthrax  

MedlinePLUS

... worried about anthrax germs being grown as a weapon. The issue of laboratory-grown B. anthracis received ... technologically difficult to use anthrax effectively as a weapon on a large scale. Types of Anthrax The ...

34

Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin  

Microsoft Academic Search

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and

Julia Y Wang; Michael H Roehrl

2005-01-01

35

The medicinal chemistry of botulinum, ricin and anthrax toxins.  

PubMed

The potential use of weapons of mass destruction (nuclear, biological or chemical) by terrorist organizations represents a major threat to world peace and safety. Only a limited number of vaccines are available to protect the general population from the medical consequences of these weapons. In addition there are major health concerns associated with a pre-exposure mass vaccination of the general population. To reduce or eliminate the impact of these terrible threats, new drugs must be developed to safely treat individuals exposed to these agents. A review of all therapeutic agents under development for the treatment of the illnesses and injuries that result from exposure to nuclear, biological or chemical warfare agents is beyond the scope of any single article. The intent here is to provide a focused review for medicinal and organic chemists of three widely discussed and easily deployed biological warfare agents, botulinum neurotoxin and ricin toxins and the bacteria Bacillus anthracis. Anthrax will be addressed because of its similarity in both structure and mechanism of catalytic activity with botulinum toxin. The common feature of these three agents is that they exhibit their biological activity via toxin enzymatic hydrolysis of a specific bond in their respective substrate molecules. A brief introduction to the history of each of the biological warfare agents is presented followed by a discussion on the mechanisms of action of each at the molecular level, and a review of current potential inhibitors under investigation. PMID:15790305

Hicks, Rickey P; Hartell, Mark G; Nichols, Daniel A; Bhattacharjee, Apurba K; van Hamont, John E; Skillman, Donald R

2005-01-01

36

Evidence against a Human Cell-Specific Role for LRP6 in Anthrax Toxin Entry  

Microsoft Academic Search

The role of the cellular protein LRP6 in anthrax toxin entry is controversial. Previous studies showed that LRP6 was important for efficient intoxication of human M2182 prostate carcinoma cells but other studies performed with cells from gene-knockout mice demonstrated no role for either LRP6 or the related LRP5 protein in anthrax toxin entry. One possible explanation for this discrepancy is

Patricia L. Ryan; John A. T. Young; Debbie Fox

2008-01-01

37

A dually active anthrax vaccine that confers protection against both bacilli and toxins  

Microsoft Academic Search

Systemic anthrax is caused by unimpeded bacillar replication and toxin secretion. We developed a dually active anthrax vaccine (DAAV) that confers simultaneous protection against both bacilli and toxins. DAAV was constructed by conjugating capsular poly--D-glutamic acid (PGA) to protective antigen (PA), converting the weakly immunogenic PGA to a potent immunogen, and synergistically enhancing the humoral response to PA. PGA-specific antibodies

Gi-Eun Rhie; Michael H. Roehrl; Michael Mourez; R. John Collier; John J. Mekalanos; Julia Y. Wang

2003-01-01

38

The Early Humoral Immune Response to Bacillus anthracis Toxins in Patients Infected with Cutaneous Anthrax  

PubMed Central

Bacillus anthracis, the causative agent of anthrax, elaborates a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF) which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin (LT) and edema toxin (ET), respectively. In this preliminary study we characterised the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF with IgG detected as early as 4 days after onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US AVA and UK AVP licensed anthrax vaccines was directed against PA.. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.

Doganay, Mehmet; Brenneman, Karen E.; Akmal, Arya; Goldman, Stanley; Galloway, Darrell R.; Mateczun, Alfred J.; Cross, Alan S.; Baillie, Leslie W.

2012-01-01

39

Potent antitumor activity of a urokinase-activated engineered anthrax toxin  

NASA Astrophysics Data System (ADS)

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

Liu, Shihui; Aaronson, Hannah; Mitola, David J.; Leppla, Stephen H.; Bugge, Thomas H.

2003-01-01

40

Anthrax.  

PubMed

Anthrax is an ancient disease associated with the plagues in biblical Egypt and modern bioterrorism. Three clinical syndromes result from exposure to anthrax spores: cutaneous,inhalational, and gastrointestinal. Cutaneous anthrax is the most common naturally occurring syndrome; inhalational anthrax is most likely to result from airborne release of spores. Prophylactic and early treatment can improve the mortality from inhalational anthrax. A vaccine is available, but has many limitations. New vaccines are currently being developed. PMID:15207306

Wenner, Kimberly A; Kenner, Julie R

2004-07-01

41

From structure to solutions: the role of basic research in developing anthrax countermeasures: Microbiology Graduate Program Seminar: Anthrax toxin.  

PubMed

Dr. John Collier traced the discoveries that elucidated the structure and function of the anthrax toxin in his talk "Anthrax Toxin," which was part of the Microbiology Graduate Program Seminar Series at Yale School of Medicine on February 23, 2012. Dr. Collier, Professor of Microbiology and Immunobiology at Harvard University, began by noting the advantages to studying anthrax pathogenesis in a biosafety level-1 lab. This designation does not merely facilitate his research, but also reflects a larger trend of basic research being leveraged to develop translational applications. Basic research on toxin structure has led to the development of a vaccine by Dr. Collier's group. Next-generation prophylactics also may stem from recent discoveries uncovering a role for cellular cofactors that mediate toxin function. Finally, basic research into the toxin substructure has facilitated efforts to change the receptor tropism to target dysregulated cells for therapeutic purposes. The urgency around biodefense agents makes the choice of research priorities a salient issue. As such, this author submits that basic research occupies a unique and lucrative niche driving clinical applications. PMID:22737057

Hardiman, Camille A

2012-06-25

42

Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin  

PubMed Central

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.

Wang, Julia Y; Roehrl, Michael H

2005-01-01

43

Anthrax  

Center for Biologics Evaluation and Research (CBER)

... However, the United States military views anthrax as a potential biological terrorism ... the licensed anthrax vaccine was conducted in US mill workers ... More results from www.fda.gov/biologicsbloodvaccines/vaccines

44

Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax  

NASA Astrophysics Data System (ADS)

The protective antigen moiety of anthrax toxin translocates the toxin's enzymic moieties to the cytosol of mammalian cells by a mechanism that depends on its ability to heptamerize and insert into membranes. We identified dominant-negative mutants of protective antigen that co-assemble with the wild-type protein and block its ability to translocate the enzymic moieties across membranes. These mutants strongly inhibited toxin action in cell culture and in an animal intoxication model, suggesting that they could be useful in therapy of anthrax.

Sellman, Bret R.; Mourez, Michael; John Collier, R.

2001-04-01

45

Anthrax Toxin-Mediated Delivery of a Cytotoxic T-Cell Epitope in vivo  

Microsoft Academic Search

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In

Jimmy D. Ballard; R. John Collier; Michael N. Starnbach

1996-01-01

46

Direct Inhibition of T-Lymphocyte Activation by Anthrax Toxins In Vivo  

PubMed Central

The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.

Comer, Jason E.; Chopra, Ashok K.; Peterson, Johnny W.; Konig, Rolf

2005-01-01

47

Anthrax toxin receptor 2a controls mitotic spindle positioning.  

PubMed

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis. PMID:23201782

Castanon, I; Abrami, L; Holtzer, L; Heisenberg, C P; van der Goot, F G; González-Gaitán, M

2012-12-02

48

Anthrax Edema Toxin Inhibits Nox1-Mediated Formation of Reactive Oxygen Species by Colon Epithelial Cells  

Microsoft Academic Search

One major route of intoxication by Bacillus anthracis (anthrax) spores is via their ingestion and subsequent uptake by the intestinal epithelium. Anthrax edema toxin (ETx) is an adenylate cyclase that causes persistent elevation of cAMP in intoxicated cells. NADPH oxidase enzymes (Nox1–Nox5, Duox1 and 2) generate reactive oxygen species (ROS) as components of the host innate immune response to bacteria,

Jun-Sub Kim; Gary M. Bokoch

2009-01-01

49

Analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action.  

PubMed

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

Ngundi, Miriam M; Meade, Bruce D; Little, Stephen F; Quinn, Conrad P; Corbett, Cindi R; Brady, Rebecca A; Burns, Drusilla L

2012-03-21

50

The Potential Contributions of Lethal and Edema Toxins to the Pathogenesis of Anthrax Associated Shock  

PubMed Central

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.

Hicks, Caitlin W.; Cui, Xizhong; Sweeney, Daniel A.; Li, Yan; Barochia, Amisha; Eichacker, Peter Q.

2011-01-01

51

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.  

PubMed Central

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin. Images

Bhatnagar, R; Friedlander, A M

1994-01-01

52

New Developments in Vaccines, Inhibitors of Anthrax Toxins, and Antibiotic Therapeutics for Bacillus anthracis  

PubMed Central

Bacillus anthracis, the causative agent responsible for anthrax infections, poses a significant biodefense threat. There is a high mortality rate associated with untreated anthrax infections; specifically, inhalation anthrax is a particularly virulent form of infection with mortality rates close to 100%, even with aggressive treatment. Currently, a vaccine is not available to the general public and few antibiotics have been approved by the FDA for the treatment of inhalation anthrax. With the threat of natural or engineered bacterial resistance to antibiotics and the limited population for whom the current drugs are approved, there is a clear need for more effective treatments against this deadly infection. A comprehensive review of current research in drug discovery is presented in this article, including efforts to improve the purity and stability of vaccines, design inhibitors targeting the anthrax toxins, and identify inhibitors of novel enzyme targets. High resolution structural information for the anthrax toxins and several essential metabolic enzymes has played a significant role in aiding the structure-based design of potent and selective antibiotics.

Beierlein, J.M.; Anderson, A.C.

2013-01-01

53

Anthrax.  

National Technical Information Service (NTIS)

Anthrax is a virulent, contagious, and potentially fatal disease. The first accounts of anthrax infection were written by the Roman poet Vergil in early antiquity. While its lethal effects were ascribed to the actions of an exotoxin over a half century ag...

A. M. Katos C. J. Hilmas J. Anderson P. T. Williams

2009-01-01

54

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins  

Microsoft Academic Search

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a

Andrei Fokine; Valorie D. Bowman; Anthony J. Battisti; Qin Li; Paul R. Chipman; Venigalla B. Rao; Michael G. Rossmann

2007-01-01

55

Tumor Cell-selective Cytotoxicity of Matrix Metalloproteinase-activated Anthrax Toxin  

Microsoft Academic Search

Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed two mutated anthrax toxin protective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by

Shihui Liu; Sarah Netzel-Arnett; Henning Birkedal-Hansen; Stephen H. Leppla

2000-01-01

56

Anthrax  

MedlinePLUS

... disease due to a type of bacteria called Bacillus anthracis . Infection in humans most often involves the ... Elsevier; 2007:chap 317. Martin GJ, Friedlander Am. Bacillus anthracis (anthrax). In: Mandell GL, Bennett JE, Dolin ...

57

Anthrax  

Center for Biologics Evaluation and Research (CBER)

... has an active vaccination program against ... Anthrax Vaccine Adsorbed (AVA) - BioThrax Package Insert ... November 16, 2010: Vaccines and Related ... More results from www.fda.gov/biologicsbloodvaccines/vaccines

58

Anthrax  

MedlinePLUS

... origin of an anthrax outbreak. Knowing the genetic fingerprint of B. anthracis might lead to gene-based ... part of that research is the functional genomic analysis of the bacterium, which should lead to new ...

59

Anthrax  

MedlinePLUS

... livestock, such as sheep, cattle, horses, goats and camels. Although rare in the United States, anthrax is ... to Controlling High Blood Pressure The Mayo Clinic Diet — Eat well. Enjoy life. Lose weight. Mayo Clinic ...

60

Fusion protein of Delta 27LFn and EFn has the potential as a novel anthrax toxin inhibitor.  

PubMed

PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Delta27LFn) and EFn. In a cell model of intoxication, fusion protein of Delta27LFn and EFn (Delta27LFn-EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Delta27LFn-EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Delta27LFn-EFn has the potential as a candidate therapeutic agent against anthrax. PMID:19332063

Kong, Yirong; Guo, Qiang; Yu, Changming; Dong, Dayong; Zhao, Jian; Cai, Chenguang; Hou, Lihua; Song, Xiaohong; Fu, Ling; Xu, Junjie; Chen, Wei

2009-03-28

61

Computational studies on molecular interactions of 6-thioguanosine analogs with anthrax toxin receptor 1.  

PubMed

Dormant endospores of Bacillus anthracis are the causative agent of anthrax, which is an acute disease for both human and animals. Anthrax has been practised as biological weapon because of two attributes: i) short duration of spore germination, and ii) lethal toxaemia of the vegetative stage. Pathogenesis is caused by the activity of edema toxin and lethal toxin. Protective antigen (PA), is an essential component of both complexes, binds to Anthrax Toxin Receptor (ATR) and mediates the lethality in mammals. The combination of vaccine and antibiotics are preferred to be effective treatment for destruction of the vegetative cell wall but could not be a successive destructor for endospores. So the present study is intended to identify the small molecules as a potential inhibitor for ATR1. 3D structure of Anthrax Toxin Receptor 1 (ATR1) was built by using the crystal structure of Anthrax Toxin Receptor 2 (ATR2) from Homo sapiens as template. Molecular docking of 6-thiogunaosine (6-TG) analogs was performed on the ATR1 model and effective inhibitor was selected based on the docking results. The docking results showed that the three residues in the ATR1 binding pocket (Phe162, Asp160, and Phe22) were essential for making hydrogen bond with the 2-(2-bromo-6-chloro-4H-purin-9(5H)-yl)- 5-(hydroxymethyl) tetrahydrofuran-3,4-diol (C(11)H(13)N(3)O(5)). The data presented here strongly indicate that the interactions of these four residues are necessary for a stronger binding of the ATR1 with C(11)H(13)N(3)O(5). Also, the study proposed C(11)H(13)N(3)O(5) as an effective inhibitor by the comparison of docking energy. PMID:23292691

Singh, Nitin K; Pakkkianathan, Britto C; Kumar, Manish; Daddam, Jayssima R; Jayavel, Sridhar; Kannan, Mani; Pillai, Girinath G; Krishnan, Muthukalingan

2013-01-08

62

Anti-toxin antibodies in prophylaxis and treatment of inhalation anthrax.  

PubMed

The CDC recommend 60 days of oral antibiotics combined with a three-dose series of the anthrax vaccine for prophylaxis after potential exposure to aerosolized Bacillus anthracis spores. The anthrax vaccine is currently not licensed for anthrax postexposure prophylaxis and has to be made available under an Investigational New Drug protocol. Postexposure prophylaxis based on antibiotics can be problematic in cases where the use of antibiotics is contraindicated. Furthermore, there is a concern that an exposure could involve antibiotic-resistant strains of B. anthracis. Availability of alternate treatment modalities that are effective in prophylaxis of inhalation anthrax is therefore highly desirable. A major research focus toward this end has been on passive immunization using polyclonal and monoclonal antibodies against B. anthracis toxin components. Since 2001, significant progress has been made in isolation and commercial development of monoclonal and polyclonal antibodies that function as potent neutralizers of anthrax lethal toxin in both a prophylactic and therapeutic setting. Several new products have completed Phase I clinical trials and are slated for addition to the National Strategic Stockpile. These rapid advances were possible because of major funding made available by the US government through programs such as Bioshield and the Biomedical Advanced Research and Development Authority. Continued government funding is critical to support the development of a robust biodefense industry. PMID:19207098

Schneemann, Anette; Manchester, Marianne

2009-02-01

63

Anthrax toxins induce shock in rats by depressed cardiac ventricular function.  

PubMed

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the microg/mL range with half-lives of 10-20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the microg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections. PMID:17520025

Watson, Linley E; Kuo, Shu-Ru; Katki, Khurshed; Dang, Tongyun; Park, Seong Kyu; Dostal, David E; Tang, Wei-Jen; Leppla, Stephen H; Frankel, Arthur E

2007-05-23

64

Preventing Voltage-dependent Gating of Anthrax Toxin Channels Using Engineered Disulfides  

PubMed Central

The channel-forming component of anthrax toxin, (PA63)7, is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded ?-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ?100 Å length of the channel's ?-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire ?-barrel.

Anderson, Damon S.; Blaustein, Robert O.

2008-01-01

65

Hemodynamic Effects of Anthrax Toxins in the Rabbit Model and the Cardiac Pathology Induced by Lethal Toxin  

PubMed Central

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.

Lawrence, William S.; Marshall, Jeffrey R.; Zavala, Diana L.; Weaver, Lori E.; Baze, Wallace B.; Moen, Scott T.; Whorton, Elbert B.; Gourley, Randy L.; Peterson, Johnny W.

2011-01-01

66

Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen  

Microsoft Academic Search

A DNA vaccine encoding the immunogenic and biologically active portion of anthrax protective antigen (PA) was constructed. Spleen cells from BALB\\/c mice immunized intramuscularly with this vaccine were stimulated to secrete IFN? and IL-4 when exposed to PA in vitro. Immunized mice also mounted a humoral immune response dominated by IgG1 anti-PA antibody production, the subclass previously shown to confer

Mi-Li Gu; Stephen H Leppla; Dennis M Klinman

1999-01-01

67

Anthrax  

MedlinePLUS

... People can get anthrax from contact with infected animals, wood, meat, or hides. It can cause three forms of disease in people. They are Cutaneous, which affects the skin. People with cuts or open sores can get it if they touch the bacteria. Inhalation, which affects the lungs. You can get ...

68

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins.  

PubMed

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc(-)Soc(-)T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63)(7) were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer. PMID:17624389

Fokine, Andrei; Bowman, Valorie D; Battisti, Anthony J; Li, Qin; Chipman, Paul R; Rao, Venigalla B; Rossmann, Michael G

2007-07-10

69

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins  

SciTech Connect

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc{sup -}Soc{sup -}T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63){sub 7} were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer.

Fokine, Andrei; Bowman, Valorie D.; Battisti, Anthony J. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Li Qin [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Chipman, Paul R. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Rao, Venigalla B. [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Rossmann, Michael G. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States)], E-mail: mr@purdue.edu

2007-10-25

70

Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge  

PubMed Central

Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics.

Crowe, Sherry R.; Ash, Linda L.; Engler, Renata J. M.; Ballard, Jimmy D.; Harley, John B.; Farris, A. Darise; James, Judith A.

2010-01-01

71

A model of anthrax toxin lethal factor bound to protective antigen  

Microsoft Academic Search

Anthrax toxin is made up of three proteins: the edema factor (EF), lethal factor (LF) enzymes, and the multifunctional protective antigen (PA). Proteolytically activated PA heptamerizes, binds the EF\\/LF enzymes, and forms a pore that allows for EF\\/LF passage into host cells. Using directed mutagenesis, we identified three LF-PA contact points defined by a specific disulfide crosslink and two pairs

D. Borden Lacy; Henry C. Lin; Roman A. Melnyk; Ora Schueler-Furman; Laura Reither; Kristina Cunningham; David Baker; R. John Collier

2005-01-01

72

Anthrax Lethal Toxin Triggers the Formation of a Membrane-Associated Inflammasome Complex in Murine Macrophages  

Microsoft Academic Search

Multiple microbial components trigger the formation of an inflammasome complex that contains pathogen- specific nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs), caspase-1, and in some cases the scaffolding protein ASC. The NLR protein Nalp1b has been linked to anthrax lethal toxin (LT)- mediated cytolysis of murine macrophages. Here we demonstrate that in unstimulated J774A.1 macrophages, caspase-1 and Nalp1b are

Adel M. Nour; Laura Santambrogio; Eric D. Boyden; E. Richard Stanley; Jurgen Brojatsch

2009-01-01

73

Cholera- and Anthrax-Like Toxins Are among Several New ADP-Ribosyltransferases  

PubMed Central

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences – including a primary sequence pattern – to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data – and we need high-throughput validation – our approach provides insight into the newest toxin ADP-ribosyltransferases.

Fieldhouse, Robert J.; Turgeon, Zachari; White, Dawn; Merrill, A. Rod

2010-01-01

74

Dissecting the urokinase activation pathway using urokinase-activated anthrax toxin.  

PubMed

Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1-254, LFn) is sufficient to allow translocation of fused "passenger" polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here, we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo. PMID:19377974

Liu, Shihui; Bugge, Thomas H; Frankel, Arthur E; Leppla, Stephen H

2009-01-01

75

[Anthrax].  

PubMed

Anthrax is a serious bacterial infection sustained by Bacillus anthracis and occurring in most mammals, especially grazing herbivors, but it can also involve humans when bacterial endospores enter the body through abrasions in the skin or by inhalation or ingestion. Human disease results from contact with infected animals or contaminated animal products, while there are no known cases of human-to-human transmission. The most common form of human anthrax is the cutaneous infection, usually curable with antimicrobial therapy and rarely leading to systemic and fatal disease; on the other hand, gastrointestinal and inhalatory forms (resulting from inhalation or ingestion of endospores) are very uncommon, but they show a mortality rate approaching 100 percent, usually related to septic and toxic shock. For centuries, anthrax has caused disease in animals and only a few cases in humans, with some outbreaks in developing countries. Today, after the new events in the United States, at least 17 nations are believed to have offensive biological weapon programs, and this old bacterial infection became a modern menace to world safety. PMID:11822090

Calza, L; Manfredi, R; Chiodo, F

2001-12-01

76

Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines.  

PubMed

Anthrax is caused by the unimpeded growth of Bacillus anthracis in the host and the secretion of toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous when used immediately following exposure to B. anthracis. Thus, we have sought to develop a vaccine or therapeutic agent that is safe and eliminates both secreted toxins and bacilli. To that end, we have previously developed a dually active vaccine by conjugating the capsular poly-gamma-d-glutamate (PGA) with PA to elicit the production of antibodies specific for both bacilli and toxins. In the present report, we describe the improved potency of anthrax vaccines through the use of a dominant-negative inhibitory (DNI) mutant to replace PA in PA or PA-PGA vaccines. When tested in mice, DNI alone is more immunogenic than PA, and DNI-PGA conjugate elicits significantly higher levels of antibodies against PA and PGA than PA-PGA conjugate. To explain the enhanced immunogenicity of DNI, we propose that the two point mutations in DNI may have improved epitopes of PA allowing better antigen presentation to helper T cells. Alternatively, these mutations may enhance the immunological processing of PA by altering endosomal trafficking of the toxin in antigen-presenting cells. Because DNI has previously been demonstrated to inhibit anthrax toxin, postexposure use of DNI-based vaccines, including conjugate vaccines, may provide improved immunogenicity and therapeutic activity simultaneously. PMID:15908368

Aulinger, Benedikt A; Roehrl, Michael H; Mekalanos, John J; Collier, R John; Wang, Julia Y

2005-06-01

77

Anthrax Lethal Toxin Downregulates Claudin-5 Expression in Human Endothelial Tight Junctions  

PubMed Central

Vascular leakage pathologies such as pleural effusion and hemorrhage are hallmarks of anthrax pathogenesis. We previously reported that anthrax lethal toxin (LT), the major virulence factor of anthrax, reduces barrier function in cultured primary human microvascular endothelial cells. Here, we show that LT-induced barrier dysfunction is accompanied by the reduced expression of the endothelial tight junction (TJ) protein claudin-5 but no change in the expression of other TJ components occludin, ZO-1, ZO-2, or the adherens junction (AJ) protein VE-cadherin. The downregulation of claudin-5 correlated temporally and dose-dependently with the reduction of transendothelial electrical resistance. LT-induced loss of claudin-5 was independent of cell death and preceded the appearance of actin stress fibers and altered AJ morphology. Pharmacological inhibition of MEK-1/2, two kinases that are proteolytically inactivated by LT, showed a similar reduction in claudin-5 expression. We found that LT reduced claudin-5 mRNA levels but did not accelerate the rate of claudin-5 degradation. Mice challenged with LT also showed significant reduction in claudin-5 expression. Together, these findings support a possible role for LT disruption of endothelial TJs in the vascular leakage pathologies of anthrax.

D'Agnillo, Felice; Williams, Matthew C.; Moayeri, Mahtab; Warfel, Jason M.

2013-01-01

78

Anthrax lethal toxin downregulates claudin-5 expression in human endothelial tight junctions.  

PubMed

Vascular leakage pathologies such as pleural effusion and hemorrhage are hallmarks of anthrax pathogenesis. We previously reported that anthrax lethal toxin (LT), the major virulence factor of anthrax, reduces barrier function in cultured primary human microvascular endothelial cells. Here, we show that LT-induced barrier dysfunction is accompanied by the reduced expression of the endothelial tight junction (TJ) protein claudin-5 but no change in the expression of other TJ components occludin, ZO-1, ZO-2, or the adherens junction (AJ) protein VE-cadherin. The downregulation of claudin-5 correlated temporally and dose-dependently with the reduction of transendothelial electrical resistance. LT-induced loss of claudin-5 was independent of cell death and preceded the appearance of actin stress fibers and altered AJ morphology. Pharmacological inhibition of MEK-1/2, two kinases that are proteolytically inactivated by LT, showed a similar reduction in claudin-5 expression. We found that LT reduced claudin-5 mRNA levels but did not accelerate the rate of claudin-5 degradation. Mice challenged with LT also showed significant reduction in claudin-5 expression. Together, these findings support a possible role for LT disruption of endothelial TJs in the vascular leakage pathologies of anthrax. PMID:23626836

D'Agnillo, Felice; Williams, Matthew C; Moayeri, Mahtab; Warfel, Jason M

2013-04-23

79

Glycogen Synthase Kinase 3 Activation Is Important for Anthrax Edema Toxin-Induced Dendritic Cell Maturation and Anthrax Toxin Receptor 2 Expression in Macrophages ?  

PubMed Central

Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3?. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocyte-derived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

Larabee, Jason L.; Maldonado-Arocho, Francisco J.; Pacheco, Sergio; France, Bryan; DeGiusti, Kevin; Shakir, Salika M.; Bradley, Kenneth A.; Ballard, Jimmy D.

2011-01-01

80

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors  

PubMed Central

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a ?-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.

Abrami, Laurence; Bischofberger, Mirko; Kunz, Beatrice; Groux, Romain; van der Goot, F. Gisou

2010-01-01

81

LRP5 and LRP6 Are Not Required for Protective Antigen–Mediated Internalization or Lethality of Anthrax Lethal Toxin  

Microsoft Academic Search

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor–related protein LRP6 has been reported to mediate

John J Young; Jennifer L Bromberg-White; Cassandra Zylstra; Joseph T Church; Elissa Boguslawski; James H Resau; Bart O Williams; Nicholas S Duesbery

2007-01-01

82

Anthrax toxin protective antigen--Insights into molecular switching from prepore to pore  

PubMed Central

The protective antigen is a key component of the anthrax toxin, as it allows entry of the enzymatic components edema factor and lethal factor into the host cell, through the formation of a membrane spanning pore. This event is absolutely critical for the pathogenesis of anthrax, and although we have yet to understand the mechanism of pore formation, recent developments have provided key insights into how this process may occur. Based on the available data, a model is proposed for the kinetic steps for protective antigen conversion from prepore to pore. In this model, the driving force for pore formation is the formation of the phi (?)-clamp, a region that forms a leak-free seal around the translocating polypeptide. Formation of the ?-clamp elicits movements within the prepore that provide steric freedom for the subsequent conformational changes required to form the membrane spanning pore.

Bann, James G

2012-01-01

83

Anthrax Lethal Toxin Suppresses Murine Cardiomyocyte Contractile Function and Intracellular Ca2+ Handling via a NADPH Oxidase-Dependent Mechanism  

Microsoft Academic Search

ObjectivesAnthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.MethodsMurine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening\\/ relengthening

Machender R. Kandadi; Yinan Hua; Heng Ma; Qun Li; Shu-ru Kuo; Arthur E. Frankel; Jun Ren

2010-01-01

84

Characterization of the native form of anthrax lethal factor for use in the toxin neutralization assay.  

PubMed

The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply. PMID:23637044

Lu, Hang; Catania, Jason; Baranji, Katalin; Feng, Jie; Gu, Mili; Lathey, Janet; Sweeny, Diane; Sanford, Hannah; Sapru, Kavita; Patamawenu, Terry; Chen, June-Home; Ng, Alan; Fesseha, Zenbework; Kluepfel-Stahl, Stefanie; Minang, Jacob; Alleva, David

2013-05-01

85

Multiplexed Detection of Anthrax-Related Toxin Genes  

PubMed Central

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Moser, Michael J.; Christensen, Deanna R.; Norwood, David; Prudent, James R.

2006-01-01

86

Monitoring of ELISA-reactive antibodies against anthrax protective antigen (PA), lethal factor (LF), and toxin-neutralising antibodies in serum of individuals vaccinated against anthrax with the PA-based UK anthrax vaccine  

Microsoft Academic Search

The human anthrax vaccines currently licensed contain the protective antigen (PA) of Bacillus anthracis as main antigen together with traces of some other bacillus components, e.g. lethal factor (LF). The present study aimed at monitoring the course of specific antibody titres against PA and LF by enzyme linked immunosorbent assays (ELISA), as well as the levels of toxin-neutralising antibodies, in

Roland Grunow; Mustafa Porsch-Özcürümez; Wolf Splettstoesser; Arno Buckendahl; Ulrike Hahn; Wolfgang Beyer; Reinhard Böhm; Maria Huber; Ulrich vd Esche; Wolfgang Bessler; Dimitrios Frangoulidis; Ernst-Jürgen Finke

2007-01-01

87

Role of macrophage oxidative burst in the action of anthrax lethal toxin.  

PubMed Central

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

Hanna, P. C.; Kruskal, B. A.; Ezekowitz, R. A.; Bloom, B. R.; Collier, R. J.

1994-01-01

88

Electrostatic Ratchet in the Protective Antigen Channel Promotes Anthrax Toxin Translocation*  

PubMed Central

Central to the power-stroke and Brownian-ratchet mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the ratchet mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of the host cell under the proton motive force (PMF). The PA-binding domains of LF and EF (LFN and EFN) possess identical folds and similar solution stabilities; however, EFN translocates ?10–200-fold slower than LFN, depending on the electrical potential (??) and chemical potential (?pH) compositions of the PMF. From an analysis of LFN/EFN chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EFN. These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a ?? or a ?pH; the second requires basic residues only when driven by a ??. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding.

Wynia-Smith, Sarah L.; Brown, Michael J.; Chirichella, Gina; Kemalyan, Gigi; Krantz, Bryan A.

2012-01-01

89

Cross-Reactivity of Anthrax and C2 Toxin: Protective Antigen Promotes the Uptake of Botulinum C2I Toxin into Human Endothelial Cells  

PubMed Central

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB7/8 type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA63) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA63 binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA63). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

Beitzinger, Christoph; Stefani, Caroline; Leuber, Michael; Flatau, Gilles; Popoff, Michel R.; Benz, Roland; Lemichez, Emmanuel

2011-01-01

90

Crystallographic studies of the anthrax lethal toxin. Final report, 1 July 1994-31 December 1996  

SciTech Connect

Protective Antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthraces, the organism responsible for anthrax. Following proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes into the cytosol. We have solved the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel b-sheets and has four domains: an N-terminal domain containing two calcium ions; a heptamerization domain containing a large flexible loop implicated in membrane insertion; a small domain of unknown function; and a C-terminal receptor-binding domain. Removal of a 20 kDa fragment from the N-terminal domain permits assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We present a model of pH-dependent membrane insertion involving formation of a porin-like membrane-spanning b barrel. These studies greatly enhance current understanding of the mechanism of anthrax intoxication, and will be useful in the design of recombinant anthrax vaccines.

Frederick, C.A.

1997-01-01

91

Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin.  

PubMed

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network. Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo. Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data suggest a role for LT in suppressing host immunity during B. anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response. PMID:12867985

Agrawal, Anshu; Lingappa, Jai; Leppla, Stephen H; Agrawal, Sudhanshu; Jabbar, Abdul; Quinn, Conrad; Pulendran, Bali

2003-07-17

92

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

PubMed Central

SUMMARY 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 specifically neutrophils, is essential to anthrax infection.

Liu, Shihui; Miller-Randolph, Sharmina; Crown, Devorah; Moayeri, Mahtab; Sastalla, Inka; Okugawa, Shu; Leppla, Stephen H.

2010-01-01

93

Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung  

PubMed Central

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.

Dumas, Eric K.; Cox, Philip M.; Fullenwider, Charles O'Connor; Nguyen, Melissa; Centola, Michael; Frank, Mark Barton; Dozmorov, Igor; James, Judith A.; Farris, A. Darise

2011-01-01

94

Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin  

Microsoft Academic Search

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the

JIMMY D. BALLARD; AMY M. DOLING; KATHRYN BEAUREGARD; R. JOHN COLLIER; MICHAEL N. STARNBACH

1998-01-01

95

Induction of Protective Immunity to Anthrax Lethal Toxin with a Nonhuman Primate Adenovirus-Based Vaccine in the Presence of Preexisting Anti-Human Adenovirus Immunity  

Microsoft Academic Search

Prevention or therapy for bioterrorism-associated anthrax infections requires rapidly acting effective vac- cines. We recently demonstrated (Y. Tan, N. R. Hackett, J. L. Boyer, and R. G. Crystal, Hum. Gene Ther. 14:1673-1682, 2003) that a single administration of a recombinant serotype 5 adenovirus (Ad) vector express- ing anthrax protective antigen (PA) provides rapid protection against anthrax lethal toxin challenge. However,

Masahiko Hashimoto; Julie L. Boyer; Neil R. Hackett; James M. Wilson; Ronald G. Crystal

2005-01-01

96

Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity ?  

PubMed Central

Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.

deCathelineau, Aimee M.; Bokoch, Gary M.

2009-01-01

97

Protective Antigen Antibody Augments Hemodynamic Support in Anthrax Lethal Toxin Shock in Canines  

PubMed Central

Background.?Anthrax-associated shock is closely linked to lethal toxin (LT) release and is highly lethal despite conventional hemodynamic support. We investigated whether protective antigen–directed monoclonal antibody (PA-mAb) treatment further augments titrated hemodynamic support. Methods and Results.?Forty sedated, mechanically ventilated, instrumented canines challenged with anthrax LT were assigned to no treatment (controls), hemodynamic support alone (protocol-titrated fluids and norepinephrine), PA-mAb alone (administered at start of LT infusion [0 hours] or 9 or 12 hours later), or both, and observed for 96 hours. Although all 8 controls died, 2 of 8 animals receiving hemodynamic support alone survived (median survival times 65 vs 85 hours, respectively; P?=?.03). PA-mAb alone at 0 hour improved survival (5 of 5 animals survived), but efficacy decreased progressively with delayed treatment (9 hours, 2 of 3 survived; 12 hours, 0 of 4 survived) (P = .004 comparing survival across treatment times). However, combined treatment increased survival irrespective of PA-mAb administration time (0 hours, 4 of 5 animals; 9 hours, 3 of 3 animals; and 12 hours, 4 of 5 animals survived) (P = .95 comparing treatment times). Compared to hemodynamic support alone, when combined over PA-mAb treatment times (0, 9, and 12 hours), combination therapy produced higher survival (P = .008), central venous pressures, and left ventricular ejection fractions, and lower heart rates, norepinephrine requirements and fluid retention (P ? .03). Conclusions.?PA-mAb may augment conventional hemodynamic support during anthrax LT-associated shock.

Barochia, Amisha V.; Cui, Xizhong; Sun, Junfeng; Solomon, Steven B.; Migone, Thi-Sau; Subramanian, G. Mani; Bolmer, Sally D.

2012-01-01

98

Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy  

PubMed Central

BACKGROUND AND PURPOSE Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction. EXPERIMENTAL APPROACH Wild-type (WT) and TLR4 knockout (TLR?/?) mice were challenged with lethal toxin (2 µg·g?1, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP–LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2? and calreticulin). KEY RESULTS In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca2+ derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells. CONCLUSION AND IMPLICATIONS Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.

Kandadi, Machender R; Frankel, Arthur E; Ren, Jun

2012-01-01

99

Proteasome Inhibitors Prevent Caspase-1-Mediated Disease in Rodents Challenged with Anthrax Lethal Toxin  

PubMed Central

NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases.

Muehlbauer, Stefan M.; Lima, Heriberto; Goldman, David L.; Jacobson, Lee S.; Rivera, Johanna; Goldberg, Michael F.; Palladino, Michael A.; Casadevall, Arturo; Brojatsch, Jurgen

2010-01-01

100

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax  

PubMed Central

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA? mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA? mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

Drysdale, Melissa; Koehler, Theresa M.; Hutt, Julie A.; Lyons, C. Rick

2012-01-01

101

Anthrax Infection  

PubMed Central

Bacillus anthracis infection is rare in developed countries. However, recent outbreaks in the United States and Europe and the potential use of the bacteria for bioterrorism have focused interest on it. Furthermore, although anthrax was known to typically occur as one of three syndromes related to entry site of (i.e., cutaneous, gastrointestinal, or inhalational), a fourth syndrome including severe soft tissue infection in injectional drug users is emerging. Although shock has been described with cutaneous anthrax, it appears much more common with gastrointestinal, inhalational (5 of 11 patients in the 2001 outbreak in the United States), and injectional anthrax. Based in part on case series, the estimated mortalities of cutaneous, gastrointestinal, inhalational, and injectional anthrax are 1%, 25 to 60%, 46%, and 33%, respectively. Nonspecific early symptomatology makes initial identification of anthrax cases difficult. Clues to anthrax infection include history of exposure to herbivore animal products, heroin use, or clustering of patients with similar respiratory symptoms concerning for a bioterrorist event. Once anthrax is suspected, the diagnosis can usually be made with Gram stain and culture from blood or surgical specimens followed by confirmatory testing (e.g., PCR or immunohistochemistry). Although antibiotic therapy (largely quinolone-based) is the mainstay of anthrax treatment, the use of adjunctive therapies such as anthrax toxin antagonists is a consideration.

Sweeney, Daniel A.; Hicks, Caitlin W.; Cui, Xizhong; Li, Yan

2011-01-01

102

Charge Requirements for Proton Gradient-driven Translocation of Anthrax Toxin*  

PubMed Central

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (?pH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ?pH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ?pH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LFN) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LFN to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LFN translocate independently of the ?pH driving force. Acidic residues markedly increase the rate of ?pH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ?pH-driven unfolding and translocation. We discuss a ?pH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the “molecular teeth” of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ?pH translocation.

Brown, Michael J.; Thoren, Katie L.; Krantz, Bryan A.

2011-01-01

103

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.  

PubMed Central

Anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death of rats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of these proteins have not been determined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacillus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm. Images

Leppla, S H

1982-01-01

104

Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge  

Microsoft Academic Search

The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce

Vijay Koya; Mahtab Moayeri; Stephen H. Leppla; Henry Daniell

2005-01-01

105

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells.  

PubMed

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to ?-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore. PMID:23716698

Slater, Louise H; Hett, Erik C; Clatworthy, Anne E; Mark, Kevin G; Hung, Deborah T

2013-05-28

106

Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen  

PubMed Central

Individually, the three protein components of anthrax toxin are nontoxic, but when they assemble, they form active holotoxin complexes. The role of the protective antigen (PA) component of the toxin is to deliver the two other enzyme components, lethal factor (LF) and edema factor (EF), across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated, and generally cell-surface activation is mediated by a furin-family protease. Activated PA can then assemble into one of two non-interconverting oligomers, a homoheptamer and homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA Domain 4 to Domain 2 with two different length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of Domain 4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin-cleavage site and show that for efficient processing, the furin site cannot be moved ~5 or 6 residues within the loop. We propose that there are different orientations of Domain 4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

Feld, Geoffrey K.; Kintzer, Alexander F.; Tang, Iok I; Thoren, Katie L.; Krantz, Bryan A.

2011-01-01

107

Protein translocation through anthrax toxin channels formed in planar lipid bilayers.  

PubMed

The 63-kDa fragment of the protective antigen (PA) component of anthrax toxin forms a heptameric channel, (PA63)7, in acidic endosomal membranes that leads to the translocation of edema factor (EF) and lethal factor (LF) to the cytosol. It also forms a channel in planar phospholipid bilayer membranes. What role does this channel play in the translocation of EF and LF? We report that after the 263-residue N-terminal piece of LF (LFN) binds to its receptor on the (PA63)7 channel and its N-terminal end enters the channel at small positive voltages to block it, LFN is translocated through the channel to the opposite side at large positive voltages, thereby unblocking it. Thus, all of the translocation machinery is contained in the (PA63)7 channel, and translocation does not require any cellular proteins. The kinetics of this translocation are S-shaped, voltage-dependent, and occur on a timescale of seconds. We suggest that the translocation process might be explained simply by electrophoresis of unfolded LFN through the channel, but the refolding of the N-terminal half of LFN as it emerges from the channel may also provide energy for moving the rest of the molecule through the channel. PMID:15377524

Zhang, Sen; Udho, Eshwar; Wu, Zhengyan; Collier, R John; Finkelstein, Alan

2004-09-17

108

Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.  

PubMed

Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter. PMID:16343527

Krantz, Bryan A; Finkelstein, Alan; Collier, R John

2005-12-01

109

Electrical graphene aptasensor for ultra-sensitive detection of anthrax toxin with amplified signal transduction.  

PubMed

Detection of the anthrax toxin, the protective antigen (PA), at the attomolar (aM) level is demonstrated by an electrical aptamer sensor based on a chemically derived graphene field-effect transistor (FET) platform. Higher affinity of the aptamer probes to PA in the aptamer-immobilized FET enables significant improvements in the limit of detection (LOD), dynamic range, and sensitivity compared to the antibody-immobilized FET. Transduction signal enhancement in the aptamer FET due to an increase in captured PA molecules results in a larger 30 mV/decade shift in the charge neutrality point (Vg,min ) as a sensitivity parameter, with the dynamic range of the PA concentration between 12 aM (LOD) and 120 fM. An additional signal enhancement is obtained by the secondary aptamer-conjugated gold nanoparticles (AuNPs-aptamer), which have a sandwich structure of aptamer/PA/aptamer-AuNPs, induce an increase in charge-doping in the graphene channel, resulting in a reduction of the LOD to 1.2 aM with a three-fold increase in the Vg,min shift. PMID:23589198

Kim, Duck-Jin; Park, Hae-Chul; Sohn, Il Yung; Jung, Jin-Heak; Yoon, Ok Ja; Park, Joon-Shik; Yoon, Moon-Young; Lee, Nae-Eung

2013-04-16

110

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR  

PubMed Central

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin-like protease on the cell surface followed by the formation of a donut-shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1-dimensional 13C-edited 1H NMR that low pH induces dissociation of the Von-Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon-13 labeled, 2-fluorohistidine labeled CMG2, consistent with studies showing that protonation of His-121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.

Rajapaksha, Maheshinie; Eichler, Jack F; Hajduch, Jan; Anderson, David E; Kirk, Kenneth L; Bann, James G

2009-01-01

111

Anthrax lethal toxin and the induction of CD4 T cell immunity.  

PubMed

Bacillus anthracis secretes exotoxins which act through several mechanisms including those that can subvert adaptive immunity with respect both to antigen presenting cell and T cell function. The combination of Protective Antigen (PA) and Lethal Factor (LF) forming Lethal Toxin (LT), acts within host cells to down-regulate the mitogen activated protein kinase (MAPK) signaling cascade. Until recently the MAPK kinases were the only known substrate for LT; over the past few years it has become evident that LT also cleaves Nlrp1, leading to inflammasome activation and macrophage death. The predicted downstream consequences of subverting these important cellular pathways are impaired antigen presentation and adaptive immunity. In contrast to this, recent work has indicated that robust memory T cell responses to B. anthracis antigens can be identified following natural anthrax infection. We discuss how LT affects the adaptive immune response and specifically the identification of B. anthracis epitopes that are both immunogenic and protective with the potential for inclusion in protein sub-unit based vaccines. PMID:23162703

Ascough, Stephanie; Ingram, Rebecca J; Altmann, Daniel M

2012-10-19

112

Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed  

Microsoft Academic Search

BACKGROUND: Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model. METHODS: Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe

Ritsuko Sawada-Hirai; Ivy Jiang; Fei Wang; Shu Man Sun; Rebecca Nedellec; Paul Ruther; Alejandro Alvarez; Diane Millis; Phillip R Morrow; Angray S Kang

2004-01-01

113

Anthrax Lethal Toxin Suppresses Murine Cardiomyocyte Contractile Function and Intracellular Ca2+ Handling via a NADPH Oxidase-Dependent Mechanism  

PubMed Central

Objectives Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties. Methods Murine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (?FFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis. Results In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ properties (PS, ± dL/dt, ?FFI), along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure. Conclusions Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca2+ through a NADPH oxidase-dependent mechanism.

Kandadi, Machender R.; Hua, Yinan; Ma, Heng; Li, Qun; Kuo, Shu-ru; Frankel, Arthur E.; Ren, Jun

2010-01-01

114

Role of the protective antigen octamer in the molecular mechanism of anthrax lethal toxin stabilization in plasma.  

PubMed

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-gamma-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood. PMID:20433851

Kintzer, Alexander F; Sterling, Harry J; Tang, Iok I; Abdul-Gader, Ali; Miles, Andrew J; Wallace, B A; Williams, Evan R; Krantz, Bryan A

2010-04-28

115

Disulfide Bonds in the Ectodomain of Anthrax Toxin Receptor 2 Are Required for the Receptor-Bound Protective-Antigen Pore to Function  

PubMed Central

Background Cell-surface receptors play essential roles in anthrax toxin action by providing the toxin with a high-affinity anchor and self-assembly site on the plasma membrane, mediating the toxin entry into cells through endocytosis, and shifting the pH threshold for prepore-to-pore conversion of anthrax toxin protective antigen (PA) to a more acidic pH, thereby inhibiting premature pore formation. Each of the two known anthrax toxin receptors, ANTXR1 and ANTXR2, has an ectodomain comprised of an N-terminal von Willebrand factor A domain (VWA), which binds PA, and an uncharacterized immunoglobulin-like domain (Ig) that connects VWA to the membrane-spanning domain. Potential roles of the receptor Ig domain in anthrax toxin action have not been investigated heretofore. Methodology/Principal Findings We expressed and purified the ANTXR2 ectodomain (R2-VWA-Ig) in E. coli and showed that it contains three disulfide bonds: one in R2-VWA and two in R2-Ig. Reduction of the ectodomain inhibited functioning of the pore, as measured by K+ release from liposomes or Chinese hamster ovary cells or by PA-mediated translocation of a model substrate across the plasma membrane. However, reduction did not affect binding of the ectodomain to PA or the transition of ectodomain-bound PA prepore to the pore conformation. The inhibitory effect depended specifically on reduction of the disulfides within R2-Ig. Conclusions/Significance We conclude that disulfide integrity within R2-Ig is essential for proper functioning of receptor-bound PA pore. This finding provides a novel venue to investigate the mechanism of anthrax toxin action and suggests new strategies for inhibiting toxin action.

Sun, Jianjun; Collier, R. John

2010-01-01

116

High-level production of a single chain antibody against anthrax toxin in Escherichia coli by high cell density cultivation  

Microsoft Academic Search

Previously, we isolated the M18 scFv, which is an affinity matured antibody against the anthrax toxin PA, and observed that\\u000a its single chain antibody (scAb) form (M18 scAb) exhibited superior stability compared to the scFv. Here, we report high cell\\u000a density cultivations for preparative scale production of M18 scAb in a 3.5 L fermenter. Briefly, a pH–stat feeding strategy\\u000a was employed

Ki Jun Jeong; Mridula Rani

117

A kinetic analysis of protein transport through the anthrax toxin channel  

PubMed Central

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell’s endosomal membrane, disrupting cellular homeostasis. (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LFN, the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LFN molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LFNs. In this paper, we show both in macroscopic and single-channel experiments that even with only one LFN bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LFN bound. We also present a simple electrodiffusion model of translocation in which LFN is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Kienker, Paul K.; Briggs, Stephen W.; Finkelstein, Alan

2011-01-01

118

65. Protective Immunity Against Anthrax Lethal Toxin in Mice Immunized with an Ad-Based Vaccine Vector Expressing Lethal Factor  

Microsoft Academic Search

Dissemination of anthrax spores in a terrorism event could result in inhalational anthrax, an infection that resulted in 45% mortality in the recent US anthrax attacks. The focus of this study is to use adenovirus gene transfer vectors as a rapidly-acting highly-defined anthrax vaccine. Bacillus anthracis, the etiologic agent of anthrax, produces two exotoxins that are associated with the fatal

Julie L. Boyer; Tiffany B. Niven; Neil R. Hackett; Ronald G. Crystal

2005-01-01

119

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity  

NASA Astrophysics Data System (ADS)

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

2000-07-01

120

Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines  

Microsoft Academic Search

Complexities of lethal challenge models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA and a lethal toxin neutralization assay (TNA) were used to measure antibody response to Protective Antigen (PA) in mice immunized once with either a commercial or a recombinant PA (rPA) vaccine formulated in-house. Even though ELISA and TNA results showed

P. M. Parreiras; L. A. Sirota; L. D. Wagner; S. L. Menzies; J. L. Arciniega

2009-01-01

121

Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin  

PubMed Central

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope—OVA257–264 from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA257–264-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA257–264 fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA257–264-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA257–264. These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

Ballard, Jimmy D.; Doling, Amy M.; Beauregard, Kathryn; Collier, R. John; Starnbach, Michael N.

1998-01-01

122

Anthrax Lethal and Edema Toxins Produce Different Patterns of Cardiovascular and Renal Dysfunction and Synergistically Decrease Survival in Canines  

PubMed Central

Background. High mortality in the 2001 US and recent European anthrax outbreaks suggests that better understanding of the effects of the toxins produced by this bacterium is needed to improve treatment. Methods and results. Here, 24-h edema (ETx) and lethal (LeTx) toxin infusions were investigated for 96 h in sedated canines receiving mechanical ventilation. The initial study compared similarly lethal doses of ETx (n=8) or LeTx (n=15) alone. ETx was 24 times less lethal than LeTx, and the median time to death in nonsurvivors (n=6 and n=9, respectively) was shorter with ETx (42 vs 67 h; P=.04). Compared with controls (n=9), both toxins decreased arterial and central venous pressures and systemic vascular resistance and increased heart rate, cardiac index, blood urea nitrogen (BUN) level, creatinine (Cr) concentration, BUN:Cr ratio, and hepatic transaminase levels (P ? .05 for toxin effect or time interaction). However, ETx stimulated early diuresis, reduced serum sodium levels, and had more pronounced vasodilatory effects, compared with LeTx, as reflected by greater or earlier central venous pressures, systemic vascular resistance, and changes in the BUN:Cr ratio (P ? .01). LeTx progressively decreased the left ventricular ejection fraction (P ? .002). In a subsequent study, a lethal dose of LeTx with an equimolar nonlethal ETx dose (n=8) increased mortality, compared with LeTx alone (n=8;P=.05). Conclusion. Shock with ETx or LeTx may require differing supportive therapies, whereas toxin antagonists should likely target both toxins.

Sweeney, Daniel A.; Cui, Xizhong; Solomon, Steven B.; Vitberg, David A.; Migone, Thi S.; Scher, Dara; Danner, Robert L.; Natanson, Charles; Subramanian, G. Mani; Eichacker, Peter Q.

2010-01-01

123

Anthrax--an overview.  

PubMed

Anthrax, a disease of mammals (including humans), is caused by a spore-forming Gram-positive bacilli called Bacillus anthracis. Anthrax is one of the oldest threats to humanity, and remains endemic in animals in many parts of the world. The incidence of anthrax has decreased in developed countries, but it remains a considerable health problem in developing countries. The disease is transmitted to humans by contact with sick animals or their products, such as wool, skin, meat etc. Capsular polypeptide and anthrax toxin are the principal virulence factors of B. anthracis. Anthrax toxin consists of three proteins called protective antigen, edema factor, and lethal factor, each of which is nontoxic but acts synergistically. Human anthrax has three major clinical forms: cutaneous, inhalational, and gastrointestinal. The diagnosis is easily established in cutaneous cases, characterized by black eschar. Severe intoxication and collapse during the course of bronchopneumonia or hemorrhagic enteritis should prompt suspicion of anthrax. Treatment with antibiotics is mandatory. If untreated, anthrax in all forms can lead to septicemia and death. Recently, considerable attention has been focused on the potential for B. anthracis to be used in acts of biological terrorism. The ease of laboratory production and its dissemination via aerosol led to its adoption by terrorists, as shown by recent events in the USA. A good knowledge of anthrax, its epidemiology, pathogenesis, clinical forms and potential as a biological weapon is essential for timely prevention and treatment. This review summarizes the current knowledge on anthrax. PMID:14586293

Oncü, Serkan; Oncü, Selcen; Sakarya, Serhan

2003-11-01

124

Regulation of Anthrax Toxin-Specific Antibody Titers by Natural Killer T Cell-Derived IL-4 and IFN?  

PubMed Central

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand ?-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFN? to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4?/? mice and IFN??/? mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing ?-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4?/? mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFN?. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFN? also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by ?-GC derivatives can be exploited to optimize anthrax vaccination.

Devera, T. Scott; Joshi, Sunil K.; Aye, Lindsay M.; Lang, Gillian A.; Ballard, Jimmy D.; Lang, Mark L.

2011-01-01

125

Tumor therapy with a urokinase plasminogen activator-activated anthrax lethal toxin alone and in combination with paclitaxel.  

PubMed

PA-U2, an engineered anthrax protective antigen that is activated by urokinase was combined with wildtype lethal factor in the treatment of Colo205 colon adenocarcinoma in vitro and B16-BL6 mouse melanoma in vitro and in vivo. This therapy was also tested in combination with the small molecule paclitaxel, based on prior reports suggesting synergy between ERK1/2 inhibition and chemotherapeutics. Colo205 was sensitive to PA-U2/LF while B16-BL6 was not. For the combination treatment of B16-BL6, paclitaxel showed a dose response in vitro, but cells remained resistant to PA-U2/LF even in the presence of paclitaxel. In vivo, each therapy slowed tumor progression, and an additive effect between the two was observed. Since LF targets tumor vasculature while paclitaxel is an antimitotic, it is possible the agents were acting against different cells in the stroma, precluding a synergistic effect. The engineered anthrax toxin PA-U2/LF warrants further development and testing, possibly in combination with an antiangiogenesis therapy such as sunitinib or sorafinib. PMID:22843210

Wein, Alexander N; Liu, Shihui; Zhang, Yi; McKenzie, Andrew T; Leppla, Stephen H

2012-07-28

126

Assembly of anthrax toxin pore: lethal-factor complexes into lipid nanodiscs.  

PubMed

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (?43%). Three-dimensional analysis of negatively stained single particles revealed the LFN -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN -PA nanodisc structures and use of advanced automated particle selection methods. PMID:23389868

Akkaladevi, N; Hinton-Chollet, L; Katayama, H; Mitchell, J; Szerszen, L; Mukherjee, S; Gogol, E P; Pentelute, B L; Collier, R J; Fisher, M T

2013-02-26

127

Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles  

Microsoft Academic Search

We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng\\/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma

Shixing Tang; Mahtab Moayeri; Zhaochun Chen; Harri Harma; Jiangqin Zhao; Haijing Hu; Robert H. Purcell; Stephen H. Leppla; Indira K. Hewlett

2009-01-01

128

Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model  

PubMed Central

Objectives The in vivo inflammatory effects of the Bacillus anthracis cell wall are unknown. We therefore investigated these effects in rats and, for comparison, those of known inflammatory stimulants, Staphylococcus aureus cell wall or lipopolysaccharide (LPS). Method and Results Sprague–Dawley rats (n = 103) were challenged with increasing B. anthracis cell wall doses (10, 20, 40, 80, or 160 mg/kg) or diluent (control) as a bolus or 24-h infusion. The three highest bolus doses were lethal (20–64% lethality rates) as were the two highest infused doses (13% with each). Comparisons among lethal or nonlethal doses on other measured parameters were not significantly different, and these were combined for analysis. Over the 24 h after challenge initiation with lethal bolus or infusion, compared to controls, ten inflammatory cytokines and NO levels were increased and circulating neutrophils and platelets decreased (P ? 0.05). Changes with lethal doses were greater than changes with nonlethal doses (P ? 0.01). Lethal bolus or infusion doses produced hypotension or hypoxemia, respectively (P ? 0.05). The effects with B. anthracis cell wall were similar to those of S. aureus cell wall or LPS. However, paradoxically administration of B. anthracis cell wall or LPS decreased the lethality of concurrently administered B. anthracis lethal toxin (P < 0.0001 and 0.04, respectively). Conclusion B. anthracis cell wall has the potential to produce inflammatory injury during anthrax infection clinically. However, understanding why cell wall or LPS paradoxically reduced lethality with lethal toxin may help understand this toxin’s pathogenic effects.

Cui, Xizhong; Su, Junwu; Li, Yan; Shiloach, Joseph; Solomon, Steven; Kaufman, Jeanne B.; Mani, Haresh; Fitz, Yvonne; Weng, Jia; Altaweel, Laith; Besch, Virginia; Eichacker, Peter Q.

2012-01-01

129

Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity.  

PubMed

We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax(®)) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18-24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P<0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66?g/mL before and 887.82?g/mL after the boost (P<0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series. PMID:24026013

Pittman, Phillip R; Fisher, Diana; Quinn, Xiaofei; Schmader, Trevor; Barrera-Oro, Julio G

2013-09-08

130

Anthrax Toxin as a Molecular Tool for Stimulation of Cytotoxic T Lymphocytes: Disulfide-Linked Epitopes, Multiple Injections, and Role of CD41 Cells  

Microsoft Academic Search

We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of

JIMMY D. BALLARD; R. JOHN COLLIER; MICHAEL N. STARNBACH

131

Germline Humanization of a Non-human Primate Antibody that Neutralizes the Anthrax Toxin, by in Vitro and in Silico Engineering  

Microsoft Academic Search

Fab 35PA83 is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part

Thibaut Pelat; Hugues Bedouelle; Anthony R. Rees; Susan J. Crennell; Marie-Paule Lefranc; Philippe Thullier

2008-01-01

132

Anthrax Toxin Receptor 1 / Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding†  

PubMed Central

Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies.

Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.

2010-01-01

133

Interlaboratory comparison of results of an anthrax lethal toxin neutralization assay for assessment of functional antibodies in multiple species.  

PubMed

The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED(50)) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED(50)s for all samples and within 7.5% of the quotients of the test sample ED(50) and the reference standard ED(50) (NF(50)s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED(50)s and 35% for the NF(50)s. These precision data suggest that the NF(50) readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision. PMID:18417668

Omland, Kristian S; Brys, April; Lansky, David; Clement, Kristin; Lynn, Freyja

2008-04-16

134

Interlaboratory Comparison of Results of an Anthrax Lethal Toxin Neutralization Assay for Assessment of Functional Antibodies in Multiple Species?  

PubMed Central

The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED50) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED50s for all samples and within 7.5% of the quotients of the test sample ED50 and the reference standard ED50 (NF50s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED50s and 35% for the NF50s. These precision data suggest that the NF50 readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision.

Omland, Kristian S.; Brys, April; Lansky, David; Clement, Kristin; Lynn, Freyja

2008-01-01

135

Identification of a Receptor-Binding Region within Domain 4 of the Protective Antigen Component of Anthrax Toxin  

PubMed Central

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity.

Varughese, Mini; Teixeira, Avelino V.; Liu, Shihui; Leppla, Stephen H.

1999-01-01

136

Both CD4+ and CD8+ T Cells Respond to Antigens Fused to Anthrax Lethal Toxin  

Microsoft Academic Search

The lethal toxin produced by Bacillus anthracis is a bipartite toxin in which the first protein, protective antigen (PA), transports the second protein, lethal factor, across the host cell membrane. We have previously shown that CD8 T-cell epitopes fused to a nontoxic derivative of lethal factor (LFn) are delivered into the host cell cytosol in a PA-dependent manner. Delivery of

Christine A. Shaw; Michael N. Starnbach

2008-01-01

137

In Vitro and In Vivo Characterization of Anthrax AntiProtective Antigen and Anti-Lethal Factor Monoclonal Antibodies after Passive Transfer in a Mouse Lethal Toxin Challenge Model To Define Correlates of Immunity  

Microsoft Academic Search

Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of

Herman F. Staats; S. Munir Alam; Richard M. Scearce; Shaun M. Kirwan; Julia Xianzhi Zhang; William M. Gwinn; Barton F. Haynes

2007-01-01

138

Rapid Vascular Responses to Anthrax Lethal Toxin in Mice Containing a Segment of Chromosome 11 from the CAST\\/Ei Strain on a C57BL\\/6 Genetic Background  

Microsoft Academic Search

Host allelic variation controls the response to B. anthracis and the disease course of anthrax. Mouse strains with macrophages that are responsive to anthrax lethal toxin (LT) show resistance to infection while mouse strains with LT non-responsive macrophages succumb more readily. B6.CAST.11M mice have a region of chromosome 11 from the CAST\\/Ei strain (a LT responsive strain) introgressed onto a

Kelsey J. Weigel; Laura Rues; Edward J. Doyle; Cassandra L. Buchheit; John G. Wood; Ryan J. Gallagher; Laura E. Kelly; Jeffrey D. Radel; Kenneth A. Bradley; Steven M. LeVine

2012-01-01

139

Combinations of monoclonal antibodies to anthrax toxin manifest new properties in neutralization assays.  

PubMed

Monoclonal antibodies (MAbs) are potential therapeutic agents against Bacillus anthracis toxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in an in vitro mouse macrophage system but did not provide significant protection in vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection both in vitro and in vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralization in vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection with B. anthracis strain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy. PMID:23509144

Pohl, Mary Ann; Rivera, Johanna; Nakouzi, Antonio; Chow, Siu-Kei; Casadevall, Arturo

2013-03-18

140

Monitoring the Kinetics of the pH-Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance.  

PubMed

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å ? barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions. PMID:23964683

Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari; Tally, Jon; McGinn-Straub, Wesley; Zhang, Na; Gao, Phillip; Gogol, E P; Pentelute, B L; Collier, R John; Fisher, Mark T

2013-09-09

141

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins  

PubMed Central

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA63)7, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA63)7 incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ?100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LFN (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LFN adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LFN in the normal direction but also can, at least partially, translocate LFN in the opposite direction.

Jennings-Antipov, Laura D.; Jakes, Karen S.; Finkelstein, Alan

2011-01-01

142

Both CD4+ and CD8+ T cells respond to antigens fused to anthrax lethal toxin.  

PubMed

The lethal toxin produced by Bacillus anthracis is a bipartite toxin in which the first protein, protective antigen (PA), transports the second protein, lethal factor, across the host cell membrane. We have previously shown that CD8(+) T-cell epitopes fused to a nontoxic derivative of lethal factor (LFn) are delivered into the host cell cytosol in a PA-dependent manner. Delivery of these antigens targets them to the intracellular major histocompatibility complex (MHC) class I processing and presentation pathway and leads to the stimulation of antigen-specific CD8(+) T cells in vivo. In this report, we describe the generation and characterization of LFn fusion proteins that include not only a CD8(+) T-cell epitope but also a CD4(+) T-cell epitope. We first show that these fusion proteins induce antigen-specific CD4(+) T-cell responses following incubation with dendritic cells in vitro or injection into mice. Stimulation of CD4(+) T cells by LFn fusion proteins does not require PA but is enhanced by PA in vitro. We also show that a single LFn fusion protein and PA can deliver antigen to both the MHC class II and the MHC class I pathways, resulting in the simultaneous induction of antigen-specific CD4(+) T cells and antigen-specific CD8(+) T cells in the same mouse. These results suggest that this toxin delivery system is capable of stimulating protective immune responses where effective immunization requires stimulation of both classes of T cells. PMID:18347032

Shaw, Christine A; Starnbach, Michael N

2008-03-17

143

Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen  

Microsoft Academic Search

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensi- tive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Cen- ters for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective anti- gen (PA) in

Conrad P. Quinn; Vera A. Semenova; Cheryl M. Elie; Sandra Romero-Steiner; Carolyn Greene; Han Li; Karen Stamey; Evelene Steward-Clark; Daniel S. Schmidt; Elizabeth Mothershed; Janet Pruckler; Stephanie Schwartz; Robert F. Benson; Leta O. Helsel; Patricia F. Holder; Scott E. Johnson; Molly Kellum; Trudy Messmer; W. Lanier Thacker; Lilah Besser; Brian D. Plikaytis; Thomas H. Taylor; Alison E. Freeman; Kelly J. Wallace; Peter Dull; Jim Sejvar; Erica Bruce; Rosa Moreno; Anne Schuchat; Jairam R. Lingappa; Nina Marano; Sandra K. Martin; John Walls; Melinda Bronsdon; George M. Carlone; Mary Bajani-Ari; David A. Ashford; David S. Stephens; Bradley A. Perkins

144

Development of a Comprehensive, Validated Pharmacophore Hypothesis for Anthrax Toxin Lethal Factor (LF) Inhibitors Using Genetic Algorithms, Pareto Scoring, and Structural Biology  

PubMed Central

Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), an 89-kDa zinc hydrolase secreted by the bacilli, is the toxin component chiefly responsible for pathogenesis and has been a popular target for rational and structure-based drug design. Although hundreds of small-molecule compounds have been designed to target the LF active site, relatively few reported inhibitors have exhibited activity in cell-based assays and no LF inhibitor is currently available to treat or prevent anthrax. This study presents a new pharmacophore map assembly, validated by experiment, designed to rapidly identify and prioritize promising LF inhibitor scaffolds from virtual compound libraries. The new hypothesis incorporates structural information from all five available LF enzyme-inhibitor complexes deposited in the Protein Data Bank (PDB) and is the first LF pharmacophore map reported to date that includes features representing interactions involving all three key subsites of the LF catalytic binding region. In a wide-ranging validation study on all 546 compounds for which published LF biological activity data exist, this model displayed strong selectivity toward nanomolar-level LF inhibitors, successfully identifying 72.1% of existing nanomolar-level compounds in an unbiased test set, while rejecting 100% of weakly active (>100 µM) compounds. In addition to its capabilities as a database searching tool, this comprehensive model points to a number of key design principles and previously unidentified ligand-receptor interactions that are likely to influence compound potency.

Chiu, Ting-Lan; Amin, Elizabeth A.

2012-01-01

145

Binding of radiolabeled Staphylococcus aureus delta-toxin to human erythrocytes.  

PubMed Central

The addition f [3H]isoleucine to a chemically defined medium resulted in the production of delta-toxin (DT) with a high specific radioactivity (0.47 microCi/mg). The purified tritium-labeled toxin ([3H]DT) was found to migrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with a molecular weight of 1,600. Upon electrofocusing, [3H]DT yielded one major peak (pI = 5.90) and two minor peaks (pI = 5.10, 6.95) of radioactivity. The percentage of [3H]DT associated with pelleted fractions of intact erythrocytes or erythrocyte ghosts remained fairly constant over a 100-fold range of toxin concentrations. Erythrocyte ghosts, however, bound more [3H]DT than did intact erythrocytes when exposed to the same concentration of toxin. Erythrocytes maintained in isotonic sucrose were more susceptible to toxin than erythrocytes suspended in saline, but did not bind more [3H]DT. The binding of [3H]DT to erythrocyte ghosts was found to be temperature dependent from 0 to 20 degrees C but was constant from 20 to 50 degrees C. Images

Nolte, F S; Kapral, F A

1981-01-01

146

Anthrax Lethal Toxin-Mediated Disruption of Endothelial VE-Cadherin Is Attenuated by Inhibition of the Rho-Associated Kinase Pathway  

PubMed Central

Systemic anthrax disease is characterized by vascular leakage pathologies. We previously reported that anthrax lethal toxin (LT) induces human endothelial barrier dysfunction in a cell death-independent manner with actin stress fiber formation and disruption of adherens junctions (AJs). In the present study, we further characterize the molecular changes in the AJ complex and investigate whether AJ structure and barrier function can be preserved by modulating key cytoskeletal signaling pathways. Here, we show that LT reduces total VE-cadherin protein and gene expression but the expression of the key linker protein beta-catenin remained unchanged. The changes in VE-cadherin expression correlated temporally with the appearance of actin stress fibers and a two-fold increase in phosphorylation of the stress fiber-associated protein myosin light chain (p-MLC) and cleavage of Rho-associated kinase-1 (ROCK-1). Co-treatment with ROCK inhibitors (H-1152 and Y27632), but not an inhibitor of MLC kinase (ML-7), blocked LT-induced p-MLC enhancement and stress fiber formation. This was accompanied by the restoration of VE-cadherin expression and membrane localization, and attenuation of the LT-induced increase in monolayer permeability to albumin. Together, these findings suggest the ROCK pathway may be a relevant target for countering LT-mediated endothelial barrier dysfunction.

Warfel, Jason M.; D'Agnillo, Felice

2011-01-01

147

Anthrax Vaccine  

MedlinePLUS

... its most common form, anthrax is a skin disease that causes skin ulcers and usually fever and fatigue. Up to 20% of these cases are fatal if untreated.Gastrointestinal Anthrax. This form of anthrax can result from eating raw or undercooked infected meat. Symptoms ...

148

Pharmacology: Screening inhibitors of anthrax lethal factor  

Microsoft Academic Search

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat

Fiorella Tonello; Michela Seveso; Oriano Marin; Michèle Mock; Cesare Montecucco

2002-01-01

149

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein  

PubMed Central

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

Goletz, Theresa J.; Klimpel, Kurt R.; Arora, Naveen; Leppla, Stephen H.; Keith, Jerry M.; Berzofsky, Jay A.

1997-01-01

150

Effect of Anthrax Immune Globulin on Response to BioThrax (Anthrax Vaccine Adsorbed) in New Zealand White Rabbits.  

PubMed

Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals. PMID:23979740

Malkevich, Nina V; Basu, Subhendu; Rudge, Thomas L; Clement, Kristin H; Chakrabarti, Ajoy C; Aimes, Ronald T; Nabors, Gary S; Skiadopoulos, Mario H; Ionin, Boris

2013-08-26

151

Secretory expression and efficient purification of recombinant anthrax toxin lethal factor with full biological activity in E. coli.  

PubMed

Lethal factor (LF), a virulence factor of Bacillus anthracis, plays key roles in anthrax pathogenesis and host-pathogen interactions. The detailed mechanisms by which LF contributes to infection are still under investigation. While these studies require pure, homogeneous and reliable LF preparations, most methods reported for production of recombinant LF (rLF) in B. anthracis or Escherichia coli either are complicated or add extra residues to the protein. In this work, we modified our previous method by codon optimization and chromatograph workflow refinement and developed an improved strategy for efficient production of rLF from the periplasm of E. coli. We were able to obtain fully functional rLF with a purity above 95% and with a considerable yield of 5 mg/L. The preparation was characterized by SDS-PAGE, Western blot, and N-terminal sequencing, and the activity was validated by intoxication of macrophages and Fischer 344 rats. Our final product is suitable for most research involving drug development and mechanism analysis of anthrax pathogenesis. PMID:23459291

Liu, Ju; Cai, Chenguang; Guo, Qiang; Zhang, Jun; Dong, Dayong; Li, Guanlin; Fu, Ling; Xu, Junjie; Chen, Wei

2013-02-28

152

Cutaneous Anthrax  

MedlinePLUS

... happen when a person handles infected animals or contaminated animal products like wool, hides, or hair. Cutaneous anthrax is most common on the head, neck, forearms, and hands. It affects the skin and tissue around the site of infection. Cutaneous anthrax is the most common ...

153

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel.  

PubMed

Entry of the enzymatic components of anthrax toxin [lethal factor (LF) and edema factor] into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA63) component to form a channel (pore) in the membrane of an acidic intracellular compartment. To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA63-binding domain of LF (LFN). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H6-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer. On the basis of these findings, we propose that the N terminus of PA63-bound LF or edema factor enters the PA63-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction. PMID:15548616

Zhang, Sen; Finkelstein, Alan; Collier, R John

2004-11-17

154

Ultra-fast pg/ml anthrax toxin (protective antigen) detection assay based on microwave-accelerated metal-enhanced fluorescence.  

PubMed

Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA. PMID:22406431

Dragan, Anatoliy I; Albrecht, Mark T; Pavlovic, Radmila; Keane-Myers, Andrea M; Geddes, Chris D

2012-03-06

155

Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice.  

National Technical Information Service (NTIS)

Bacillus cereus G9241 was isolated from a welder with a pulmonary anthrax-like illness. The organism contains two megaplasmids, pBCXOl and pBC218. These plasmids are analogous to the Baci/11.1s anthracis Ames plasmids pXOl and pX02 that encode anthrax tox...

A. M. Keane-Myers F. Alem J. M. Vergis J. R. Palmer M. K. Wilson

2011-01-01

156

Antigen Delivered by Anthrax Lethal Toxin Induces the Development of Memory CD8+ T Cells That Can Be Rapidly Boosted and Display Effector Functions?  

PubMed Central

Memory CD8+ T cells are essential for protective immunity against many intracellular pathogens; therefore, stimulation of this population of cells is an important goal of vaccination. We have previously shown that a detoxified derivative of Bacillus anthracis anthrax lethal toxin (LT) can deliver heterologous CD8+ T-cell epitopes to the major histocompatibility complex class I processing and presentation pathway of murine host cells and that immunization of mice with these LT-antigen fusion proteins leads to the induction of antigen-specific CD8+ T cells. In this report we extend these findings to include a detailed characterization of the phenotypic and functional properties of the T cells stimulated by the LT-based system. We found that after an initial period of expansion and contraction, antigen-specific CD8+ T cells differentiated into a pool of memory cells that produced gamma interferon and displayed in vivo cytotoxic activity. The transition to memory cells appeared to be quite rapid based on an analysis of the phenotypic marker CD127 and the effectiveness of a booster immunization administered early after the initial immunization. We also investigated the composition of the memory T-cell pool induced by this system and found that while one immunization induced a mixture of effector memory T cells (CD62Llow) and central memory T cells (CD62Lhigh), a second immunization preferentially elevated the effector memory T-cell frequency. Finally, we demonstrated that mice that received prime-boost immunizations of LT-antigen proteins were more protected in a Listeria monocytogenes challenge model than mice that received only one immunization.

Shaw, Christine A.; Starnbach, Michael N.

2008-01-01

157

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance  

PubMed Central

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921- 942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.

1990-01-01

158

Anthrax: pre-publication and special issue  

NSDL National Science Digital Library

This special topics Webpage from Nature contains two pre-publication research papers and a collection of articles, news stories, and commentary from Nature's archive. The two pre-pubs, Bradley et al.'s "Identification of the cellular receptor for anthrax toxin" and "Crystal structure of the anthrax lethal factor" by Pannifer et al., should be useful to researchers and scientists working on treatments for anthrax. The two other feature articles here, "Designing a polyvalent inhibitor of anthrax toxin" by Mourez et al. and "Genomics and future biological weapons: the need for preventive action by the biomedical community," by Fraser et al., come from October issues of Nature Biotechnology and Nature Genetics respectively. Interested members of the general public should find the collection of Nature news stories, which cover a range of issues related to bioweapons and defense, a worthwhile read. All material is available in HTML or .pdf formats.

2001-01-01

159

Neutralizing Antibodies and Persistence of Immunity following Anthrax Vaccination  

Microsoft Academic Search

Anthrax toxin consists of protective antigen (PA) and two toxic components, lethal factor (LF) and edema factor (EF). PA binds to mammalian cellular receptors and delivers the toxic components to the cytoplasm. PA is the primary antigenic component of the current anthrax vaccine. Immunity is due to the generation of antibodies that prevent the PA-mediated internalization of LF and EF.

James F. Hanson; Sarah C. Taft; Alison A. Weiss

2006-01-01

160

Neutron-based sterilization of anthrax contamination.  

PubMed

With the anthrax threat becoming a reality, it is very important to have an effective way to sterilize areas contaminated by anthrax. Anthrax spores are the dormant form of the anthrax bacteria. They can germinate in tissues, producing new bacteria that release lethal toxins. Neutrons can be a powerful tool in our defense against anthrax contamination. Neutrons are elementary particles that have no charge, which allows them to be very penetrating, killing the anthrax spores on the surface and inside the containers. So neutrons have an advantage over other forms of radiation if deep penetration is required to kill biological organisms. A Cf neutron source allows for a low cost method of decontamination. It emits most neutrons in the 100 keV to 2 MeV energy regions, and a neutron in this energy region is 20 times more deadly than electrons or gamma rays in killing anthrax spores. If we just consider the first neutron collision with anthrax spores and that all the anthrax spores will not survive at the dose level above 2.0 x 10 Gy, our calculations show that a 0.5-g Cf neutron source within 20 min can generate 1.11 x 10 m fluence neutrons, which is good enough to kill the anthrax spores on the sample. An experimental confirmation of the above results may prove that to achieve 1.11 x 10 m fluence neutrons on the anthrax spore sample, the neutron irradiation time may be reduced dramatically or the Cf neutron source reduced to 0.1 g level or even less. The aim of this paper is to evaluate a feasible way to sterilize the anthrax contamination by using a Cf neutron source. Presently, we are mainly concentrating on the theoretical estimation of neutron fluence to see if the Cf neutron source can deliver enough neutron irradiation dose to kill the anthrax spores. Our future work will focus on experimental confirmation and Monte Carlo simulation by using Geant4 or MCNP codes. At that time, we will consider the effects of the real experimental setup, the shielding materials, the exact chemical components, and the biological structures of anthrax spores. We also need to consider the ways of carrying the anthrax spores, and this includes surface contamination, inside an envelope, or hidden in sealed metal containers and luggage. PMID:16607173

Liu, Bin; Wang, Qingfei

2006-05-01

161

Scientists Report New Lead in How Anthrax Kills Cells  

Cancer.gov

For years scientists have known that anthrax bacillus produces a toxin containing a deadly protein called lethal factor. However, researchers have never been able to identify how lethal factor kills cells.

162

Inhalational anthrax.  

PubMed

Inhalational anthrax is a lethal infection acquired from the inhalation of Bacillus anthracis, a pathogen classified as a Category A bioterrorist agent by the Centers for Disease Control and Prevention. The recent 2001 attack in which weaponized spores were delivered by mail to several US cities exposed our vulnerability to bioterrorism, and taught us important lessons in the timely diagnosis of this devastating disease. It is clear that patient mortality is significantly decreased by early recognition and immediate administration of antibiotic therapy. Unfortunately, the nonspecific clinical presentation is often misinterpreted as a flu-like illness and confirmatory microbiologic tests may take up to 24 hours. Radiologic manifestations, however, are distinctive and may prove essential in directing appropriate clinical care in the critical early hours of inhalational anthrax. PMID:17110848

Frazier, Aletta Ann; Franks, Teri J; Galvin, Jeffrey R

2006-11-01

163

Inhalational Anthrax.  

PubMed

Until recently, inhalational anthrax was a medical curiosity in both the Western medical literature and clinical practice. The post-September 11, 2001 outbreak of this disease in the eastern United States that spread through the mail, however, instantly changed the appreciation of this disease and the appreciation of biological terrorism/warfare in general. The microbiology, epidemiology, clinical, and therapeutic/preventative aspects of this entity, classically known as "wool sorter's disease" are highlighted in this review. PMID:12015917

Cullamar, Erwin Kurt; Lutwick, Larry I.

2002-06-01

164

Vaccines: countering anthrax: vaccines and immunoglobulins.  

PubMed

Anthrax spores rank as the leading threat among bioweapons. This article reviews the accumulated evidence for immunization, either active or passive, to counter the malicious release of anthrax spores. The key protective factor in current anthrax vaccines for humans is a protein called protective antigen, which allows ingress of toxins into cells. The US vaccine is licensed to prevent anthrax, regardless of the route of exposure. Its dosing schedule is cumbersome and somewhat painful (shortcomings that may be resolved by ongoing clinical studies). It can be prescribed with the confidence commensurate with dozens of human safety studies and experience in 1.8 million recent vaccinees. For post-exposure prophylaxis, combining antibiotic prophylaxis and active immunization before illness onset may offer the best combination of prompt and sustained protection, especially for people who inhale large doses of spores. To treat anthrax infection, passive immunization using a polyclonal or monoclonal antibody product may offer important clinical benefit, especially if the anthrax bacteria are resistant to multiple antibiotics. PMID:18171228

Grabenstein, John D

2008-01-01

165

Anthrax Vaccine: A Review.  

National Technical Information Service (NTIS)

Anthrax is a zoonotic disease, primarily of ruminants, that is caused by Bacillus anthracis. The three most common forms of anthrax are cutaneous, inhalational, and gastrointestinal. In the 1870s, Robert Koch cultured B anthracis and first established the...

J. D. Grabenstein

2003-01-01

166

Effect of Temperature and Drug Therapy on Anthrax Intoxication.  

National Technical Information Service (NTIS)

In contrast to other known bacterial toxins and venoms, challenge with anthrax toxin resulted in a hypothermia which is as great as 14-15C in animals stressed by holdings at 4C. In comparison with animals held at room temperature following challenge, mean...

F. Klein R. E. Lincoln B. G. Mahlandt J. O. Dodds J. S. Walker

1966-01-01

167

Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin  

Microsoft Academic Search

Bacillus anthracis is the etiological agent of anthrax and the bacterium produces a tripartite anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA represents the binding domain of the toxin and acts in concert with either LF, a metalloprotease, or EF, an adenylate cyclase, to form lethal toxin (LeTx) or edema toxin (EdTx), respectively.

R. Sapra; S. P. Gaucher; J. S. Lachmann; G. M. Buffleben; G. S. Chirica; J. E. Comer; J. W. Peterson; A. K. Chopra; A. K. Singh

2006-01-01

168

Serotherapy of Anthrax.  

National Technical Information Service (NTIS)

The use of serum for anthrax has more justification than the use of neosalvarsan. The anthrax serum should be used in all serious cases. In view of the possibility of sepsis in the median and mild forms of anthrax, it is sometimes necessary to utilize ser...

B. G. Popkova L. A. Abramovich

1968-01-01

169

ANTHRAX TECHNICAL ASSISTANCE DOCUMENT  

EPA Science Inventory

The Anthrax TAD was developed as an Interim Draft Final technical resource in November 2003. It is specifically for response to an actual or suspected terrorist release of anthrax (i.e., it is not intended for response to anthrax in agricultural settings.). The TAD was provided ...

170

Biological Terrorism The Anthrax Scare of 2001  

NSDL National Science Digital Library

In the weeks following the September 11, 2001, terrorist attacks on the World Trade Center and the Pentagon, anthrax-laced envelopes were mailed to individuals in government and the news media. Thousands were treated for exposure, and five people were killed. At the same time, scientists solved the last remaining pieces of the anthrax puzzle and the mechanism of infection of the anthrax toxin is now well understood. Developed for a second-semester biochemistry course, this case presents students with a wealth of biochemical, microbiological, and immunological material to analyze. It also explores important societal issues related to national preparedness against bioterrorist attacks, funding for biodefense research, and the use and misuse of antibiotic therapy.

Cornely, Kathleen A.

2005-01-01

171

Cys-Cys Cross-Linking Shows Contact between the N-Terminus of Lethal Factor and Phe427 of the Anthrax Toxin Pore  

PubMed Central

Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.

2011-01-01

172

A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen  

Microsoft Academic Search

Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA.

Nicholas Kushner; Dong Zhang; Neal Touzjian; Max Essex; Judy Lieberman; Yichen Lu

2003-01-01

173

Study of Immunization against Anthrax with the Purified Recombinant Protective Antigen of Bacillus anthracis  

Microsoft Academic Search

Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a

YOGENDRA SINGH; BRUCE E. IVINS; STEPHEN H. LEPPLA; S. Army; Fort Detrick

1998-01-01

174

Anthrax capsule vaccine protects against experimental infection  

Microsoft Academic Search

Efficacy of a poly-?-d-glutamic acid anthrax capsule vaccine was assessed in a mouse model of infection. Capsule by itself was protective against lethal challenge with a toxin?, capsule+Bacillus anthracis strain. Conjugation of capsule to bovine serum albumin resulted in enhanced IgG anti-capsule antibodies measured by ELISA, but completely abrogated the protection. The protective unconjugated capsule vaccine elicited significantly higher IgM

Donald J. Chabot; Angelo Scorpio; Steven A. Tobery; Stephen F. Little; Sarah L. Norris; Arthur M. Friedlander

2004-01-01

175

Rapid Vascular Responses to Anthrax Lethal Toxin in Mice Containing a Segment of Chromosome 11 from the CAST/Ei Strain on a C57BL/6 Genetic Background  

PubMed Central

Host allelic variation controls the response to B. anthracis and the disease course of anthrax. Mouse strains with macrophages that are responsive to anthrax lethal toxin (LT) show resistance to infection while mouse strains with LT non-responsive macrophages succumb more readily. B6.CAST.11M mice have a region of chromosome 11 from the CAST/Ei strain (a LT responsive strain) introgressed onto a LT non-responsive C57BL/6J genetic background. Previously, B6.CAST.11M mice were found to exhibit a rapid inflammatory reaction to LT termed the early response phenotype (ERP), and displayed greater resistance to B. anthracis infection compared to C57BL/6J mice. Several ERP features (e.g., bloat, hypothermia, labored breathing, dilated pinnae vessels) suggested vascular involvement. To test this, Evan’s blue was used to assess vessel leakage and intravital microscopy was used to monitor microvascular blood flow. Increased vascular leakage was observed in lungs of B6.CAST.11M mice compared to C57BL/6J mice 1 hour after systemic administration of LT. Capillary blood flow was reduced in the small intestine mesentery without concomitant leukocyte emigration following systemic or topical application of LT, the latter suggesting a localized tissue mechanism in this response. Since LT activates the Nlrp1b inflammasome in B6.CAST.11M mice, the roles of inflammasome products, IL-1? and IL-18, were examined. Topical application to the mesentery of IL-1? but not IL-18 revealed pronounced slowing of blood flow in B6.CAST.11M mice that was not present in C57BL/6J mice. A neutralizing anti-IL-1? antibody suppressed the slowing of blood flow induced by LT, indicating a role for IL-1? in the response. Besides allelic differences controlling Nlrp1b inflammasome activation by LT observed previously, evidence presented here suggests that an additional genetic determinant(s) could regulate the vascular response to IL-1?. These results demonstrate that vessel leakage and alterations to blood flow are part of the rapid response in mice resistant to B. anthracis infection.

Weigel, Kelsey J.; Rues, Laura; Doyle, Edward J.; Buchheit, Cassandra L.; Wood, John G.; Gallagher, Ryan J.; Kelly, Laura E.; Radel, Jeffrey D.; Bradley, Kenneth A.; LeVine, Steven M.

2012-01-01

176

Anthrax toxin protective antigen integrates poly-?-d-glutamate and pH signals to sense the optimal environment for channel formation  

PubMed Central

Many toxins assemble into oligomers on the surface of cells. Local chemical cues signal and trigger critical rearrangements of the oligomer, inducing the formation of a membrane-fused or channel state. Bacillus anthracis secretes two virulence factors: a tripartite toxin and a poly-?-d-glutamic acid capsule (?-DPGA). The toxin’s channel-forming component, protective antigen (PA), oligomerizes to create a prechannel that forms toxic complexes upon binding the two other enzyme components, lethal factor (LF) and edema factor (EF). Following endocytosis into host cells, acidic pH signals the prechannel to form the channel state, which translocates LF and EF into the host cytosol. We report ?-DPGA binds to PA, LF, and EF, exhibiting nanomolar avidity for the PA prechannel oligomer. We show PA channel formation requires the pH-dependent disruption of the intra-PA domain-2–domain-4 (D2-D4) interface. ?-DPGA stabilizes the D2-D4 interface, preventing channel formation both in model membranes and cultured mammalian cells. A 1.9-Å resolution X-ray crystal structure of a D2-D4-interface mutant and corresponding functional studies reveal how stability at the intra-PA interface governs channel formation. We also pinpoint the kinetic pH trigger for channel formation to a residue within PA’s membrane-insertion loop at the inter-PA D2-D4 interface. Thus, ?-DPGA may function as a chemical cue, signaling that the local environment is appropriate for toxin assembly but inappropriate for channel formation.

Kintzer, Alexander F.; Tang, Iok I; Schawel, Adam K.; Brown, Michael J.; Krantz, Bryan A.

2012-01-01

177

Use of Recombinant DNA Techniques for the Production of a More Effective Anthrax Vaccine.  

National Technical Information Service (NTIS)

The overall goal of the present research is to construct a safe and effective human anthrax vaccine using recombinant DNA techniques. During the course of this contract, we have isolated and characterized each of the Bacillus anthracis toxin genes. Althou...

D. L. Robertson

1988-01-01

178

Anthrax: an update.  

PubMed

Anthrax is a zoonotic disease caused by Bacillus anthracis. It is potentially fatal and highly contagious disease. Herbivores are the natural host. Human acquire the disease incidentally by contact with infected animal or animal products. In the 18th century an epidemic destroyed approximately half of the sheep in Europe. In 1900 human inhalational anthrax occured sporadically in the United States. In 1979 an outbreak of human anthrax occured in Sverdlovsk of Soviet Union. Anthrax continued to represent a world wide presence. The incidence of the disease has decreased in developed countries as a result of vaccination and improved industrial hygiene. Human anthrax clinically presents in three forms, i.e. cutaneous, gastrointestinal and inhalational. About 95% of human anthrax is cutaneous and 5% is inhalational. Gastrointestinal anthrax is very rare (less than 1%). Inhalational form is used as a biological warefare agent. Penicillin, ciprofloxacin (and other quinolones), doxicyclin, ampicillin, imipenem, clindamycin, clarithromycin, vancomycin, chloramphenicol, rifampicin are effective antimicrobials. Antimicrobial therapy for 60 days is recommended. Human anthrax vaccine is available. Administration of anti-protective antigen (PA) antibody in combination with ciprofloxacin produced 90%-100% survival. The combination of CPG-adjuvanted anthrax vaccine adsorbed (AVA) plus dalbavancin significantly improved survival. PMID:23569822

Kamal, Sm; Rashid, Akm M; Bakar, Ma; Ahad, Ma

2011-12-01

179

Antibodies Against Anthrax: Mechanisms of Action and Clinical Applications  

PubMed Central

B. anthracis is a bioweapon of primary importance and its pathogenicity depends on its lethal and edema toxins, which belong to the A-B model of bacterial toxins, and on its capsule. These toxins are secreted early in the course of the anthrax disease and for this reason antibiotics must be administered early, in addition to other limitations. Antibodies (Abs) may however neutralize those toxins and target this capsule to improve anthrax treatment, and many Abs have been developed in that perspective. These Abs act at various steps of the cell intoxication and their mechanisms of action are detailed in the present review, presented in correlation with structural and functional data. The potential for clinical application is discussed for Abs targeting each step of entry, with four of these molecules already advancing to clinical trials. Paradoxically, certain Abs may also enhance the lethal toxin activity and this aspect will also be presented. The unique paradigm of Abs neutralizing anthrax toxins thus exemplifies how they may act to neutralize A-B toxins and, more generally, be active against infectious diseases.

Froude, Jeffrey W.; Thullier, Philippe; Pelat, Thibaut

2011-01-01

180

Potentiation of an anthrax DNA vaccine with electroporation  

Microsoft Academic Search

DNA vaccines are a promising method of immunization against biothreats and emerging infections because they are relatively easy to design, manufacture, store and distribute. However, immunization with DNA vaccines using conventional delivery methods often fails to induce consistent, robust immune responses, especially in species larger than the mouse. Intramuscular (i.m.) delivery of a plasmid encoding anthrax toxin protective antigen (PA)

D. Hannaman; E. Nolana; B. Ellefsen; G. Nakamura; L. Chau; O. Tellez; S. Little; R. Bernard

2008-01-01

181

Researchers Compare Anthrax Genomes  

NSF Publications Database

... 703) 292-8440 mhenkart@nsf.gov Researchers Compare Anthrax Genomes In a pioneering use of genomics ... In the recent TIGR study, the scientists compared information gained from a previous investigation ...

182

What Is Anthrax?  

MedlinePLUS

... anthrax could be spread and used as a weapon. Although this is a frightening thought, the government and police are working on ways to protect us. In the meantime, it's important not to panic over ...

183

Effect of 2-Fluorohistidine Labeling of the Anthrax Protective Antigen on Stability, Pore Formation, and Translocation †  

Microsoft Academic Search

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly

D. Shyamali Wimalasena; John C. Cramer; Blythe E. Janowiak; Stephen J. Juris; Roman A. Melnyk; D. Eric Anderson; Kenneth L. Kirk; R. John Collier; James G. Bann

2007-01-01

184

Cutaneous anthrax associated with microangiopathic hemolytic anemia and coagulopathy in a 7-month-old infant.  

PubMed

A 7-month-old infant with cutaneous anthrax developed severe systemic illness despite early treatment with antibiotics. The infant displayed severe microangiopathic hemolytic anemia with renal involvement, coagulopathy, and hyponatremia. These findings are unusual with cutaneous anthrax, but have been described in illness resulting from spider toxin and may delay correct diagnosis. The systemic manifestations of the disease persisted for nearly a month despite corticosteroid therapy, but resolved. PMID:11851579

Freedman, Abigail; Afonja, Olubunmi; Chang, Mary Wu; Mostashari, Farzad; Blaser, Martin; Perez-Perez, Guillermo; Lazarus, Herb; Schacht, Robert; Guttenberg, Jane; Traister, Michael; Borkowsky, William

2002-02-20

185

Characterization of the human immune response to the UK anthrax vaccine  

Microsoft Academic Search

The anthrax bipartite lethal toxin (protective antigen (PA) and lethal factor (LF))-specific antibody responses of humans receiving the UK licensed anthrax vaccine were determined. The PA-specific IgG response peaked two weeks post immunization and fell back to pre-boost levels by week 12. The heterogeneity of the host population modulated the extent of the PA-specific antibody response. Significantly lower levels of

Les Baillie; Tim Townend; Nicki Walker; Ulla Eriksson; Diane Williamson

2004-01-01

186

Prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity.  

PubMed

The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab')2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines. PMID:16988263

Vitale, Laura; Blanset, Diann; Lowy, Israel; O'Neill, Thomas; Goldstein, Joel; Little, Stephen F; Andrews, Gerard P; Dorough, Gary; Taylor, Ronald K; Keler, Tibor

2006-10-01

187

Evaluation of intravenous anthrax immune globulin for treatment of inhalation anthrax.  

PubMed

Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a component of anthrax toxins. Anthrivig (human anthrax immunoglobulin), also known as AIGIV, derived from plasma of humans immunized with BioThrax (anthrax vaccine adsorbed), is under development for the treatment of toxemia following exposure to anthrax spores. The pharmacokinetics (PK) of AIGIV was assessed in naive animals and healthy human volunteers, and the efficacy of AIGIV was assessed in animals exposed via inhalation to aerosolized B. anthracis spores. In the clinical study, safety, tolerability, and PK were evaluated in three dose cohorts (3.5, 7.1, and 14.2 mg/kg of body weight of anti-PA IgG) with 30 volunteers per cohort. The elimination half-life of AIGIV in rabbits, nonhuman primates (NHPs), and humans following intravenous infusion was estimated to be approximately 4, 12, and 24 days, respectively, and dose proportionality was observed. In a time-based treatment study, AIGIV protected 89 to 100% of animals when administered 12 h postexposure; however, a lower survival rate of 39% was observed when animals were treated 24 h postexposure, underscoring the need for early intervention. In a separate set of studies, animals were treated on an individual basis upon detection of a clinical sign or biomarker of disease, namely, a significant increase in body temperature (SIBT) in rabbits and presence of PA in the serum of NHPs. In these trigger-based intervention studies, AIGIV induced up to 75% survival in rabbits depending on the dose and severity of toxemia at the time of treatment. In NHPs, up to 33% survival was observed in AIGIV-treated animals. (The clinical study has been registered at ClinicalTrials.gov under registration no. NCT00845650.). PMID:23979731

Mytle, Nutan; Hopkins, Robert J; Malkevich, Nina V; Basu, Subhendu; Meister, Gabriel T; Sanford, Daniel C; Comer, Jason E; Van Zandt, Kristopher E; Al-Ibrahim, Mohamed; Kramer, William G; Howard, Cris; Daczkowski, Nancy; Chakrabarti, Ajoy C; Ionin, Boris; Nabors, Gary S; Skiadopoulos, Mario H

2013-08-26

188

Airing Out Anthrax.  

National Technical Information Service (NTIS)

The AiroCide TiO2 is an air-purifier that kills 93.3 percent of airborne pathogens that pass through it, including Bacillus anthraci, more commonly known as anthrax. It is essentially a spinoff of KES Science & Technology, Inc.'s Bio-KES system, a highly ...

2002-01-01

189

Contracting for Anthrax Vaccine.  

National Technical Information Service (NTIS)

This audit was requested by Congressman Walter B. Jones to review the financial and contractual relationship between the DoD and BioPort Corporation, the sole U.S. manufacturer of the anthrax vaccine. Specifically, Congressman Jones requested we review th...

2000-01-01

190

Anthrax Toxin Entry into Polarized Epithelial Cells  

Microsoft Academic Search

applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane

KATHRYN E. BEAUREGARD; SUSAN WIMER-MACKIN; R. JOHN COLLIER

1999-01-01

191

Protective antigen composite nanofibers as a transdermal anthrax vaccine.  

PubMed

Anthrax, a disease caused by the gram positive bacteria Bacillus anthracis, has become an increasing threat to public health in the last several years, due to its use as an agent of biological warfare. The currently utilized human anthrax vaccine, which confers immunity through the host antibody recognition of protective antigen (PA), requires a three dose regimen and annual booster shots after the initial vaccination to maintain its efficacy. The long term goal of this project is to produce an anthrax vaccine that is capable of delivering protective antigen through human skin. The novel method for transdermal vaccine delivery that we propose utilizes the high surface area to volume ratio offered by protein-containing nanofiber membranes, prepared by the electrospinning technique. Research has already been undertaken to study the effect the main virulent agent of anthrax, lethal toxin (LT), has on a human monocytic cell line, Monomac 6 cells (MM6). Lethal toxin is said to comprise of a Zn2+ -dependent metalloprotease known as lethal factor (LF), and a binding protein known as protective antigen. The successful encapsulation of the protective antigen within the nanofibrous membrane was analyzed with the use of an in vitro MM6 assay. The assay was designed to ensure the functionality of PA through the harsh environment of the electrospinning process. Quantitative analysis of IL-6 cytokine production by lipopolysaccharide (LPS) stimulated MM6 cells in the presence of LF and PA provided proof that PA retained its biological activity through the process of electrospinning. This finding provides an innovative platform for the development of a transdermal anthrax vaccine. PMID:19162840

Knockenhauer, Kevin E; Sawicka, Katarzyna M; Roemer, Elizabeth J; Simon, Sanford R

2008-01-01

192

Immune Responses to Bacillus anthracis Protective Antigen in Patients with Bioterrorism?Related Cutaneous or Inhalation Anthrax  

Microsoft Academic Search

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for 11 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected

Vera Semenova; Han Li; Shane Crotty; Carolyn Greene; John Glidewell; Rafi Ahmed

2004-01-01

193

Identification of new dominant-negative mutants of anthrax protective antigen using directed evolution.  

PubMed

The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment. PMID:22948605

Wu, Gaobing; Feng, Chunfang; Cao, Sha; Guo, Aizhen; Liu, Ziduo

2012-09-05

194

Anthrax eTool: Protecting the Worksite against Terrorism  

MedlinePLUS

... Home : Anthrax Credits What is anthrax? What is Bacillus anthracis ? How can I be exposed to Bacillus anthracis ? What are the symptoms of anthrax? What ... can I prevent anthrax infection? Additional information on Bacillus anthracis and anthrax Who is at risk? Anthrax ...

195

Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates.  

PubMed

Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be a much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec(-) (unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo(-) (unable to form oligomers), and DNI (Dominant Negative Inhibitory, unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PAtoxin-neutralizing activity. In contrast, Oligo(-)-immunized mice had high levels of anti-PA IgG but lower titers of toxin-neutralizing activity, suggesting that Oligo(-) mutation sites may overlap with critical protective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA non-functional but also simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure vaccination. PMID:18192092

Yan, Ming; Roehrl, Michael H; Basar, Emre; Wang, Julia Y

2007-12-26

196

Lethal Factor Toxemia and Anti-Protective Antigen Antibody Activity in Naturally Acquired Cutaneous Anthrax  

PubMed Central

Cutaneous anthrax outbreaks occurred in Bangladesh from August to October 2009. As part of the epidemiological response and to confirm anthrax diagnoses, serum samples were collected from suspected case patients with observed cutaneous lesions. Anthrax lethal factor (LF), anti-protective antigen (anti-PA) immunoglobulin G (IgG), and anthrax lethal toxin neutralization activity (TNA) levels were determined in acute and convalescent serum of 26 case patients with suspected cutaneous anthrax from the first and largest of these outbreaks. LF (0.005–1.264 ng/mL) was detected in acute serum from 18 of 26 individuals. Anti-PA IgG and TNA were detected in sera from the same 18 individuals and ranged from 10.0 to 679.5 ?g/mL and 27 to 593 units, respectively. Seroconversion to serum anti-PA and TNA was found only in case patients with measurable toxemia. This is the first report of quantitative analysis of serum LF in cutaneous anthrax and the first to associate acute stage toxemia with subsequent antitoxin antibody responses.

Quinn, Conrad P.; Beesley, Cari A.; Gallegos-Candela, Maribel; Marston, Chung K.; Cronin, Li X.; Lins, Renato C.; Stoddard, Robyn A.; Li, Han; Schiffer, Jarad; Hossain, M. Jahangir; Chakraborty, Apurba; Rahman, Mahmudur; Luby, Stephen P.; Shieh, Wun-Ju; Zaki, Sherif; Barr, John R.; Hoffmaster, Alex R.

2011-01-01

197

Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease.  

PubMed

Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans; however, the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homologue of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbours bpsX-H, B. cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in Bacillus anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-d-?-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease. PMID:21371137

Oh, So-Young; Budzik, Jonathan M; Garufi, Gabriella; Schneewind, Olaf

2011-03-03

198

Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease  

PubMed Central

Summary Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans, however the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homolog of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbors bpsX-H, Bacillus cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in B. anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-D-?-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease.

Oh, So-Young; Budzik, Jonathan M.; Garufi, Gabriella; Schneewind, Olaf

2012-01-01

199

A Comparative Study of Anthrax Bacteriophages.  

National Technical Information Service (NTIS)

A study of the morphology of negative colonies of various anthrax bacteriophages showed that according to morphology the negative colonies of anthrax bacteriophages included all the known types described in other bacteriophages. On the basis of the study ...

E. N. Levina V. R. Arkhipova

1968-01-01

200

From Structure to Solutions: The Role of Basic Research in Developing Anthrax Countermeasures  

PubMed Central

Dr. John Collier traced the discoveries that elucidated the structure and function of the anthrax toxin in his talk “Anthrax Toxin,” which was part of the Microbiology Graduate Program Seminar Series at Yale School of Medicine on February 23, 2012. Dr. Collier, Professor of Microbiology and Immunobiology at Harvard University, began by noting the advantages to studying anthrax pathogenesis in a biosafety level-1 lab. This designation does not merely facilitate his research, but also reflects a larger trend of basic research being leveraged to develop translational applications. Basic research on toxin structure has led to the development of a vaccine by Dr. Collier’s group. Next-generation prophylactics also may stem from recent discoveries uncovering a role for cellular cofactors that mediate toxin function. Finally, basic research into the toxin substructure has facilitated efforts to change the receptor tropism to target dysregulated cells for therapeutic purposes. The urgency around biodefense agents makes the choice of research priorities a salient issue. As such, this author submits that basic research occupies a unique and lucrative niche driving clinical applications.

Hardiman, Camille A.

2012-01-01

201

GASTROINTESTINAL ANTHRAX: REVIEW OF NINE PATIENTS  

Microsoft Academic Search

Background - Gastrointestinal anthrax is quite rare and often fatal. Here we report the clinical presentation and outcome of nine patients with this disease. Methods - The medical records of all patients with documented anthrax admitted to our university hospitals from 1975 to 2000 were reviewed. Nine patients were found to have gastrointestinal anthrax. Data regarding the clinical presentation, incubation

Shohreh Beheshti; Gholam-Reza Rezaian; Sasan Afifi; Sheema Rezaian

2003-01-01

202

Anthrax Vaccine: What You Need to Know  

MedlinePLUS

... disease. Nursing mothers may safely be given anthrax vaccine. 5 What are the risks from anthrax vaccine? Like any medicine, a vaccine could cause a ... Anthrax is a very serious disease, and the risk of serious harm from the vaccine is extremely small. Mild problems • Reactions on the ...

203

Anthrax protective antigen delivered by Salmonella enterica serovar Typhi Ty21a protects mice from a lethal anthrax spore challenge.  

PubMed

Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified the B. anthracis protective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of the Escherichia coli alpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain, Salmonella enterica serovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores of B. anthracis 7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time. PMID:19179420

Osorio, Manuel; Wu, Yanping; Singh, Sunil; Merkel, Tod J; Bhattacharyya, Siba; Blake, Milan S; Kopecko, Dennis J

2009-01-29

204

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice.  

PubMed

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA. PMID:22507985

Uchida, Makoto; Harada, Toshihiko; Enkhtuya, Jargalsaikhan; Kusumoto, Akiko; Kobayashi, Yoshiyasu; Chiba, Shiori; Shyaka, Anselme; Kawamoto, Keiko

2012-04-09

205

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax.  

PubMed

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax. PMID:12963063

Rivera, Victor R; Merrill, Gerald A; White, Jill A; Poli, Mark A

2003-10-01

206

Genetic Vaccines for Anthrax Based on Recombinant Adeno-associated Virus Vectors  

Microsoft Academic Search

Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction

Te-Hui Liu; Jon Oscherwitz; Bruce Schnepp; Jana Jacobs; Fen Yu; Kemp B Cease; Philip R Johnson

2009-01-01

207

Microneedle-Based Intradermal Delivery of the Anthrax Recombinant Protective Antigen Vaccine  

Microsoft Academic Search

The recombinant protective antigen (rPA) of Bacillus anthracis is a promising anthrax vaccine. We compared serum immunoglobulin G levels and toxin-neutralizing antibody titers in rabbits following delivery of various doses of vaccine by microneedle-based intradermal (i.d.) delivery or intramuscular (i.m.) injection using conventional needles. Intradermal delivery required less antigen to induce levels of antibody similar to those produced via i.m.

John A. Mikszta; John P. Dekker; Noel G. Harvey; Cheryl H. Dean; John M. Brittingham; Joanne Huang; Vincent J. Sullivan; Beverly Dyas; Chad J. Roy; Robert G. Ulrich

2006-01-01

208

Primary involvement of pharynx and peyer's patch in inhalational and intestinal anthrax.  

PubMed

Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis. PMID:17542645

Glomski, Ian J; Piris-Gimenez, Alejandro; Huerre, Michel; Mock, Michèle; Goossens, Pierre L

2007-06-01

209

Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function  

PubMed Central

The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A.; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M.; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin

2012-01-01

210

Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase  

NASA Astrophysics Data System (ADS)

Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

Koo, Han-Mo; Vanbrocklin, Matt; McWilliams, Mary Jane; Leppla, Stephan H.; Duesbery, Nicholas S.; Vande Woude, George F.

2002-03-01

211

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis  

PubMed Central

Objective Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. Methods Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. Results To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. Conclusion Neutralizing-antibody titers can be used as a surrogate marker.

Chun, Jeong-Hoon; Choi, On-Jee; Cho, Min-Hee; Hong, Kee-Jong; Seong, Won Keun; Oh, Hee-Bok; Rhie, Gi-Eun

2012-01-01

212

Oculocutaneous anthrax: detection and treatment  

PubMed Central

Anthrax, a zoonotic disease that primarily affects herbivores, has received recent attention as a potential agent of bioterrorism. We report a patient who presented with a 4-day history of pain, watering and difficulty in opening the left upper and lower eyelids, and fever. Clinical examination revealed brawny nonpitting edema with serosanguinous discharge. The history of the death of his sheep 1 week prior to the illness provided the clue to the diagnosis. Although standard cultures of the blood and the serous fluid from the lesion were negative, probably as a result of prior treatment, the diagnosis of cutaneous anthrax was made by a polymerase chain reaction (PCR) test of the serous fluid. Serial photographs demonstrating resolution of the lesion with appropriate antibiotic therapy are presented.

David, Sarada; Peter, Jayanthi; Raju, Renu; Padmaja, P; Mohanraj, Promila

2010-01-01

213

Clinical impressions of anthrax from the 2006 outbreak in Saskatchewan  

PubMed Central

Clinical signs and carcass traits observed during the 2006 Saskatchewan anthrax outbreak were largely consistent with those previously published, except for cutaneous anthrax and anthrax mastitis in cows, and subcutaneous edema in bulls and horses. Failure of blood to clot was the most reliable indicator of anthrax in carcasses.

Himsworth, Chelsea G.; Argue, Connie K.

2009-01-01

214

Anthrax: Exposure to Hides/Drums  

MedlinePLUS

... purchased in Haiti, where anthrax is common. Another fatal case of inhalation anthrax occurred in Scotland in 2006. The patient, who may have had increased susceptibility to infectious diseases, became sick as a result of using or handling contaminated hide drums from ...

215

Anthrax vaccine efficacy in golden Syrian hamsters  

Microsoft Academic Search

The efficacy of a licensed human anthrax vaccine (anthrax vaccine adsorbed, AVA) was tested in golden Syrian hamsters against a virulent Bacillus anthracis spore challenge. Groups of golden Syrian hamsters were vaccinated at either 0 and 4 weeks or 0, 4 and 8 weeks, then challenged subcutaneously (s.c.) at 10 weeks with spores of various B. anthracis isolates. Although ELISA

P. F Fellows; M. K Linscott; S. F Little; P Gibbs; B. E Ivins

2002-01-01

216

Molecular basis for improved anthrax vaccines  

Microsoft Academic Search

The current vaccine for anthrax has been licensed since 1970 and was developed based on the outcome of human trials conducted in the 1950s. This vaccine, known as anthrax vaccine adsorbed (AVA), consists of a culture filtrate from an attenuated strain of Bacillus anthracis adsorbed to aluminum salts as an adjuvant. This vaccine is considered safe and effective, but is

Robert N. Brey

2005-01-01

217

Anthrax in America 2001-2003.  

PubMed Central

Anthrax caused by Bacillus anthracis in humans is rare. Two recent outbreaks that were intentionally caused occurred among postal employees, politicians, and journalists in the United States. This has caused tremendous fear, and our experience with these "anthrax incidents" has changed our views on the natural history of this disease in people. In this paper, we review the lifecycle and biology of this micro-organism. Anthrax that occurs from a weaponized form of this micro-organism has a specific clinical presentation that requires a suspicion of anthrax exposure to be diagnosed. New methods of testing for anthrax have been developed and may simplify diagnosis in the future. The range of illness caused by B. anthracis from the molecular level to the clinical symptoms is discussed. We also review the diagnostic criteria and differential diagnosis as well as treatment of this condition.

Joshi, Shivang G.; Cymet, Holly Berkovits; Kerkvliet, Gary; Cymet, Tyler

2004-01-01

218

Anthrax outbreaks in Bangladesh, 2009-2010.  

PubMed

During August 2009-October 2010, a multidisciplinary team investigated 14 outbreaks of animal and human anthrax in Bangladesh to identify the etiology, pathway of transmission, and social, behavioral, and cultural factors that led to these outbreaks. The team identified 140 animal cases of anthrax and 273 human cases of cutaneous anthrax. Ninety one percent of persons in whom cutaneous anthrax developed had history of butchering sick animals, handling raw meat, contact with animal skin, or were present at slaughtering sites. Each year, Bacillus anthracis of identical genotypes were isolated from animal and human cases. Inadequate livestock vaccination coverage, lack of awareness of the risk of anthrax transmission from animal to humans, social norms and poverty contributed to these outbreaks. Addressing these challenges and adopting a joint animal and human health approach could contribute to detecting and preventing such outbreaks in the future. PMID:22492157

Chakraborty, Apurba; Khan, Salah Uddin; Hasnat, Mohammed Abul; Parveen, Shahana; Islam, M Saiful; Mikolon, Andrea; Chakraborty, Ranjit Kumar; Ahmed, Be-Nazir; Ara, Khorsed; Haider, Najmul; Zaki, Sherif R; Hoffmaster, Alex R; Rahman, Mahmudur; Luby, Stephen P; Hossain, M Jahangir

2012-04-01

219

Anthrax Outbreaks in Bangladesh, 2009-2010  

PubMed Central

During August 2009–October 2010, a multidisciplinary team investigated 14 outbreaks of animal and human anthrax in Bangladesh to identify the etiology, pathway of transmission, and social, behavioral, and cultural factors that led to these outbreaks. The team identified 140 animal cases of anthrax and 273 human cases of cutaneous anthrax. Ninety one percent of persons in whom cutaneous anthrax developed had history of butchering sick animals, handling raw meat, contact with animal skin, or were present at slaughtering sites. Each year, Bacillus anthracis of identical genotypes were isolated from animal and human cases. Inadequate livestock vaccination coverage, lack of awareness of the risk of anthrax transmission from animal to humans, social norms and poverty contributed to these outbreaks. Addressing these challenges and adopting a joint animal and human health approach could contribute to detecting and preventing such outbreaks in the future.

Chakraborty, Apurba; Khan, Salah Uddin; Hasnat, Mohammed Abul; Parveen, Shahana; Islam, M. Saiful; Mikolon, Andrea; Chakraborty, Ranjit Kumar; Ahmed, Be-Nazir; Ara, Khorsed; Haider, Najmul; Zaki, Sherif R.; Hoffmaster, Alex R.; Rahman, Mahmudur; Luby, Stephen P.; Hossain, M. Jahangir

2012-01-01

220

A chimeric protein that functions as both an anthrax dual-target antitoxin and a trivalent vaccine.  

PubMed

Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PA(F427D). In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax. PMID:20713663

Wu, Gaobing; Hong, Yuzhi; Guo, Aizhen; Feng, Chunfang; Cao, Sha; Zhang, Cheng-Cai; Shi, Ruiping; Tan, Yadi; Liu, Ziduo

2010-08-16

221

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice  

PubMed Central

Background Photocatalysis of titanium dioxide (TiO2) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host.

Huang, Hsin-Hsien; Wong, Ming-Show; Lin, Hung-Chi; Chang, Hsin-Hou

2009-01-01

222

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.  

PubMed

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone. PMID:23658392

Ionin, Boris; Hopkins, Robert J; Pleune, Brett; Sivko, Gloria S; Reid, Frances M; Clement, Kristin H; Rudge, Thomas L; Stark, Gregory V; Innes, Alison; Sari, Suha; Guina, Tina; Howard, Cris; Smith, Jeffrey; Swoboda, M Lisa; Vert-Wong, Ekaterina; Johnson, Virginia; Nabors, Gary S; Skiadopoulos, Mario H

2013-05-08

223

Anthrax vaccine-induced antibodies provide cross-species prediction of survival to aerosol challenge.  

PubMed

Because clinical trials to assess the efficacy of vaccines against anthrax are not ethical or feasible, licensure for new anthrax vaccines will likely involve the Food and Drug Administration's "Animal Rule," a set of regulations that allow approval of products based on efficacy data only in animals combined with immunogenicity and safety data in animals and humans. U.S. government-sponsored animal studies have shown anthrax vaccine efficacy in a variety of settings. We examined data from 21 of those studies to determine whether an immunological bridge based on lethal toxin neutralization activity assay (TNA) can predict survival against an inhalation anthrax challenge within and across species and genera. The 21 studies were classified into 11 different settings, each of which had the same animal species, vaccine type and formulation, vaccination schedule, time of TNA measurement, and challenge time. Logistic regression models determined the contribution of vaccine dilution dose and TNA on prediction of survival. For most settings, logistic models using only TNA explained more than 75% of the survival effect of the models with dose additionally included. Cross-species survival predictions using TNA were compared to the actual survival and shown to have good agreement (Cohen's ? ranged from 0.55 to 0.78). In one study design, cynomolgus macaque data predicted 78.6% survival in rhesus macaques (actual survival, 83.0%) and 72.6% in rabbits (actual survival, 64.6%). These data add support for the use of TNA as an immunological bridge between species to extrapolate data in animals to predict anthrax vaccine effectiveness in humans. PMID:22972844

Fay, Michael P; Follmann, Dean A; Lynn, Freyja; Schiffer, Jarad M; Stark, Gregory V; Kohberger, Robert; Quinn, Conrad P; Nuzum, Edwin O

2012-09-12

224

Anthrax and Smallpox: Comparison of Two Outbreaks.  

National Technical Information Service (NTIS)

Partial contents: Anthrax and Smallpox: Comparison of Two Outbreaks, Late Diagnosis, 1979 Sverdleovsk Epidemic, Sources of Evidence, Research Findings, Soviet Public Health Response, Structural Sources of Late Diagnosis, Solutions to Late Diagnosis.

J. Guillemin

2002-01-01

225

FDA Workshop: Anthrax Vaccines: Bridging Correlates Of ...  

Center for Biologics Evaluation and Research (CBER)

Text Version... ANTHRAX VACCINES: BRIDGING CORRELATES OF PROTECTION IN ... then potentially be bridged to humans. ... bridge data from animal studies to ... More results from www.fda.gov/downloads/biologicsbloodvaccines/newsevents

226

FDA APPROVES LICENSE SUPPLEMENTS FOR ANTHRAX ...  

Center for Drug Evaluation (CDER)

... In addition, each lot of anthrax vaccine undergoes thorough testing for purity, potency ... and Research releases it based on the results of these tests. ... More results from www.fda.gov/drugs/emergencypreparedness/bioterrorismanddrugpreparedness

227

Anthrax vaccines: present status and future prospects.  

PubMed

The management of anthrax remains a top priority among the biowarfare/bioterror agents. It was the Bacillus anthracis spore attack through the US mail system after the September 11, 2001, terrorist attacks in the USA that highlighted the potential of B. anthracis as a bioterrorism agent and the threat posed by its deliberate dissemination. These attacks invigorated the efforts toward understanding the anthrax pathogenesis and development of more comprehensive medical intervention strategies for its containment in case of both natural disease and manmade, accidental or deliberate infection of a non-suspecting population. Currently, efforts are directed toward the development of safe and efficacious vaccines as well as intervention tools for controlling the disease in the advanced fulminant stage when toxemia has already developed. This work presents an overview of the current understanding of anthrax pathogenesis and recent advances made, particularly after 2001, for the successful management of anthrax and outlines future perspectives. PMID:23984963

Kaur, Manpreet; Singh, Samer; Bhatnagar, Rakesh

2013-08-01

228

Pediatric Labeling for Medical Countermeasures: Anthrax ...  

Center for Biologics Evaluation and Research (CBER)

Text VersionPage 1. Pediatric Labeling for Medical Countermeasures: Anthrax Examples John Alexander, MD, MPH Medical Team Leader, ... More results from www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials

229

List of Contractors to Support Anthrax Remediation  

SciTech Connect

This document responds to a need identified by private sector businesses for information on contractors that may be qualified to support building remediation efforts following a wide-area anthrax release.

Judd, Kathleen S.; Lesperance, Ann M.

2010-05-14

230

Gastrointestinal anthrax: clinical experience in 5 cases  

PubMed Central

Background: Bacillus anthracis may usually cause three forms of anthrax: inhalation, gastrointestinal and cutaneous. The gastrointestinal (GI) anthrax develops after eating contaminated meat. Thus, in this paper were report 5 cases of intestinal anthrax. Case Presentation: We report a case series of intestinal anthrax, with history of consumption of raw or poorly cooked liver of sheep. One patient was female and 4 were males with the age range between 17 and 26 years. All patients were admitted with abdominal pain, nausea, and vomiting. Examination revealed abdominal distention on the right lower quadrant or diffuse tenderness. Laboratory examination in all patients showed leukocytosis with polymorphonuclear of >80%. Because of the unclear and questionable diagnosis, exploratory laparotomy was performed on several patients, invariably showing an abundant yellowish and thick ascitic fluid, soft hypertrophied mesenteric lymph nodes (3-5 cm) mostly in the ileocecal region, and substantial edema involving one segment of small bowel, cecum or ascending colon. Anthrax was diagnosed on the epidemiologic basis (ingestion history of half cooked liver of sheep) or microbiologic (microscopy with bacterial culture) and pathologic testing (post surgery in four patients or autopsy in one patient). With appropriate treatment, 4 survived and one patient died. Conclusion: Gastrointestinal anthrax is characterized by rapid onset, fever, ascitis and septicemia. The symptoms can mimic those seen in an acute surgical abdomen. Rapid diagnosis and prompt initiation of antibiotic therapy and then exploratory laparotomy (right hemicolectomy) are keys to survival.

Maddah, Ghodratollah; Abdollahi, Abbas; Katebi, Mehrdad

2013-01-01

231

Gastrointestinal anthrax: clinical experience in 5 cases.  

PubMed

Background: Bacillus anthracis may usually cause three forms of anthrax: inhalation, gastrointestinal and cutaneous. The gastrointestinal (GI) anthrax develops after eating contaminated meat. Thus, in this paper were report 5 cases of intestinal anthrax. Case Presentation: We report a case series of intestinal anthrax, with history of consumption of raw or poorly cooked liver of sheep. One patient was female and 4 were males with the age range between 17 and 26 years. All patients were admitted with abdominal pain, nausea, and vomiting. Examination revealed abdominal distention on the right lower quadrant or diffuse tenderness. Laboratory examination in all patients showed leukocytosis with polymorphonuclear of >80%. Because of the unclear and questionable diagnosis, exploratory laparotomy was performed on several patients, invariably showing an abundant yellowish and thick ascitic fluid, soft hypertrophied mesenteric lymph nodes (3-5 cm) mostly in the ileocecal region, and substantial edema involving one segment of small bowel, cecum or ascending colon. Anthrax was diagnosed on the epidemiologic basis (ingestion history of half cooked liver of sheep) or microbiologic (microscopy with bacterial culture) and pathologic testing (post surgery in four patients or autopsy in one patient). With appropriate treatment, 4 survived and one patient died. Conclusion: Gastrointestinal anthrax is characterized by rapid onset, fever, ascitis and septicemia. The symptoms can mimic those seen in an acute surgical abdomen. Rapid diagnosis and prompt initiation of antibiotic therapy and then exploratory laparotomy (right hemicolectomy) are keys to survival. PMID:24009958

Maddah, Ghodratollah; Abdollahi, Abbas; Katebi, Mehrdad

2013-01-01

232

Multicomponent anthrax toxin display and delivery using bacteriophage T4  

Microsoft Academic Search

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to

Sathish B. Shivachandra; Qin Li; Kristina K. Peachman; Gary R. Matyas; Stephen H. Leppla; Carl R. Alving; Mangala Rao; Venigalla B. Rao

2007-01-01

233

Hepatic Subcellular Distribution of (Tritium)T-2 Toxin.  

National Technical Information Service (NTIS)

Hepatic subcellular distribution of (3H)T-2 toxin. The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with (3H)T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%,...

J. G. Pace M. R. Watts

1989-01-01

234

Anthrax Vaccine Powder Formulations for Nasal Mucosal Delivery.  

National Technical Information Service (NTIS)

Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complet...

G. E. Jiang S. B. Joshi L. J. Peek D. T. Brandau J. Huang

2005-01-01

235

Laboratories Face Crackdown in Wake of Anthrax Scare.  

ERIC Educational Resources Information Center

Explores the after-effects on college laboratories of the anthrax mail scare; scientists say the anthrax scare justifies tougher rules on biological agents, but some fear that Congress may go too far. (EV)

Southwick, Ron

2001-01-01

236

Modified anthrax fusion proteins deliver HIV antigens through MHC Class I and II pathways.  

PubMed

T cell-based HIV vaccine candidates have focused on eliciting both CD4- and CD8-mediated responses. One challenge in vaccine development is the successful introduction and presentation of exogenous antigen to elicit an immune response. Modified bacterial toxins have been studied extensively as intracellular delivery agents because of their unique capability to translocate antigen across the cell membrane without affecting cell viability. Modified anthrax toxin lethal factor (LFn) fusion protein is able to effectively induce anti-HIV cytotoxic T lymphocytes in the absence of protective antigen (PA) and is being evaluated as a vaccine candidate. Here we describe, for the first time, the processing and presentation of LFn fusion proteins by the MHC Class II pathway. The ability of LFn--HIV to induce both CD8- and CD4-mediated responses may have relevance in current approaches to vaccine design. Furthermore, the translocation and presentation of antigens occurs in the absence of PA, which proposes a modified molecular mechanism of antigen presentation by the anthrax toxin model. Additionally, we found that LFn--HIV is specific and sensitive in detecting HIV-specific CD4(+) and CD8(+) T cell responses in T cell assays, further broadening the value of this antigen delivery system as a useful immunologic tool. PMID:15964481

McEvers, K; Elrefaei, M; Norris, P; Deeks, S; Martin, J; Lu, Y; Cao, H

2005-04-15

237

Toxin Production  

Microsoft Academic Search

Staphylococci have developed a highly regulated system of toxin production that researchers are only beginning to understand. These toxin control systems respond to a wide range of environmental conditions, from catabolite and oxygen concentrations to bacterial cell density. Understanding the mechanisms of toxin regulation and production and how these systems might be manipulated to prevent toxin production will greatly enhance

Jeremy M. Yarwood; Patrick M. Schlievert

238

Anthrax  

MedlinePLUS

... potential to be used as a weapon of bioterrorism; this occurred in 2001 with an attack on ... Animal shearers Tanners In the case of a bioterrorism attack, anyone exposed to B. anthracis is at ...

239

Anthrax  

Center for Drug Evaluation (CDER)

... Enter Search terms Search. Search the FDA Archive. Home; Food; Drugs; Medical Devices; ... Safety of Long Term Therapy with Penicillin and Penicillin ... More results from www.fda.gov/drugs/emergencypreparedness/bioterrorismanddrugpreparedness

240

anthrax.  

National Technical Information Service (NTIS)

The report contains material for a lecture and also some supplementary points that go beyond the limits of a popular lecture, but which may be of interest to the lecturer and could be used by him, in part, in answers to some of the audience's questions. (...

A. V. Mashkov

1968-01-01

241

Anthrax: has the clinical milieu changed since 2001?  

PubMed Central

Since the anthrax attacks of 2001 (Amerithrax), several important improvements in the knowledge of Bacillus anthracis and the clinical condition it causes have occurred. While much remains to be known about the optimal management of anthrax patients, several approaches that were not widely utilized, available, or known in 2001 would be used in the treatment of critically ill anthrax patients in 2012.

Adalja, Amesh A.

2012-01-01

242

Anthrax: a continuing concern in the era of bioterrorism  

Microsoft Academic Search

Anthrax, a potentially fatal infection, is a virulent and highly contagious disease. It is caused by a gram-positive, toxigenic, spore-forming bacillus: Bacillus anthracis. For centuries, anthrax has caused disease in animals and, although uncommonly, in humans throughout the world. Descriptions of this naturally occurring disease begin in antiquity. Anthrax is primarily a disease of herbivores, which are infected by ingestion

STEFAN RIEDEL

243

Human-derived, plant-produced monoclonal antibody for the treatment of anthrax.  

PubMed

The unpredictable nature of bio-terrorism compels us to develop medical countermeasures that will enable authorities to treat individuals exposed to agents such as anthrax. We report the feasibility of producing a protective, human-derived, monoclonal antibody directed against the protective antigen (PA) of Bacillus anthracis in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting antibody was able to neutralize toxin activity in vitro and in vivo at a comparable level to that seen for its hybridoma-produced counterpart. PMID:15755575

Hull, Anna K; Criscuolo, Carolyn J; Mett, Vadim; Groen, Herman; Steeman, Wilma; Westra, Hans; Chapman, Gail; Legutki, Bart; Baillie, Les; Yusibov, Vidadi

2005-03-18

244

Cell-wall preparation containing poly-?-D-glutamate covalently linked to peptidoglycan, a straightforward extractable molecule, protects mice against experimental anthrax infection.  

PubMed

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-?-D-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-?-D-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain. PMID:23122993

Candela, Thomas; Dumetz, Fabien; Tosi-Couture, Evelyne; Mock, Michèle; Goossens, Pierre L; Fouet, Agnès

2012-11-01

245

[Selected research problems of anthrax vaccine development].  

PubMed

The threat of bioterrorism with B. anthracis against civilian population is one of major concern. After successful bioterroristic attack in 2001 in US renewed research interest has prompted in the development of new and more effective vaccine against anthrax. There are two licensed vaccines against anthrax--AVA-Bio-Thrax US and UK--sterile culture filtrate prepared by alum precipitation. Both vaccines are based on PA antigen. There are several concerns regarding PA based vaccines. They require six sc injections and yearly booster, high rates of local reaction after vaccination is observed, the immunity is not long lasting, vaccination do not protect animals against different strains of B. anthracis. New strategies in the development of anthrax vaccines have been presented (recombinant PA, subunits vaccine, mutants, conjugated). Using proteomic approaches new antigens have been also identified as candidates for future vaccines. More effective and easy to perform methods of vaccination have been reviewed. PMID:20120948

Zakowska, Dorota; Kocik, Janusz; Bartoszcze, Micha?

2009-01-01

246

Sverdlovsk revisited: Modeling human inhalation anthrax  

PubMed Central

Several models have been proposed for the dose–response function and the incubation period distribution for human inhalation anthrax. These models give very different predictions for the severity of a hypothetical bioterror attack, when an attack might be detected from clinical cases, the efficacy of medical intervention and the requirements for decontamination. Using data from the 1979 accidental atmospheric release of anthrax in Sverdlovsk, Russia, and limited nonhuman primate data, this paper eliminates two of the contending models and derives parameters for the other two, thereby narrowing the range of models that accurately predict the effects of human inhalation anthrax. Dose–response functions that exhibit a threshold for infectivity are contraindicated by the Sverdlovsk data. Dose-dependent incubation period distributions explain the 10-day median incubation period observed at Sverdlovsk and the 1- to 5-day incubation period observed in nonhuman primate experiments.

Wilkening, Dean A.

2006-01-01

247

A synthetic peptide vaccine directed against the 2ß2-2ß3 loop of domain 2 of protective antigen protects rabbits from inhalation anthrax.  

PubMed

The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax. PMID:20696862

Oscherwitz, Jon; Yu, Fen; Cease, Kemp B

2010-08-09

248

Is new always better than old?: The development of human vaccines for anthrax.  

PubMed

Anthrax is caused by a Gram-positive aerobic spore-forming bacillus called Bacillus anthracis. Although primarily a disease of animals, it can also infect man, sometimes with fatal consequences. As a result of concerns over the illicit use of this organism, considerable effort is focused on the development of therapies capable of conferring protection against anthrax. while effective concerns over the toxicity of the current vaccines have driven the development of second-generation products. Recombinant Protective Antigen (rPA), the nontoxic cell-binding component of anthrax lethal toxin, is the principal immunogen of the vaccines currently undergoing human clinical trials. While these new vaccines are likely to show reduced side effects they will still require multiple needle based dosing and the inclusion of the adjuvant alum which will make them expensive to administer and stockpile. To address these issues, researchers are seeking to develop vaccine formulations capable of stimulating rapid protection following needle-free injection which are stable at room temperature to facilitate stockpiling and mass vaccination programs. Recent concerns over the potential use of molecular biology to engineer vaccine resistant strains has prompted investigators to identify additional vaccine targets with which to extend the spectrum of protection conferred by rPA. While the injection of research dollars has seen a dramatic expansion of the anthrax vaccine field it is sobering to remember that work to develop the current second generation vaccines began around the time of the first gulf war. Almost two decades and millions of dollars later we still do not have a replacement vaccine and even when we do some argue that the spectrum of protection that it confers will not be as broad as the vaccine it replaces. If we are to respond effectively to emerging biological threats we need to develop processes that generate protective vaccines in a meaningful time frame and yield products in months not decades! PMID:19786839

Baillie, Leslie W

2009-12-09

249

Validation of an anti-PA-ELISA for the potency testing of anthrax vaccine in mice.  

PubMed

The potency test for the anthrax vaccine currently licensed for human use in the United States (Anthrax Vaccine Adsorbed) involves the protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis. Lethal challenge tests entail the use of specialized containment facilities for the safe and secure handling of the challenge strain. This potential difficulty, plus humane considerations, have prompted us to investigate non-lethal, alternative immunogenicity assays that could be considered as potency tests not only for the current vaccine, but also for vaccines under development. Immunogenicity tests will require suitable measurement of an antibody response to relevant antigens, by methods such as enzyme linked immunosorbent assay (ELISA) or a toxin neutralization assay. Any assay chosen for this purpose should be adequately validated and reproducible by other laboratories. Validation of an analytical procedure requires the demonstration that the assay is suitable for its intended purpose. The objective of this work was to study the performance of an anti-PA-ELISA designed to assess the antibody response to anthrax vaccines in mice. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH), and we have established the working range of the assay (37-1159 EU/mL) on the bases of the following parameters: linearity (20-1159 EU/mL; r2=0.99; p-value=0.21), accuracy (91-118% recovery), precision (< or =20%CV, repeatability; < or =9 and < or =21%CV, intermediate precision per day and per analyst, respectively), detection limit (5 EU/mL), and quantification limit (37 EU/mL). We believe that assay specificity and the above characteristics are adequate to allow this ELISA to be considered for use in a mouse immunogenicity (potency) test of anthrax vaccines, and for the standardization of reagents. PMID:15536047

Pombo, María; Berthold, Inge; Gingrich, Elise; Jaramillo, María; Leef, Mary; Sirota, Lev; Hsu, Henry; Arciniega, Juan

2004-09-01

250

Acceleration of epithelial cell syndecan-1 shedding by anthrax hemolytic virulence factors  

PubMed Central

Background It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice. Results The isolated anthrax hemolytic proteins AnlB (sphingomyelinase) and AnlO (cholesterol-binding pore-forming factor), as well as ClnA (B. cereus homolog of B. anthracis phosphatidyl choline-preferring phospholipase C) cause accelerated shedding of Synd1 and E-cadherin from epithelial cells and compromise epithelial barrier integrity within a few hours. In comparison with hemolysins in a similar range of concentrations, anthrax lethal toxin (LT) also accelerates shedding albeit at slower rate. Individual components of LT, lethal factor and protective antigen are inactive with regard to shedding. Inhibition experiments favor a hypothesis that activities of tested bacterial shedding inducers converge on the stimulation of cytoplasmic tyrosine kinases of the Syk family, ultimately leading to activation of cellular sheddase. Both LT and AnlO modulate ERK1/2 and p38 MAPK signaling pathways, while JNK pathway seems to be irrelevant to accelerated shedding. Accelerated shedding of Synd1 also takes place in DBA/2 mice challenged with Bacillus anthracis (Sterne) spores. Elevated levels of shed ectodomain are readily detectable in circulation after 24 h. Conclusion The concerted acceleration of shedding by several virulence factors could represent a new pathogenic mechanism contributing to disruption of epithelial or endothelial integrity, hemorrhage, edema and abnormal cell signaling during anthrax infection.

Popova, Taissia G; Millis, Bryan; Bradburne, Chris; Nazarenko, Svetlana; Bailey, Charles; Chandhoke, Vikas; Popov, Serguei G

2006-01-01

251

A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen  

PubMed Central

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR?STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.

Gawlik, Katarzyna; Remacle, Albert G.; Shiryaev, Sergey A.; Golubkov, Vladislav S.; Ouyang, Mingxing; Wang, Yingxiao; Strongin, Alex Y.

2010-01-01

252

Serodiagnosis of Human Cutaneous Anthrax in India Using an Indirect Anti-Lethal Factor IgG Enzyme-Linked Immunosorbent Assay  

PubMed Central

Anthrax, caused by Bacillus anthracis, is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (anti-LF) IgG in human serum samples. A panel of 203 human serum samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from areas of India where anthrax is nonendemic, 44 samples from controls from an area of India where anthrax is endemic, and 16 patients with a disease confirmed not to be anthrax were evaluated with an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 ELISA unit (EU), with a 95% confidence interval (CI) of 0.120 to 0.151 EU. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89 to 100%) and 97.39% (95% CI, 93.44 to 99.28%), respectively, at a cutoff value of 0.375 EU, as decided by receiver operating characteristic (ROC) curve analysis. The likelihood ratio was found to be 49.98. The positive predictive value (PPV), negative predictive value (NPV), efficiency, and Youden's index (J) for reliability of the assay were 92.5%, 100%, 98.02%, and 0.97, respectively. The false-positive predictive rate and false-negative predictive rate of the assay were 2.61% and 0%. The assay could be a very useful tool for early diagnosis of cutaneous anthrax cases, as antibodies against LF appear much earlier than those against other anthrax toxins in human serum samples.

Ghosh, N.; Tomar, I.; Lukka, H.

2013-01-01

253

Serodiagnosis of human cutaneous anthrax in India using an indirect anti-lethal factor IgG enzyme-linked immunosorbent assay.  

PubMed

Anthrax, caused by Bacillus anthracis, is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (anti-LF) IgG in human serum samples. A panel of 203 human serum samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from areas of India where anthrax is nonendemic, 44 samples from controls from an area of India where anthrax is endemic, and 16 patients with a disease confirmed not to be anthrax were evaluated with an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 ELISA unit (EU), with a 95% confidence interval (CI) of 0.120 to 0.151 EU. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89 to 100%) and 97.39% (95% CI, 93.44 to 99.28%), respectively, at a cutoff value of 0.375 EU, as decided by receiver operating characteristic (ROC) curve analysis. The likelihood ratio was found to be 49.98. The positive predictive value (PPV), negative predictive value (NPV), efficiency, and Youden's index (J) for reliability of the assay were 92.5%, 100%, 98.02%, and 0.97, respectively. The false-positive predictive rate and false-negative predictive rate of the assay were 2.61% and 0%. The assay could be a very useful tool for early diagnosis of cutaneous anthrax cases, as antibodies against LF appear much earlier than those against other anthrax toxins in human serum samples. PMID:23269414

Ghosh, N; Tomar, I; Lukka, H; Goel, A K

2012-12-26

254

Millipede toxin  

MedlinePLUS

... toxin) if they are threatened or if you handle them roughly. Millipedes can squirt toxin several inches ... Schwartz RA. Arthropod bites and stings. In: Wolff K, Goldsmith LA, Katz SI, et al., eds. Fitzpatrick’s ...

255

[Recombinant antibodies for medical protection against bioterrorism agents: the example of anthrax].  

PubMed

Recombinant antibodies are a highly successful class of therapeutic molecules, they are well adapted for use against bio-weapons (BW) as they act immediately, are often synergistic with other therapeutic molecules, have a long half-life and are well tolerated. Anthrax is regarded at high risk of being used as BW, and its pathogenic properties depend on toxins, which might be neutralized by antibodies. These toxins are made of three different types of sub-units (PA, LF, EF). Several anti-PA have been developed, including an original approach by our team. We have developed an anti-LF, as recommended by experts. Our anti-PA antibody, and to a lesser extend our anti-LF antibody, will be presented here. PMID:20950579

Thullier, Philippe; Pelat, Thibault; Paucod, Jean-Charles; Vidal, Dominique

2010-04-08

256

Genetic Vaccines for Anthrax Based on Recombinant Adeno-associated Virus Vectors  

PubMed Central

Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments encoding activated PA (PA63), or LF, were synthesized and cloned into optimized rAAV1 vectors containing a human cytomegalovirus (hCMV) promoter and synthetic optimized leader. Serum from rabbits immunized intramuscularly with rAAV1/PA (monovalent), rAAV1/LF (monovalent), rAAV1/PA + rAAV1/LF (bivalent), or rAAV1/enhanced green fluorescent protein (control) exhibited substantial PA- and LF-specific antibody responses at 4 weeks by both western blot (> 1:10,000 dilution) and enzyme-linked immunosorbent assay (ELISA) (mean end-point titer: 32,000–260,000), and contained anthrax LeTx–neutralizing activity in vitro, with peak titers approximating those of a rabbit hyperimmune antisera raised against soluble PA and LF. Compared to the monovalent groups (rAAV1/PA or rAAV1/LF), the bivalent group (rAAV1/PA + rAAV1/LF) exhibited marginally higher ELISA and neutralization activity with dual specificity for both PA and LF. The finding of robust neutralizing antibody responses after a single injection of these rAAV1-based vectors supports their further development as candidate anthrax vaccines.

Liu, Te-Hui; Oscherwitz, Jon; Schnepp, Bruce; Jacobs, Jana; Yu, Fen; Cease, Kemp B; Johnson, Philip R

2008-01-01

257

76 FR 34994 - Vaccine To Protect Children From Anthrax-Public Engagement Workshop  

Federal Register 2010, 2011, 2012, 2013

...DEPARTMENT OF HEALTH AND HUMAN SERVICES Vaccine To Protect Children From Anthrax--Public...Biodefense Science Board's (NBSB) Anthrax Vaccine (AV) Working Group (WG) will hold...workshop on July 7, 2011, to discuss vaccine to protect children from anthrax....

2011-06-15

258

Increased long-term immunity to Bacillus anthracis protective antigen in mice immunized with a CIA06B-adjuvanted anthrax vaccine.  

PubMed

Anthrax is an acute infectious disease caused by Bacillus anthracis. We previously reported that the adjuvant CIA06B, which consists of TLR4 agonist CIA05 and aluminum hydroxide (alum), enhanced the immune response to anthrax protective antigen (PA) in mice. This study was carried out to determine whether CIA06B can enhance long-term immune responses to PA in mice. BALB/c mice were immunized intramuscularly three times at 2-week intervals with recombinant PA alone or PA combined with alum or CIA06B. At 8 and 24 weeks post-immunization, the immunological responses including serum anti-PA IgG antibody titer, toxin-neutralizing antibody titer, splenic cytokine secretion and the frequency of PA-specific memory B cells were assessed. Compared with mice injected with PA alone or PA plus alum, mice injected with PA plus CIA06B had higher titers of serum anti-PA IgG antibodies, and higher frequencies of PA-specific memory B cells and interferon-? secreting cells. Furthermore, anti-PA antibodies induced by CIA06B were more effective in neutralizing anthrax toxin. These results demonstrated that CIA06B is capable of providing long-term immunity when used as an adjuvant in a PA-based anthrax vaccine. PMID:23440578

Wui, Seo Ri; Han, Ji Eun; Kim, Yeon Hee; Rhie, Gi-eun; Lee, Na Gyong

2013-02-26

259

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*  

PubMed Central

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

Zakharova, Maria Yu.; Kuznetsov, Nikita A.; Dubiley, Svetlana A.; Kozyr, Arina V.; Fedorova, Olga S.; Chudakov, Dmitry M.; Knorre, Dmitry G.; Shemyakin, Igor G.; Gabibov, Alexander G.; Kolesnikov, Alexander V.

2009-01-01

260

Sverdlovsk Anthrax Outbreak: An Educational Case Study  

Microsoft Academic Search

In April and May of 1979 an Anthrax epidemic broke out in the city of Sverdlovsk (now Ekaterinburg) in the former Soviet Union. Sixty-four people were reported to have died from the outbreak, although there is still debate concerning the actual number of victims. While Soviet officials initially attributed this outbreak to contaminated meat, the US Government maintained that the

S. J. Steele; G. van der Vink

2002-01-01

261

Inhalation Anthrax: Dose Response and Risk Analysis  

PubMed Central

The notion that inhalation of a single Bacillus anthracis spore is fatal has become entrenched nearly to the point of urban legend, in part because of incomplete articulation of the scientific basis for microbial risk assessment, particularly dose-response assessment. Risk analysis (ie, risk assessment, risk communication, risk management) necessitates transparency: distinguishing scientific facts, hypotheses, judgments, biases in interpretations, and potential misinformation. The difficulty in achieving transparency for biothreat risk is magnified by misinformation and poor characterization of both dose-response relationships and the driving mechanisms that cause susceptibility or resistance to disease progression. Regrettably, this entrenchment unnecessarily restricts preparedness planning to a single response scenario: decontaminate until no spores are detectable in air, water, or on surfaces—essentially forcing a zero-tolerance policy inconsistent with the biology of anthrax. We present evidence about inhalation anthrax dose-response relationships, including reports from multiple studies documenting exposures insufficient to cause inhalation anthrax in laboratory animals and humans. The emphasis of the article is clarification about what is known from objective scientific evidence for doses of anthrax spores associated with survival and mortality. From this knowledge base, we discuss the need for future applications of more formal risk analysis processes to guide development of alternative non-zero criteria or standards based on science to inform preparedness planning and other risk management activities.

Thran, Brandolyn; Morse, Stephen S.; Hugh-Jones, Martin; Massulik, Stacey

2008-01-01

262

Anthrax Attack! A Case on Bioterrorism  

NSDL National Science Digital Library

This case study presents a fictitious bio-terrorist plan to release anthrax in the United States. Students are assigned character roles and, through research, role-playing, and teamwork, develop a plan to minimize or avert the attack. The case is appropriate for courses designed for health professionals, general biology courses, and social science courses.

Mergenhagen, Kari A.

2003-01-01

263

Anthrax Attacks, Biological Terrorism and Preventive Responses.  

National Technical Information Service (NTIS)

The recent anthrax attacks represent a fundamental shift in the nature of the biological terrorism threat. Fortunately, the scope and magnitude of this shift is far less devastating than the events of September 11th. As we face this new phase of biologica...

J. Parachini

2001-01-01

264

Murine Aerosol Challenge Model of Anthrax  

Microsoft Academic Search

The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores

Crystal L. Loving; Mary Kennett; Gloria M. Lee; Vanessa K. Grippe; Tod J. Merkel

2007-01-01

265

Edema Toxin Impairs Anthracidal Phospholipase A2 Expression by Alveolar  

Microsoft Academic Search

Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced

Macrophages Benoit Raymond; Dominique Leduc; Lucas Ravaux; Ronan Le Goffic; Thomas Candela; Michel Raymondjean; Pierre Louis Goossens; Lhousseine Touqui

266

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins  

SciTech Connect

Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

1981-11-01

267

Anthrax: a continuing concern in the era of bioterrorism  

PubMed Central

Anthrax, a potentially fatal infection, is a virulent and highly contagious disease. It is caused by a gram-positive, toxigenic, spore-forming bacillus: Bacillus anthracis. For centuries, anthrax has caused disease in animals and, although uncommonly, in humans throughout the world. Descriptions of this naturally occurring disease begin in antiquity. Anthrax is primarily a disease of herbivores, which are infected by ingestion of spores from the soil. With the advent of modern microbiology, Pasteur developed the first successful anthrax vaccine in 1881. The incidence of the disease has continually decreased since the late 19th century, and animal vaccination programs drastically reduced the animal mortality from the disease. However, anthrax spores continue to be documented in soil samples from throughout the world. Research on anthrax as a biological weapon began more than 80 years ago, and today at least 17 nations are believed to have offensive biological weapons programs that include anthrax. Recent events in the USA have shown how society is affected by both hoax and real threats of anthrax bioweapons. This fourth article in the series on weapons of biowarfare/bioterrorism summarizes the historical background of anthrax as well as clinical and laboratory information useful for bioterrorism preparedness.

2005-01-01

268

Antibody response to a delayed booster dose of anthrax vaccine and botulinum toxoid.  

PubMed

We evaluated the prevalence and concentration of serum antibodies 18-24 months after primary inoculation with anthrax and botulinum vaccines, and assessed the reactogenicity and immunogenicity of a significantly delayed booster dose of these vaccines. Five hundred and eight male active-duty military personnel received one, two or three inoculations with anthrax vaccine and/or botulinum toxoid in 1990/1991 in preparation for Operations Desert Shield/Desert Storm. Subjects were vaccinated with the licensed anthrax vaccine, adsorbed (AVA) and pentavalent (ABCDE) botulinum toxoid (PBT) BB-IND 3723. Anthrax protective antigen (PA) IgG antibody was measured in serum using an immunocapture enzyme-linked immunosorbent assay (ELISA). A mouse neutralization test was used to determine the titer of Clostridium botulinum type A antitoxin in serum samples. The prevalence of anti-PA IgG was 30% in individuals 18-24 months after priming with one, two or three doses of AVA. After boosting, 99% of volunteers had detectable anti-PA IgG; only two individuals failed to respond. The prevalence of antibodies against botulinum toxin type A was 28% 18-24 months after initial priming. Following boosting, 99% of volunteers had serum titers >0.02IU/ml, and 97% responded with titers > or =0.25IU/ml. Systemic reactions to booster vaccinations could not be specifically ascribed to one or the other vaccine, but were generally mild and of brief duration. Forty-five percent of volunteers reported one or more systemic reactions over the course of 7 days. Injection site reactions of any kind occurred in 25% of AVA recipients and in 16% of PBT recipients; persistence of local reactions beyond 7 days was infrequent. While the kinetics and durability of immune responses must be studied, these findings suggest that booster doses of anthrax vaccine and botulinum toxoid sufficient to stimulate a robust anamnestic response may be given at times distant from receipt of the primary inoculations. PMID:11972980

Pittman, Phillip R; Hack, Dallas; Mangiafico, Joseph; Gibbs, Paul; McKee, Kelly T; Friedlander, Arthur M; Sjogren, Maria H

2002-05-15

269

Bacillus anthracis and the Pathogenesis of Anthrax  

Microsoft Academic Search

\\u000a Bacillus anthracis is the causative agent of anthrax, a disease of animals that is transmissible to humans. Because B. anthracis forms spores that can be aerosolized and sprayed with the intent to kill, this pathogen can also be viewed as an agent of\\u000a biological warfare and bioterrorism (1). The accidental release of spores into the air in Sverdlosk, Russia, and

Dominique M. Missiakas; Olaf Schneewind

270

A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein  

PubMed Central

Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

Rogers, Michael S.; Cryan, Lorna M.; Habeshian, Kaiane A.; Bazinet, Lauren; Caldwell, Thomas P.; Ackroyd, P. Christine; Christensen, Kenneth A.

2012-01-01

271

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers  

PubMed Central

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

Fischer, Audrey; Holden, Matthew A.; Pentelute, Brad L.; Collier, R. John

2011-01-01

272

Modeling the host response to inhalation anthrax  

PubMed Central

Inhalation anthrax, an often fatal infection, is initiated by endospores of the bacterium Bacillus anthracis that are introduced into the lung. To better understand the pathogenesis of an inhalation anthrax infection, we propose a two compartment mathematical model which takes into account the documented early events of such an infection. Anthrax spores, once inhaled, are readily taken up by alveolar phagocytes which then migrate rather quickly out of the lung and into the thoracic/mediastinal lymph nodes. En route, these spores germinate to become vegetative bacteria. In the lymph nodes, the bacteria kill the host cells and are released into the extracellular environment where they can be disseminated into the blood stream and grow to a very high level, often resulting in death of the infected person. Using this framework as the basis of our model, we explore the probability of survival of an infected individual. This is dependent on several factors, such as the rate of migration and germination events and treatment with antibiotics.

Day, Judy; Friedman, Avner; Schlesinger, Larry S

2011-01-01

273

Modeling the incubation period of inhalational anthrax.  

PubMed

Ever since the pioneering work of Philip Sartwell, the incubation period distribution for infectious diseases is most often modeled using a lognormal distribution. Theoretical models based on underlying disease mechanisms in the host are less well developed. This article modifies a theoretical model originally developed by Brookmeyer and others for the inhalational anthrax incubation period distribution in humans by using a more accurate distribution to represent the in vivo bacterial growth phase and by extending the model to represent the time from exposure to death, thereby allowing the model to be fit to nonhuman primate time-to-death data. The resulting incubation period distribution and the dose dependence of the median incubation period are in good agreement with human data from the 1979 accidental atmospheric anthrax release in Sverdlovsk, Russia, and limited nonhuman primate data. The median incubation period for the Sverdlovsk victims is 9.05 (95% confidence interval = 8.0-10.3) days, shorter than previous estimates, and it is predicted to drop to less than 2.5 days at doses above 10(6) spores. The incubation period distribution is important because the left tail determines the time at which clinical diagnosis or syndromic surveillance systems might first detect an anthrax outbreak based on early symptomatic cases, the entire distribution determines the efficacy of medical intervention-which is determined by the speed of the prophylaxis campaign relative to the incubation period-and the right tail of the distribution influences the recommended duration for antibiotic treatment. PMID:18556642

Wilkening, Dean A

2008-06-12

274

The anthrax attacks 10 years later.  

PubMed

Ten years ago, just weeks after the September 11 attacks, the United States experienced a deliberate act of bioterrorism. Through use of the postal service, anthrax spores were widely disseminated, including to homes, the Senate, and major newsrooms, resulting in morbidity and mortality and effectively disrupting our way of life and revealing our vulnerability. Even though such attacks had been the subject of much writing and had been planned for, detection of and the appropriate response to an attack with an agent from the so-called "Category 'A' List" had only been considered in theoretical terms. What transpired during the following difficult weeks, including how public health and federal government agencies performed, has been both praised and criticized. An intertwined epidemiologic and criminal investigation of such magnitude was unprecedented in U.S. history. To address the question of whether we as a nation are now better prepared for future threats involving biologic agents, it is important to learn from the lessons of the 2001 anthrax attacks, including the critical role of clinicians in surveillance. As physicians involved in diagnosing anthrax in the index case and alerting authorities, we offer our perspective on these events a decade after their occurrence. PMID:21969275

Bush, Larry M; Perez, Maria T

2011-10-03

275

Murine aerosol challenge model of anthrax.  

PubMed

The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis. PMID:17353290

Loving, Crystal L; Kennett, Mary; Lee, Gloria M; Grippe, Vanessa K; Merkel, Tod J

2007-03-12

276

Epidemiologic Investigations of Bioterrorism-Related Anthrax, New Jersey, 2001  

Microsoft Academic Search

At least four Bacillus anthracis-containing envelopes destined for New York City and Washington, D.C. were processed at the Trenton Processing and Distribution Center (PDC) on September 18 and October 9, 2001. When cutaneous anthrax was confirmed in a Trenton postal worker, the PDC was closed. Four cutaneous and two inhalational anthrax cases were identified. Five patients were hospitalized; none died.

Carolyn M. Greene; Jennita Reefhuis; Christina Tan; Anthony E. Fiore; Susan Goldstein; Michael J. Beach; Stephen C. Redd; David Valiante; Gregory Burr; James Buehler; Robert W. Pinner; Eddy Bresnitz; Beth P. Bell

2002-01-01

277

Absence of Mycoplasma Contamination in the Anthrax Vaccine  

PubMed Central

Mycoplasma contamination of the licensed anthrax vaccine administered to military personnel has been suggested as a possible cause of Persian Gulf illness. Vaccine samples tested by nonmilitary laboratories were negative for viable mycoplasma and mycoplasma DNA and did not support its survival. Mycoplasma contamination of anthrax vaccine should not be considered a possible cause of illness.

Hart, Mary Kate; Del Giudice, Richard A.

2002-01-01

278

Anthrax Vaccine Debate: A Medical Review for Commanders.  

National Technical Information Service (NTIS)

There are two distinct yet related aspects to the debate over the safety and efficacy of the anthrax vaccine. An assessment of the clinical safety and efficacy of the anthrax vaccine. The policy level decision to vaccinate military personnel based on inte...

R. A. Hersack

2001-01-01

279

Why Do UK Military Personnel Refuse the Anthrax Vaccination?  

Microsoft Academic Search

The purpose of this study was to understand the reasons why some UK military personnel refused the anthrax vaccination. Data were collected from 5,302 members of the UK Armed Forces who had been deployed to Iraq since 2003 and had been offered the anthrax vaccination. As part of a larger questionnaire, information was collected on acceptance or refusal of the

Dominic Murphy; Theresa Marteau; Matthew Hotopf; Roberto J. Rona; Simon Wessely

2008-01-01

280

Proteolytic Activation of Bacterial Toxins by Eukaryotic Cells Is Performed by Furin and by Additional Cellular Proteases  

Microsoft Academic Search

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR)andcleavagesitemutantsofPA,eachadministeredwithamodifiedformofanthraxtoxinlethalfactor (the N terminus of lethal factor fused to PE domain III),

VALERY M. GORDON; KURT R. KLIMPEL; NAVEEN ARORA; MARLON A. HENDERSON; ANDSTEPHEN H. LEPPLA

281

Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores  

PubMed Central

In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed with Escherichia coli heat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development.

Peachman, Kristina K.; Li, Qin; Matyas, Gary R.; Shivachandra, Sathish B.; Lovchik, Julie; Lyons, Rick C.; Alving, Carl R.; Rao, Venigalla B.

2012-01-01

282

Anthrax vaccine antigen-adjuvant formulations completely protect New Zealand white rabbits against challenge with Bacillus anthracis Ames strain spores.  

PubMed

In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed with Escherichia coli heat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development. PMID:22089245

Peachman, Kristina K; Li, Qin; Matyas, Gary R; Shivachandra, Sathish B; Lovchik, Julie; Lyons, Rick C; Alving, Carl R; Rao, Venigalla B; Rao, Mangala

2011-11-16

283

Bacillus cereus G9241 S-Layer Assembly Contributes to the Pathogenesis of Anthrax-Like Disease in Mice  

PubMed Central

Bacillus cereus G9241, the causative agent of anthrax-like disease, harbors virulence plasmids encoding anthrax toxins as well as hyaluronic acid (HA) and B. cereus exopolysaccharide (BPS) capsules. B. cereus G9241 also harbors S-layer genes, including homologs of Bacillus anthracis surface array protein (Sap), extractable antigen 1 (EA1), and the S-layer-associated proteins (BSLs). In B. anthracis, S-layer proteins and BSLs attach via their S-layer homology domains (SLH) to the secondary cell wall polysaccharide (SCWP) in a manner requiring csaB, a predicted ketalpyruvate transferase. Here we used a genetic approach to analyze B. cereus G9241 S-layer assembly and function. Variants lacking the csaB gene synthesized SCWP but failed to retain Sap, EA1, and BSLs in the bacterial envelope. The B. cereus G9241 csaB mutant assembled capsular polysaccharides but displayed an increase in chain length relative to the wild-type strain. This phenotype is likely due to its inability to deposit BslO murein hydrolase at divisional septa. During growth under capsule-inducing conditions, B. cereus G9241 assembled BSLs (BslA and BslO) and the Sap S-layer protein, but not EA1, in the envelope. Finally, csaB-mediated assembly of S-layer proteins and BSLs in B. cereus G9241 contributes to the pathogenesis of anthrax-like disease in mice.

Wang, Ya-Ting; Oh, So-Young; Hendrickx, Antoni P. A.; Lunderberg, J. M.

2013-01-01

284

Bacillus cereus G9241 S-layer assembly contributes to the pathogenesis of anthrax-like disease in mice.  

PubMed

Bacillus cereus G9241, the causative agent of anthrax-like disease, harbors virulence plasmids encoding anthrax toxins as well as hyaluronic acid (HA) and B. cereus exopolysaccharide (BPS) capsules. B. cereus G9241 also harbors S-layer genes, including homologs of Bacillus anthracis surface array protein (Sap), extractable antigen 1 (EA1), and the S-layer-associated proteins (BSLs). In B. anthracis, S-layer proteins and BSLs attach via their S-layer homology domains (SLH) to the secondary cell wall polysaccharide (SCWP) in a manner requiring csaB, a predicted ketalpyruvate transferase. Here we used a genetic approach to analyze B. cereus G9241 S-layer assembly and function. Variants lacking the csaB gene synthesized SCWP but failed to retain Sap, EA1, and BSLs in the bacterial envelope. The B. cereus G9241 csaB mutant assembled capsular polysaccharides but displayed an increase in chain length relative to the wild-type strain. This phenotype is likely due to its inability to deposit BslO murein hydrolase at divisional septa. During growth under capsule-inducing conditions, B. cereus G9241 assembled BSLs (BslA and BslO) and the Sap S-layer protein, but not EA1, in the envelope. Finally, csaB-mediated assembly of S-layer proteins and BSLs in B. cereus G9241 contributes to the pathogenesis of anthrax-like disease in mice. PMID:23204457

Wang, Ya-Ting; Oh, So-Young; Hendrickx, Antoni P A; Lunderberg, J M; Schneewind, Olaf

2012-11-30

285

A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.  

PubMed

The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax. PMID:21270531

Mett, Vadim; Chichester, Jessica A; Stewart, Michelle L; Musiychuk, Konstantin; Bi, Hong; Reifsnyder, Carolyn J; Hull, Anna K; Albrecht, Mark T; Goldman, Stanley; Baillie, Les W J; Yusibov, Vidadi

2011-01-01

286

BICHAT GUIDELINES* FOR THE CLINICAL MANAGEMENT OF ANTHRAX AND BIOTERRORISM-RELATED ANTHRAX  

Microsoft Academic Search

The spore-forming Bacillus anthracis must be considered as one of the most serious potential biological weapons. The recent cases of anthrax caused by a deliberate release reported in 2001 in the United States point to the necessity of early recognition of this disease. Infection in humans most often involves the skin, and more rarely the lungs and the gastrointestinal tract.

P Bossi; A Tegnell; A Baka; F Van Loock; J Hendriks; A Werner; H Maidhof; G Gouvras

287

Botulinum toxin.  

PubMed

Botulinum toxin is regarded as the most lethal substance known. It is estimated that the human LD50 for inhalation botulism is 1 to 3 nanograms of toxin/kilogram body mass. Although only three cases of inhalational botulism have been described, an understanding of the pathophysiology of food-borne outbreaks, wound botulism, and infant botulism, and their therapies, enables the medical community to plan treatment in the event of an aerosol release of botulinum toxin. Antitoxin, vaccine, and F(ab')2 immune fragment therapies are discussed as adjuncts to supportive therapy. PMID:16168317

Horowitz, B Zane

2005-10-01

288

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy.  

PubMed

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827-34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax. PMID:18805452

Baillie, Leslie W J; Rodriguez, Ana L; Moore, Stephen; Atkins, Helen S; Feng, Chiguang; Nataro, James P; Pasetti, Marcela F

2008-09-19

289

Rapid generation of an anthrax immunotherapeutic from goats using a novel non-toxic muramyl dipeptide adjuvant  

PubMed Central

Background There is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections. Methods Using recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication. Results Anti-PA83 IgG conferred 100% protection at 7.5 ?g in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo. Conclusion The protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations.

Kelly, Cassandra D; O'Loughlin, Chris; Gelder, Frank B; Peterson, Johnny W; Sower, Laurie E; Cirino, Nick M

2007-01-01

290

Marine Toxins  

MedlinePLUS

... by marine toxins? General guidelines for safe seafood consumption: Although any person eating fish or shellfish containing ... same food safety regulations as seafood for human consumption. Back to Top What is the government doing ...

291

Hepatic Subcellular Distribution of (3H)T-2 Toxin.  

National Technical Information Service (NTIS)

The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with (3 tritium)T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile (53%), perfusate (38), and liver (7%). Livers we...

J. G. Pace M. R. Watts

1989-01-01

292

Mucosal immunization with attenuated Salmonella Typhi expressing anthrax PA83 primes monkeys for accelerated serum antibody responses to parenteral PA83 vaccine  

PubMed Central

Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was genetically engineered for stable plasmid-based expression of protective antigen of anthrax toxin (PA83) fused with the export protein ClyA (ClyA-PA83). The priming potential of CVD 908-htrA expressing ClyA-PA83 was assessed in 12 rhesus and 20 cynomolgus macaques immunized mucosally (intranasally) on days 0 and 14. A parenteral boost with purified PA83 plus alum was given to rhesus macaques on days 42 and 225; cynomolgus monkeys were boosted only once, 3 months after priming, with either PA or licensed anthrax vaccine (Biothrax®). Monkeys primed with S. Typhi expressing ClyA-PA83 developed high levels of serum toxin neutralization activity (TNA) antibodies (> 1.3 ×103 ED50), 7 days after boosting, while unprimed controls lacked serum TNA (0 ED50). The success in non-human primates of this anthrax vaccine strategy based on heterologous mucosal prime followed by parenteral subunit vaccine boost paves the way for clinical trials.

Galen, James E.; Chinchilla, Magaly; Pasetti, Marcela F.; Wang, Jin Yuan; Zhao, Licheng; Arciniega-Martinez, Ivonne; Silverman, David J.; Levine, Myron M.

2008-01-01

293

Investigation of new dominant-negative inhibitors of anthrax protective antigen mutants for use in therapy and vaccination.  

PubMed

The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 microg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1 beta (IL-1 beta) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections. PMID:19620345

Cao, Sha; Guo, Aizhen; Liu, Ziduo; Tan, Yadi; Wu, Gaobing; Zhang, Chengcai; Zhao, Yaxing; Chen, Huanchun

2009-07-20

294

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia.  

PubMed

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ?Ames strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies. PMID:22767539

Cote, C K; Kaatz, L; Reinhardt, J; Bozue, J; Tobery, S A; Bassett, A D; Sanz, P; Darnell, S C; Alem, F; O'Brien, A D; Welkos, S L

2012-07-05

295

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia  

PubMed Central

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ?Ames strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Cote, C. K.; Kaatz, L.; Reinhardt, J.; Bozue, J.; Tobery, S. A.; Bassett, A. D.; Sanz, P.; Darnell, S. C.; Alem, F.; O'Brien, A. D.

2012-01-01

296

The Glucocorticoid Receptor: A Revisited Target for Toxins  

PubMed Central

The hypothalamic-pituitary-adrenal (HPA) axis activation and glucocorticoid responses are critical for survival from a number of bacterial, viral and toxic insults, demonstrated by the fact that removal of the HPA axis or GR blockade enhances mortality rates. Replacement with synthetic glucocorticoids reverses these effects by providing protection against lethal effects. Glucocorticoid resistance/insensitivity is a common problem in the treatment of many diseases. Much research has focused on the molecular mechanism behind this resistance, but an area that has been neglected is the role of infectious agents and toxins. We have recently shown that the anthrax lethal toxin is able to repress glucocorticoid receptor function. Data suggesting that the glucocorticoid receptor may be a target for a variety of toxins is reviewed here. These studies have important implications for glucocorticoid therapy.

Marketon, Jeanette I. Webster; Sternberg, Esther M.

2010-01-01

297

Interferon Protects Mice Against Inhalation Anthrax  

PubMed Central

Interferons (IFNs) play a role in innate immunity during many viral, bacterial, and protozoal infections. With the increasing threat of bioterrorist attacks with Bacillus anthracis, its high lethality, and the limited effectiveness of antibiotics, alternative treatments are being studied. Antibodies to protective antigen (PA) are promising, as is IFN. During many bacterial infections, production of and protection by IFNs has been reported, including B. anthracis in vitro. In vivo, we find that (1) the type I IFN inducer, Poly-ICLC, strongly and rapidly protects mice; (2) the protection is IFN-mediated since recombinant murine IFN-? can protect, and protection by Poly-ICLC is abrogated in IFN type I receptor knockout mice. The greatest protection by Poly-ICLC was conferred by intra-nasal treatment. A delay in death was observed with the intramuscular route alone, but was not significant. Together, the results suggest the IFN defense could protect mice, up to 60%, against lethal inhalational anthrax, and thus have important medical implications for therapy of human anthrax.

Walberg, Kristin; Poast, Joyce; Schwartz, Barbara; Izotova, Lara; Pestka, Sidney; Peterson, J.W.

2008-01-01

298

Interferon protects mice against inhalation anthrax.  

PubMed

Interferons (IFNs) play a role in innate immunity during many viral, bacterial, and protozoal infections. With the increasing threat of bioterrorist attacks with Bacillus anthracis, its high lethality, and the limited effectiveness of antibiotics, alternative treatments are being studied. Antibodies to protective antigen (PA) are promising, as is IFN. During many bacterial infections, production of and protection by IFNs has been reported, including B. anthracis in vitro. In vivo, we find that (1) the type I IFN inducer, Poly-ICLC, strongly and rapidly protects mice; (2) the protection is IFN-mediated since recombinant murine IFN-beta can protect, and protection by Poly-ICLC is abrogated in IFN type I receptor knockout mice. The greatest protection by Poly-ICLC was conferred by intranasal treatment. A delay in death was observed with the intramuscular route alone, but was not significant. Together, the results suggest the IFN defense could protect mice, up to 60%, against lethal inhalational anthrax, and thus have important medical implications for therapy of human anthrax. PMID:18778201

Walberg, Kristin; Baron, Samuel; Poast, Joyce; Schwartz, Barbara; Izotova, Lara; Pestka, Sidney; Peterson, J W

2008-10-01

299

Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities  

Microsoft Academic Search

\\u000a Purpose  The current anthrax vaccine adsorbed (AVA) was originally licensed for the prevention of cutaneous anthrax infection. It has\\u000a many drawbacks, including the requirement for multiple injections and subsequent annual boosters. Thus, an easily administrable\\u000a and efficacious anthrax vaccine is needed to prevent the most lethal form of anthrax infection, inhalation anthrax. We propose\\u000a to develop a nasal anthrax vaccine using

Brian R. Sloat; Zhengrong Cui

2006-01-01

300

BOTULINUM TOXIN  

PubMed Central

Botulinum toxin, one of the most poisonous biological substances known, is a neurotoxin produced by the bacterium Clostridium botulinum. C. botulinum elaborates eight antigenically distinguishable exotoxins (A, B, C1, C2, D, E, F and G). All serotypes interfere with neural transmission by blocking the release of acetylcholine, the principal neurotransmitter at the neuromuscular junction, causing muscle paralysis. The weakness induced by injection with botulinum toxin A usually lasts about three months. Botulinum toxins now play a very significant role in the management of a wide variety of medical conditions, especially strabismus and focal dystonias, hemifacial spasm, and various spastic movement disorders, headaches, hypersalivation, hyperhidrosis, and some chronic conditions that respond only partially to medical treatment. The list of possible new indications is rapidly expanding. The cosmetological applications include correction of lines, creases and wrinkling all over the face, chin, neck, and chest to dermatological applications such as hyperhidrosis. Injections with botulinum toxin are generally well tolerated and side effects are few. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice.

Nigam, P K; Nigam, Anjana

2010-01-01

301

Differential Effects of Linezolid and Ciprofloxacin on Toxin Production by Bacillus anthracis in an In Vitro Pharmacodynamic System  

PubMed Central

Bacillus anthracis causes anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxin-producing ?Sterne strain of B. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibitor, while ciprofloxacin is not. Since toxins are produced only by vegetative B. anthracis, the effect of linezolid and ciprofloxacin on toxin production is of interest. The effect of simulated clinical regimens of ciprofloxacin and linezolid on the vegetative and spore populations and on toxin production was examined in an in vitro pharmacodynamic model over 15 days by using the toxin-producing Sterne strain of B. anthracis. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. With ciprofloxacin therapy, the total Sterne population consisted of spores. With linezolid therapy, >90% of the population was vegetative B. anthracis. With ciprofloxacin therapy, toxin was first detectable at 3 h and remained detectable for at least 5 h. Toxin was never detected with linezolid therapy. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. However, the B. anthracis population was primarily spores with ciprofloxacin therapy and was primarily vegetative bacteria with linezolid therapy. Toxin production was detected for at least 5 h with ciprofloxacin therapy but was never detected with linezolid treatment. Linezolid may have an advantage over ciprofloxacin for the treatment of B. anthracis infections.

VanScoy, Brian D.; Heine, Henry S.; Liu, Weiguo; Abshire, Terry; Holman, Kari; Kulawy, Robert; Brown, David L.; Drusano, George L.

2012-01-01

302

DIRECT MEASUREMENT OF ADSORPTION OF RADIOLABELED COMPOUNDS  

Microsoft Academic Search

A new apparatus and technique permit direct and continuous measurement ; of radiolabeled compounds adsorbed from solution onto clean solid surfaces. Thin ; films of metals are deposited under high vacuum onto the surface of a mica window ; and contacted with the solution containing the radiolabeled compound; adsorption ; is measured by a counter tube below the window. Clean

D. C. Walker; H. E. Jr. Ries

1962-01-01

303

Detecting anthrax in the mail by coherent Raman microspectroscopy.  

PubMed

In this report, we show the collection of spatial information through a turbid medium by coherent Raman microspectroscopic imaging. In particular, the technique is capable of identifying anthrax endospores inside a sealed paper envelope. PMID:22215594

Arora, Rajan; Petrov, Georgi I; Yakovlev, Vladislav V; Scully, Marlan O

2012-01-03

304

Anthrax Vaccine. Is It Safe. Does It Work.  

National Technical Information Service (NTIS)

Anthrax Vaccine Adsorbed (AVA) was licensed in 1970 to provide protection against infection with Bacillus anthracis. AVA was initially administered on a limited basis, primarily to protect veterinarians and workers processing animal products such as hair ...

B. L. Strom J. S. Durch L. L. Zwanziger L. M. Joellenbeck

2002-01-01

305

Detecting anthrax in the mail by coherent Raman microspectroscopy  

PubMed Central

In this report, we show the collection of spatial information through a turbid medium by coherent Raman microspectroscopic imaging. In particular, the technique is capable of identifying anthrax endospores inside a sealed paper envelope.

Arora, Rajan; Petrov, Georgi I.; Yakovlev, Vladislav V.; Scully, Marlan O.

2012-01-01

306

Update on the Use of Doxycycline for Anthrax Exposure  

Center for Drug Evaluation (CDER)

... any antibiotic for prevention of anthrax without the specific advice of a physician and a clear indication that exposure to the organism may have ... More results from www.fda.gov/drugs/drugsafety/postmarketdrugsafetyinformationforpatientsandproviders

307

Beyond Anthrax: Confronting the Future Biological Weapons Threat.  

National Technical Information Service (NTIS)

The threat to national security posed by classical biological agents such as smallpox and anthrax is severe and the federal government's response is thus far incomplete. But this current threat pales in comparison to the potential scourges of the future. ...

2004-01-01

308

Identification of anthrax-specific signature sequence from Bacillus anthracis  

NASA Astrophysics Data System (ADS)

The primary objective was to identify and clone novel chromosomal DNA fragments for use as B. anthracis-specific markers. Towards this goal, 300 random primers (RAPD technology, randomly amplified polymorphic DNA) were screened to identify polymorphic loci on the anthrax chromosome. Five such DNA fragments uniquely amplifying from anthrax chromosome were identified and isolated. These fragments were cloned in pCR vector and sequenced. Database (genebank) analysis of one of the cloned probe, VRTC899, revealed the presence of specific chromosomal DNA probe, Ba813 from anthrax. This prove also contains flanking DNA with no homology to known sequences. Availability of signature DNA probes for detection of antrax-causing agent in environmental samples is critical for field application of DNA-based sensor technologies. In conclusion, we have demonstrated application of RAPD technology for identification of anthrax-specific signature sequences. This strategy can be extended to identify signature sequences from other BW agents.

Rastogi, Vipin K.; Cheng, Tu-chen

2001-08-01

309

Bridging anthrax PEP animal protection data to humans  

Center for Biologics Evaluation and Research (CBER)

Text Version... in humans comparable to the immune response achieved in animals that were protected by the vaccine). 11. Anthrax Vaccines: Efficacy Testing and ... More results from www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials

310

Search for a New-Generation Human Anthrax Vaccine,  

National Technical Information Service (NTIS)

Anthrax is a disease primarily of herbivores, but humans can become infected through contact with infected animals or animal products. The etiological agent, Bacillus anthracis, possesses two primary virulence factors: a poly-D-glumatic acid capsule and a...

B. E. Ivins

1987-01-01

311

Immunogenicity of Chemical Anthrax Vaccine in Experiments on Sheep.  

National Technical Information Service (NTIS)

Results of the study of the immunogenic effectiveness of the improved chemical anthrax vaccine for sheep support earlier published data on checking the immunogenicity of the alum antigen on sheep. It was established that a single inoculation with chemical...

N. I. Aleksandrov

1965-01-01

312

[Post anthrax vaccine delayed hypersensitivity. II--delayed hypersensitivity in humans vaccinated against anthrax].  

PubMed

To detect cell immunity characterized by delayed postvaccination hypersensitivity to anthrax in man and assess its dynamics, vaccination using unencapsulated live anthrax vaccine was performed in 668 healthy volunteers. Vaccination was performed either by scarification (n = 172), subcutaneous injection (n = 202), or low-dose (n = 202) or high-dose (n = 83) inhalation. The anthraxin intradermal tests were performed in each patient at various times during the year following vaccination (D7, D15, D90, D180, D365). This study confirm that, regardless of the mode of administration, the vaccine induces cell-mediated immunity in man, as determined by positive anthraxine skin test. The incidence of positive tests decreases with time regardless of the mode of vaccination. After one year, the test remained positive in 34.8% of subjects vaccinated by subcutaneous injection, 37.5% vaccinated by low-dose inhalation, 34.2% vaccinated by high-dose inhalation, and 22.4% vaccinated by scarification. These findings are in agreement with those obtained in clinical epidemiological studies documenting the effectiveness of encapsulated live anthrax vaccine in man. PMID:7934778

Shlyakhov, E; Rubinstein, E

1994-01-01

313

[Delayed hypersensitivity after anthrax vaccination. I--Study of guinea pigs vaccinated against anthrax].  

PubMed

To evaluate delayed hypersensitivity after anthrax vaccination, an Anthraxin skin test was performed in 682 guinea pigs at various times after immunization with veterinary unencapsulated active anthrax vaccine. Results were compared with those obtained in unimmunized control guinea pigs (n = 216), in guinea pigs that received a non-immunizing dose of live vaccine (n = 183) and in guinea pigs inoculated with inactivated vaccine (n = 120). Anthraxin skin tests were positive in the first postvaccination days. The incidence and intensity of positive tests peaked between two weeks and one month after vaccination and then gradually decreased during the first year. Study of resistance of guinea pigs to an inoculum at a lethal dose of a virulent strain of Bacillus anthracis showed a close correlation between positive tests and resistance. These findings demonstrate development of cell-mediated immunity after anthrax vaccination. The Anthraxin skin test should have practical applications for the production of vaccines and for evaluation of the immune status of vaccinated livestock [corrected]. PMID:8196523

Shlyakhov, E; Rubinstein, E

1994-01-01

314

Epidemic Anthrax in the Eighteenth Century, the Americas  

Microsoft Academic Search

Anthrax has been described as a veterinary disease of minor importance to clinical medicine, causing occasional occupational infections in single cases or clusters. Its potential for rapid and widespread epi- demic transmission under natural circumstances has not been widely appreciated. A little-known 1770 epi- demic that killed 15,000 people in Saint-Domingue (modern Haiti) was probably intestinal anthrax. The epidemic spread

David M. Morens

315

Botulinum Toxin.  

National Technical Information Service (NTIS)

Botulism is a disease caused by anaerobic, spore-forming bacteria found in soil. Disease results from the actions of chemical toxins produced by these bacteria. The most common forms of human botulism include foodborne, infant, and wound. The main etiolog...

A. M. Katos J. Anderson M. Krasna P. T. Williams W. Burrows

2009-01-01

316

Legionella Toxin.  

National Technical Information Service (NTIS)

The presence of a Legionella pneumophila toxin has been clinically suspected since 1978. A common 3,400 molecular weight protein has been found in the cell free extracts of sonicated L. pneumophila organisms serotype 1, as well as L. dumoffii, L. bozemani...

K. W. Hedlund

1981-01-01

317

Emergency response to an anthrax attack  

PubMed Central

We developed a mathematical model to compare various emergency responses in the event of an airborne anthrax attack. The system consists of an atmospheric dispersion model, an age-dependent dose–response model, a disease progression model, and a set of spatially distributed two-stage queueing systems consisting of antibiotic distribution and hospital care. Our results underscore the need for the extremely aggressive and timely use of oral antibiotics by all asymptomatics in the exposure region, distributed either preattack or by nonprofessionals postattack, and the creation of surge capacity for supportive hospital care via expanded training of nonemergency care workers at the local level and the use of federal and military resources and nationwide medical volunteers. The use of prioritization (based on disease stage and/or age) at both queues, and the development and deployment of modestly rapid and sensitive biosensors, while helpful, produce only second-order improvements.

Wein, Lawrence M.; Craft, David L.; Kaplan, Edward H.

2003-01-01

318

Radiolabeled antibodies for cancer imaging and therapy.  

PubMed

Radiolabeled antibodies were studied first for tumor detection by single-photon imaging, but FDG PET stopped these developments. In the meantime, radiolabeled antibodies were shown to be effective in the treatment of lymphoma. Radiolabeling techniques are well established and radiolabeled antibodies are a clinical and commercial reality that deserves further studies to advance their application in earlier phase of the diseases and to test combination and adjuvant therapies including radiolabeled antibodies in hematological diseases. In solid tumors, more resistant to radiations and less accessible to large molecules such as antibodies, clinical efficacy remains limited. However, radiolabeled antibodies used in minimal or small-size metastatic disease have shown promising clinical efficacy. In the adjuvant setting, ongoing clinical trials show impressive increase in survival in otherwise unmanageable tumors. New technologies are being developed over the years: recombinant antibodies and pretargeting approaches have shown potential in increasing the therapeutic index of radiolabeled antibodies. In several cases, clinical trials have confirmed preclinical studies. Finally, new radionuclides, such as lutetium-177, with better physical properties will further improve the safety of radioimmunotherapy. Alpha particle and Auger electron emitters offer the theoretical possibility to kill isolated tumor cells and microscopic clusters of tumor cells, opening the perspective of killing the last tumor cell, which is the ultimate challenge in cancer therapy. Preliminary preclinical and preliminary clinical results confirm the feasibility of this approach. PMID:22907380

Barbet, Jacques; Bardiès, Manuel; Bourgeois, Mickael; Chatal, Jean-François; Chérel, Michel; Davodeau, François; Faivre-Chauvet, Alain; Gestin, Jean-François; Kraeber-Bodéré, Françoise

2012-01-01

319

Anthrax Vaccine as a Component of the Strategic National Stockpile: A Dilemma for Homeland Security.  

National Technical Information Service (NTIS)

The author explains how past problems with the Defense Department anthrax vaccine currently affect Department of Homeland Security and Department of Health and Human Service policy. The departments included the BioThrax(Registered) anthrax vaccine in the ...

T. L. Rempfer

2009-01-01

320

Military Hospitalizations Among Deployed US Service Members Following Anthrax Vaccination, 1998-2001.  

National Technical Information Service (NTIS)

Safety concerns have confronted the Department of Defense Anthrax Vaccine Immunization Program since inception in 1998. To determine if anthrax vaccination was associated with an increased risk of hospitalization, a historical cohort study utilizing pre- ...

L. Z. Wang P. A. Sato R. J. Reed T. C. Smith T. S. Wells

2006-01-01

321

Differential contribution of Bacillus anthracis toxins to pathogenicity in two animal models.  

PubMed

The virulence of Bacillus anthracis, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of the pag, lef, and cya genes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing a pag deletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion of pag or even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism of B. anthracis in the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation of B. anthracis virulence in general and in particular for the design of relevant next-generation anthrax vaccines. PMID:22585968

Levy, Haim; Weiss, Shay; Altboum, Zeev; Schlomovitz, Josef; Glinert, Itai; Sittner, Assa; Shafferman, Avigdor; Kobiler, David

2012-05-14

322

Screen-printed fluorescent sensors for rapid and sensitive anthrax biomarker detection.  

PubMed

Since the 2001 anthrax attacks, efforts have focused on the development of an anthrax detector with rapid response and high selectivity and sensitivity. Here, we demonstrate a fluorescence sensor for detecting anthrax biomarker with high sensitivity and selectivity using a screen-printing method. A lanthanide-ethylenediamine tetraacetic acid complex was printed on a flexible polyethersulfone film. Screen-printing deposition of fluorescent detecting moieties produced fluorescent patterns that acted as a visual alarm against anthrax. PMID:23518174

Lee, Inkyu; Oh, Wan-Kyu; Jang, Jyongsik

2013-03-14

323

The ecology of anthrax spores: tough but not invincible.  

PubMed Central

Bacillus anthracis is the causative agent of anthrax, a serious and often fatal disease of wild and domestic animals. Central to the persistence of anthrax in an area is the ability of B. anthracis to form long-lasting, highly resistant spores. Understanding the ecology of anthrax spores is essential if one hopes to control epidemics. Studies on the ecology of anthrax have found a correlation between the disease and specific soil factors, such as alkaline pH, high moisture, and high organic content. Researchers initially suggested that these factors influenced vegetative anthrax bacilli. However, subsequent research has shown that vegetative cells of B. anthracis have very specific nutrient and physiological requirements and are unlikely to survive outside a host. Review of the properties of spores of B. anthracis and other Bacillus species suggests that the specific soil factors linked to epidemic areas reflect important environmental conditions that aid the anthrax spores in causing epidemics. Specifically, high levels of calcium in the soil may help to maintain spore vitality for prolonged periods, thereby increasing the chance of spores encountering and infecting a new host. Cycles of runoff and evaporation may collect spores dispersed from previous epidemics into storage areas, thereby concentrating them. Uptake of large doses of viable spores from storage areas by susceptible animals, via altered feeding or breeding behavior, may then allow the bacterium to establish infection and cause a new epidemic. Literature search for this review was done by scanning the Life Sciences Collection 1982-1994 using the keywords "anthrax" and "calcium and spore." Images Figure 1.

Dragon, D C; Rennie, R P

1995-01-01

324

Clinical Presentation of Inhalational Anthrax Following Bioterrorism Exposure Report of 2 Surviving Patients  

Microsoft Academic Search

The use of anthrax as a weapon of biological terrorism has moved from theory to reality in recent weeks. Following processing of a letter containing anthrax spores that had been mailed to a US senator, 5 cases of inhalational anthrax have occurred among postal workers employed at a major postal facility in Wash- ington, DC. This report details the clinical

Thom A. Mayer; Susan Bersoff-Matcha; Cecele Murphy; James Earls; Scott Harper; Denis Pauze; Michael Nguyen; Jonathan Rosenthal; Donald Cerva; Glenn Druckenbrod; Dan Hanfling; Naaz Fatteh; Anthony Napoli; Ashna Nayyar; Elise L. Berman

325

Laboratory Aspects of Bioterrorism-related Anthrax - from Identification to Molecular Subtyping to Microbial Forensics  

Microsoft Academic Search

During the bioterrorism-associated anthrax investigation of 2001 in the United States, 11 patients were diagnosed with inhalational anthrax and 11 more with the cutaneous forms of the disease. Over 125,000 specimens were processed at laboratories of the Laboratory Response Network including those at the Centers for Disease Control and Prevention. Although the 2001 anthrax investigation initially began as a public

Tanja Popoviæ; Mindy Glass

2003-01-01

326

Botulinum Toxins  

Microsoft Academic Search

\\u000a In minute doses, purified BTX-A injected into facial muscles causes temporary chemodenervation. Partial or complete paralysis\\u000a of selected muscles of facial expression reduces hyperdynamic wrinkles, improves the position or shape of the brow and mouth,\\u000a and even contours the face. There are discussions on the mechanisms of action of botulinum toxin, clinical effects, anatomy,\\u000a indications, facial assessment, and general techniques.

Peter M. Prendergast

327

Recent developments in monoclonal antibody radiolabeling techniques  

SciTech Connect

Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

Srivastava, S.C.; Mease, R.C.

1989-01-01

328

Public response to an anthrax attack: reactions to mass prophylaxis in a scenario involving inhalation anthrax from an unidentified source.  

PubMed

An attack with Bacillus anthracis ("anthrax") is a known threat to the United States. When weaponized, it can cause inhalation anthrax, the deadliest form of the disease. Due to the rapid course of inhalation anthrax, delays in initiation of antibiotics may decrease survival chances. Because a rapid response would require cooperation from the public, there is a need to understand the public's response to possible mass dispensing programs. To examine the public's response to a mass prophylaxis program, this study used a nationally representative poll of 1,092 adults, supplemented by a targeted focus on 3 metropolitan areas where anthrax attacks occurred in 2001: New York City (n=517), Washington, DC (n=509), and Trenton/Mercer County, NJ (n=507). The poll was built around a "worst-case scenario" in which cases of inhalation anthrax are discovered without an identified source and the entire population of a city or town is asked to receive antibiotic prophylaxis within a 48-hour period. Findings from this poll provide important signs of public willingness to comply with public health recommendations for obtaining antibiotics from a dispensing site, although they also indicate that public health officials may face several challenges to compliance, including misinformation about the contagiousness of inhalation anthrax; fears about personal safety in crowds; distrust of government agencies to provide sufficient, safe, and effective medicine; and hesitation about ingesting antibiotic pills after receiving them. In general, people living in areas where anthrax attacks occurred in 2001 had responses similar to those of the nation as a whole. PMID:21819225

SteelFisher, Gillian; Blendon, Robert; Ross, Laura J; Collins, Blanche C; Ben-Porath, Eran N; Bekheit, Mark M; Mailhot, Johanna R

2011-08-05

329

Reduced Expression of CD45 Protein-tyrosine Phosphatase Provides Protection against Anthrax Pathogenesis*S?  

PubMed Central

The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.

Panchal, Rekha G.; Ulrich, Ricky L.; Bradfute, Steven B.; Lane, Douglas; Ruthel, Gordon; Kenny, Tara A.; Iversen, Patrick L.; Anderson, Arthur O.; Gussio, Rick; Raschke, William C.; Bavari, Sina

2009-01-01

330

Update: Cutaneous anthrax in a laboratory worker--Texas, 2002.  

PubMed

On April 5, 2002, CDC reported a case of suspected cutaneous anthrax in a worker at laboratory A who had been processing environmental samples for Bacillus anthracis in support of CDC investigations of the 2001 bioterrorist attacks in the United States. Since the initial report, the worker had serial serology performed at the CDC laboratory. A greater than fourfold rise from baseline in the concentration of immunoglobulin G to protective antigen was demonstrated. The peak antibody level was observed 7-8 weeks after the onset of symptoms, and the time course and levels of detectable antibodies were consistent with those seen in other cases of cutaneous anthrax. On the basis of case definitions developed during the recent investigation, these additional findings confirm this as a case of cutaneous anthrax. This case brings the number of anthrax cases identified in the United States since October 3, 2001, to 23, including 11 inhalation and 12 cutaneous (eight confirmed and four suspected). This is the first laboratory-acquired case of anthrax associated with the recent investigation. PMID:12064454

2002-06-01

331

COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro.  

PubMed

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein-protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins. In this study, we have used LFnDTA and its associated ADP-ribosyltransferase activity (DTA) to determine the requirements for LF translocation from the lumen of endosomal vesicles to the external medium in vitro. Although low-level release of LFnDTA from enriched endosomal vesicles occurs in the absence of added factors, translocation was enhanced by the addition of cytosolic proteins and ATP to the reaction mixture. We show by GST-LFn pull-down assays that LFn specifically interacts with at least zeta-COP and beta-COP of the COPI coatomer complex. Immunodepletion of COPI coatomer complex and associated proteins from cytosolic extracts blocks in vitro LFnDTA translocation. Translocation may be reconstituted by the addition of partially purified bovine COPI to the translocation assay mixture. Taken together, these data suggest that the delivery of LF to the cytosol requires either COPI coatomer complex or a COPI subcomplex for translocation from the endosomal lumen. This facilitated delivery appears to use a mechanism that is analogous to that of DT entry. PMID:18356299

Tamayo, Alfred G; Bharti, Ajit; Trujillo, Carolina; Harrison, Robert; Murphy, John R

2008-03-20

332

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.  

PubMed

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies. PMID:22815152

Wagner, Leslie; Verma, Anita; Meade, Bruce D; Reiter, Karine; Narum, David L; Brady, Rebecca A; Little, Stephen F; Burns, Drusilla L

2012-07-18

333

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant  

PubMed Central

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Wagner, Leslie; Verma, Anita; Meade, Bruce D.; Reiter, Karine; Narum, David L.; Brady, Rebecca A.; Little, Stephen F.

2012-01-01

334

COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro  

PubMed Central

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein–protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins. In this study, we have used LFnDTA and its associated ADP-ribosyltransferase activity (DTA) to determine the requirements for LF translocation from the lumen of endosomal vesicles to the external medium in vitro. Although low-level release of LFnDTA from enriched endosomal vesicles occurs in the absence of added factors, translocation was enhanced by the addition of cytosolic proteins and ATP to the reaction mixture. We show by GST-LFn pull-down assays that LFn specifically interacts with at least ?-COP and ?-COP of the COPI coatomer complex. Immunodepletion of COPI coatomer complex and associated proteins from cytosolic extracts blocks in vitro LFnDTA translocation. Translocation may be reconstituted by the addition of partially purified bovine COPI to the translocation assay mixture. Taken together, these data suggest that the delivery of LF to the cytosol requires either COPI coatomer complex or a COPI subcomplex for translocation from the endosomal lumen. This facilitated delivery appears to use a mechanism that is analogous to that of DT entry.

Tamayo, Alfred G.; Bharti, Ajit; Trujillo, Carolina; Harrison, Robert; Murphy, John R.

2008-01-01

335

Anthrax in wildlife in the Luangwa Valley, Zambia.  

PubMed

An abnormally high mortality among hippos (Hippopotamus amphibius) in the Luangwa River valley between June and November 1987 and estimated to number more than 4000 deaths was attributed to anthrax. Several other species, particularly Cape buffalo (Syncerus caffer) and elephant (Loxodonta africana), appear to have been affected. A smaller outbreak of anthrax in hippos occurred between August and September 1988, approximately 100 km up-river. A field study was arranged in August 1989 to assess the extent of environmental contamination by Bacillus anthracis and the risks to people in the area, to study possible methods of control and to equip local laboratory staff for continued monitoring of the disease. The study confirmed the enzootic status of the region. The characteristics of the outbreaks of anthrax in 1987 and 1988, and the results of the field study are described. PMID:1907048

Turnbull, P C; Bell, R H; Saigawa, K; Munyenyembe, F E; Mulenga, C K; Makala, L H

1991-04-27

336

Sverdlovsk Anthrax Outbreak: An Educational Case Study  

NASA Astrophysics Data System (ADS)

In April and May of 1979 an Anthrax epidemic broke out in the city of Sverdlovsk (now Ekaterinburg) in the former Soviet Union. Sixty-four people were reported to have died from the outbreak, although there is still debate concerning the actual number of victims. While Soviet officials initially attributed this outbreak to contaminated meat, the US Government maintained that the outbreak was due to a leakage from a biological weapons facility. We have created and implemented an undergraduate educational exercise based on the forensic analysis of this event. Students were provided case data of the victims, area satellite images and meteorological data. One goal of the exercise was for students to reconstruct the most probable scenario of events through valid inference based on the limited information and uncertainties associated with the data set. Another goal was to make students sensitive to issues of biological weapons and bioterrorism. The exercise was highly rated by students even before the events of September 11. There is a clear need to educate students, particularly in the sciences, to be aware of the signatures of terrorist activities. Evidence of terrorist activities is more likely to appear from unintended discoveries than from active intelligence gathering. We believe our national security can be enhanced by sensitizing those that monitor the natural environment to the signatures of terrorist activities through the types of educational exercises that we have developed.

Steele, S. J.; van der Vink, G.

2002-05-01

337

Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.  

PubMed

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases. PMID:23479355

Cryan, Lorna M; Habeshian, Kaiane A; Caldwell, Thomas P; Morris, Meredith T; Ackroyd, P Christine; Christensen, Kenneth A; Rogers, Michael S

2013-03-11

338

Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen  

PubMed Central

Summary The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity single chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here we report the high resolution X-ray structures of three high affinity single chain antibodies in the 14B7 family; 14B7 and two high affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization and on the effect of various mutations on antibody affinity and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.

Leysath, Clinton E.; Monzingo, Arthur F.; Maynard, Jennifer A.; Barnett, Jason; Georgiou, George; Iverson, Brent L.

2009-01-01

339

Agar-Gel Precipitin Technique in Anthrax Antibody Determinations1  

PubMed Central

A modification of the agar-gel precipitation inhibition technique of Thorne and Belton for detecting anthrax antibodies reduces inconsistency of visually determined end points on the same sera observed by different technicians. Determination of the minimal reacting concentrations of the anthrax antigen and antibody reagents, modifications of the visualization apparatus, methods for combining reagents, and length of incubation periods contribute to the ease of the end-point determinations and the uniformity of results. When compared with the previous technique, the modified procedure is less time-consuming while retaining satisfactory reproducibility, simplicity, specificity, and sensitivity. Images FIG. 1 FIG. 2

Ray, John G.; Kadull, Paul J.

1964-01-01

340

Anthrax LFn-PA Hybrid Antigens: Biochemistry, Immunogenicity, and Protection Against Lethal Ames Spore Challenge in Rabbits.  

PubMed

We describe a novel hybrid anthrax toxin approach that incorporates multiple components into a single vaccine product. The key domains of protective antigen (PA) and lethal factor (LF) that may be critical for inducing protective immunity are combined into one recombinant molecule. Two LF N-terminal domain-PA hybrids, one with wild-type PA and another with furin cleavage-minus PA, were expressed in E. coli and purified in a native form. Both the hybrids bind to the extracellular domain of the host receptor, CMG2; the wild-type hybrid can be cleaved by furin exposing the LF interacting domain, allowing it to oligomerize into lethal toxin as well as translocation pore-like complexes. The hybrid antigens are immunogenic in Dutch-belted rabbits, eliciting strong PA-specific and LF-specific antibodies. However, the lethal toxin neutralizing antibody titers are 3-7 times lower than those elicited by PA-alum. The hybrid antigens conferred 100% (6/6) protection in rabbits challenged intranasally with a 100 LD(50) dose of Bacillus anthracis Ames strain spores. PMID:20390054

Li, Qin; Peachman, Kristina K; Sower, Laurie; Leppla, Stephen H; Shivachandra, Sathish B; Matyas, Gary R; Peterson, Johnny W; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

2009-01-01

341

Radiolabelled RGD peptides for imaging and therapy.  

PubMed

Imaging of angiogenesis has become increasingly important with the rising use of targeted antiangiogenic therapies like bevacizumab (Avastin). Non-invasive assessment of angiogenic activity is in this respect interesting, e.g. for response assessment of such targeted antiangiogenic therapies. One promising approach of angiogenesis imaging is imaging of specific molecular markers of the angiogenic cascade like the integrin ?(v)?(3). For molecular imaging of integrin expression, the use of radiolabelled peptides is still the only approach that has been successfully translated into the clinic. In this review we will summarize the current data on imaging of ?(v)?(3) expression using radiolabelled RGD peptides with a focus on tracers already in clinical use. A perspective will be presented on the future clinical use of radiolabelled RGD peptides including an outlook on potential applications for radionuclide therapy. PMID:22388629

Gaertner, F C; Kessler, H; Wester, H-J; Schwaiger, M; Beer, A J

2012-02-01

342

Recombinant Protective Antigen Anthrax Vaccine Improves Survival when Administered as a Postexposure Prophylaxis Countermeasure with Antibiotic in the New Zealand White Rabbit Model of Inhalation Anthrax  

PubMed Central

Inhalation anthrax is a potentially lethal form of disease resulting from exposure to aerosolized Bacillus anthracis spores. Over the last decade, incidents spanning from the deliberate mailing of B. anthracis spores to incidental exposures in users of illegal drugs have highlighted the importance of developing new medical countermeasures to protect people who have been exposed to “anthrax spores” and are at risk of developing disease. The New Zealand White rabbit (NZWR) is a well-characterized model that has a pathogenesis and clinical presentation similar to those seen in humans. This article reports how the NZWR model was adapted to evaluate postexposure prophylaxis using a recombinant protective antigen (rPA) vaccine in combination with an oral antibiotic, levofloxacin. NZWRs were exposed to multiples of the 50% lethal dose (LD50) of B. anthracis spores and then vaccinated immediately (day 0) and again on day 7 postexposure. Levofloxacin was administered daily beginning at 6 to 12 h postexposure for 7 treatments. Rabbits were evaluated for clinical signs of disease, fever, bacteremia, immune response, and survival. A robust immune response (IgG anti-rPA and toxin-neutralizing antibodies) was observed in all vaccinated groups on days 10 to 12. Levofloxacin plus either 30 or 100 ?g rPA vaccine resulted in a 100% survival rate (18 of 18 per group), and a vaccine dose as low as 10 ?g rPA resulted in an 89% survival rate (16 of 18) when used in combination with levofloxacin. In NZWRs that received antibiotic alone, the survival rate was 56% (10 of 18). There was no adverse effect on the development of a specific IgG response to rPA in unchallenged NZWRs that received the combination treatment of vaccine plus antibiotic. This study demonstrated that an accelerated two-dose regimen of rPA vaccine coadministered on days 0 and 7 with 7 days of levofloxacin therapy results in a significantly greater survival rate than with antibiotic treatment alone. Combination of vaccine administration and antibiotic treatment may be an effective strategy for treating a population exposed to aerosolized B. anthracis spores.

Bourdage, James S.; Williamson, E. Diane; Duchars, Matthew; Fuerst, Thomas R.; Fusco, Peter C.

2012-01-01

343

Recombinant protective antigen anthrax vaccine improves survival when administered as a postexposure prophylaxis countermeasure with antibiotic in the New Zealand white rabbit model of inhalation anthrax.  

PubMed

Inhalation anthrax is a potentially lethal form of disease resulting from exposure to aerosolized Bacillus anthracis spores. Over the last decade, incidents spanning from the deliberate mailing of B. anthracis spores to incidental exposures in users of illegal drugs have highlighted the importance of developing new medical countermeasures to protect people who have been exposed to "anthrax spores" and are at risk of developing disease. The New Zealand White rabbit (NZWR) is a well-characterized model that has a pathogenesis and clinical presentation similar to those seen in humans. This article reports how the NZWR model was adapted to evaluate postexposure prophylaxis using a recombinant protective antigen (rPA) vaccine in combination with an oral antibiotic, levofloxacin. NZWRs were exposed to multiples of the 50% lethal dose (LD(50)) of B. anthracis spores and then vaccinated immediately (day 0) and again on day 7 postexposure. Levofloxacin was administered daily beginning at 6 to 12 h postexposure for 7 treatments. Rabbits were evaluated for clinical signs of disease, fever, bacteremia, immune response, and survival. A robust immune response (IgG anti-rPA and toxin-neutralizing antibodies) was observed in all vaccinated groups on days 10 to 12. Levofloxacin plus either 30 or 100 ?g rPA vaccine resulted in a 100% survival rate (18 of 18 per group), and a vaccine dose as low as 10 ?g rPA resulted in an 89% survival rate (16 of 18) when used in combination with levofloxacin. In NZWRs that received antibiotic alone, the survival rate was 56% (10 of 18). There was no adverse effect on the development of a specific IgG response to rPA in unchallenged NZWRs that received the combination treatment of vaccine plus antibiotic. This study demonstrated that an accelerated two-dose regimen of rPA vaccine coadministered on days 0 and 7 with 7 days of levofloxacin therapy results in a significantly greater survival rate than with antibiotic treatment alone. Combination of vaccine administration and antibiotic treatment may be an effective strategy for treating a population exposed to aerosolized B. anthracis spores. PMID:22695155

Leffel, Elizabeth K; Bourdage, James S; Williamson, E Diane; Duchars, Matthew; Fuerst, Thomas R; Fusco, Peter C

2012-06-13

344

Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein  

PubMed Central

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M.; McDonald, Karen A.

2011-01-01

345

Induction of neutralizing antibody responses to anthrax protective antigen by using influenza virus vectors: implications for disparate immune system priming pathways.  

PubMed

Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1') were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of alpha-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties. PMID:20504926

Langley, William A; Bradley, Konrad C; Li, Zhu-Nan; Smith, Mary Ellen; Schnell, Matthias J; Steinhauer, David A

2010-05-26

346

Induction of Neutralizing Antibody Responses to Anthrax Protective Antigen by Using Influenza Virus Vectors: Implications for Disparate Immune System Priming Pathways?  

PubMed Central

Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1?) were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of ?-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.

Langley, William A.; Bradley, Konrad C.; Li, Zhu-Nan; Smith, Mary Ellen; Schnell, Matthias J.; Steinhauer, David A.

2010-01-01

347

Bacillus cereus food poisoning and its toxins.  

PubMed

The genus Bacillus includes members that demonstrate a wide range of diversity from physiology and ecological niche to DNA sequence and gene regulation. The species of most interest tend to be known for their pathogenicity and are closely linked genetically. Bacillus anthracis causes anthrax, and Bacillus thuringiensis is widely used for its insecticidal properties but has also been associated with foodborne disease. Bacillus cereus causes two types of food poisoning, the emetic and diarrheal syndromes, and a variety of local and systemic infections. Although in this review we provide information on the genus and a variety of species, the primary focus is on the B. cereus strains and toxins that are involved in foodborne illness. B. cereus produces a large number of potential virulence factors, but for the majority of these factors their roles in specific infections have not been established. To date, only cereulide and the tripartite hemolysin BL have been identified specifically as emetic and diarrheal toxins, respectively. Nonhemolytic enterotoxin, a homolog of hemolysin BL, also has been associated with the diarrheal syndrome. Recent findings regarding these and other putative enterotoxins are discussed. PMID:15771198

Schoeni, Jean L; Wong, Amy C Lee

2005-03-01

348

Anthrax 2001: Observations on the Medical and Public Health Response  

Microsoft Academic Search

HIS ARTICLE DESCRIBES ASPECTS of the medical and public health response to the 2001 anthrax attacks based on interviews with individuals who were directly involved in the response. It has been more than 18 months since B. anthracis spores were discovered in let- ters sent through the U.S. postal system. The specific purpose and perpetrator(s) of these attacks remain un-

Elin Gursky; Thomas V. Inglesby; Tara O'Toole

2003-01-01

349

Studies on Anthrax Infections in Immunized Guinea Pigs.  

National Technical Information Service (NTIS)

Groups of guinea pigs immunized with 1 or 3 doses of anthrax protective antigen were challenged with 2 different strains of Bacillus anthracis at 2,4 and 6 weeks after immunization. Few animals survived challenge with strain NH-6, regardless of immunizati...

M. K. Ward V. G. McGann A. L. Hogge M. L. Huff R. G. Kanode

1964-01-01

350

Detection and Sterilization of Anthrax Spores by Microwave Radiation.  

National Technical Information Service (NTIS)

The goal of this program is to investigate the ability of microwave and millimeter-wave radiation to detect and destroy biological agents, with a particular focus on anthrax spores. Dry constituents, such as dipicolinic acid (DPA), can be placed within si...

N. C. Luhmann C. W. Domier A. Wan M. Johnson

2005-01-01

351

Anthrax vaccine: short-term safety experience in humans  

Microsoft Academic Search

Bacillus anthracis is the major terrorist and biological warfare agent of concern to civilian and military medical planners. The licensed anthrax vaccine, adsorbed (AVA) is believed to be an effective prophylactic medical countermeasure against this threat. Our objective in this report was to expand the safety database for this vaccine by assessing data on self-reported, short-term safety of AVA during

Phillip R Pittman; Paul H Gibbs; Timothy L Cannon; Arthur M Friedlander

2001-01-01

352

[Four cases of cutaneous anthrax in Diyarbakir, Turkey].  

PubMed

Anthrax which is a rare disease in developed countries, is still a serious public health problem in countries like Turkey where livestock is common. In this report, four cases of cutaneous anthrax detected in Kirkira village of Diyarbakir, Southeast Anatolia, Turkey, were presented. Three female and one male patients were admitted to our hospital with the complaints of skin lesions and high fever lasting for 10 days. Their history indicated that they injured their fingers during slaughtering of a dead cow meat. All patients had irregular edged necrotic vesiculobullous lesions on the erythematous and edematous base on their hand fingers, developed in 1 week following the contact. There was no systemic finding and the laboratory findings were within normal limits. Typical bamboo cane shaped gram-positive bacilli were observed on the Gram stained smears prepared from the vesicular lesions. Aerobic cultures in blood agar media revealed typical R type colonies, gray in color, creased, granulated and 2-3 mm in diameter within 24 hours of incubation. In one patient although the lesion was typical and characteristic gram-positive bacilli were detected in the Gram stained smears, no growth was seen in the cultures. The isolates (n= 3) were identified as Bacillus anthracis by conventional microbiological methods, and also confirmed by Vitek 2 (BioMerieux, France) automated identification system. Antibiotic susceptibility tests were performed by disc diffusion method according to the CLSI guidelines. The isolates were found susceptible to penicillin G, ampicillin, erythromycin, amikacin, chloramphenicol, tetracycline, vancomycin and ciprofloxacin. All of the patients were treated successfully with penicillin or ciprofloxacin accompanied by topical wound care. In the last years several case series of anthrax were reported especially from the East and Southeastern Anatolia regions of Turkey. These four cutaneous anthrax cases from Diyarbakir, Turkey were reported to withdraw attention to anthrax in that specific area. It was concluded that in areas where anthrax is endemic to educate people under risk, to take the necessary preventive measures and to rule out anthrax in the differential diagnosis of cases presenting with typical ulcers and had contact with animals or their products, are of crucial importance for the early initiation of appropriate treatment which would decrease related morbidity and mortality. PMID:23971932

Turhano?lu, Nezire Mine; Bay?nd?r Bilman, Fulya; Kutlu Yürüker, Safiye

2013-07-01

353

Strong Mucosal and Systemic Immunities Induced by Nasal Immunization with Anthrax Protective Antigen Protein Incorporated in Liposome–Protamine–DNA Particles  

Microsoft Academic Search

\\u000a Purpose  The very lengthy and complicated dosing schedule of the current anthrax vaccine adsorbed, which was licensed in the USA for\\u000a the prevention of cutaneous anthrax infection, calls for the development of an efficacious and easily administrable vaccine\\u000a to prevent against the most lethal form of anthrax infection, the inhalation anthrax. We propose to develop a nasal anthrax\\u000a vaccine using anthrax

Brian R. Sloat; Zhengrong Cui

2006-01-01

354

A case of septicaemic anthrax in an intravenous drug user  

PubMed Central

Background In 2000, Ringertz et al described the first case of systemic anthrax caused by injecting heroin contaminated with anthrax. In 2008, there were 574 drug related deaths in Scotland, of which 336 were associated with heroin and or morphine. We report a rare case of septicaemic anthrax caused by injecting heroin contaminated with anthrax in Scotland. Case Presentation A 32 year old intravenous drug user (IVDU), presented with a 12 hour history of increasing purulent discharge from a chronic sinus in his left groin. He had a tachycardia, pyrexia, leukocytosis and an elevated C-reactive protein (CRP). He was treated with Vancomycin, Clindamycin, Ciprofloxacin, Gentamicin and Metronidazole. Blood cultures grew Bacillus anthracis within 24 hours of presentation. He had a computed tomography (CT) scan and magnetic resonance imagining (MRI) of his abdomen, pelvis and thighs performed. These showed inflammatory change relating to the iliopsoas and an area of necrosis in the adductor magnus. He underwent an exploration of his left thigh. This revealed chronically indurated subcutaneous tissues with no evidence of a collection or necrotic muscle. Treatment with Vancomycin, Ciprofloxacin and Clindamycin continued for 14 days. Negative Pressure Wound Therapy (NPWT) device was applied utilising the Venturi™ wound sealing kit. Following 4 weeks of treatment, the wound dimensions had reduced by 77%. Conclusions Although systemic anthrax infection is rare, it should be considered when faced with severe cutaneous infection in IVDU patients. This case shows that patients with significant bacteraemia may present with no signs of haemodynamic compromise. Prompt recognition and treatment with high dose IV antimicrobial therapy increases the likelihood of survival. The use of simple wound therapy adjuncts such as NPWT can give excellent wound healing results.

2011-01-01

355

Generation and characterization of radiolabeled diesel exhaust.  

PubMed

To evaluate the potential health risks associated with increased use of diesel engines, information is needed on the biological fate of inhaled diesel exhaust components. Appropriately radiolabeled exhaust produced by burning radiolabeled fuel could be used to gain this information. The purpose of this study was to characterize different radiolabeled diesel exhausts with respect to their potential use in studies of the biological fate of exhaust carbon particles and particle-associated organic compounds (particle extracts). A single-cylinder diesel engine was used to burn diesel fuel containing trace amounts of 14C-labeled hexadecane, dotriacontane, benzene, phenanthrene or benzo(a)pyrene. Greater than 98% of the 14C in all additives was converted to volatile materials upon combustion. The remainder was distributed in varying amounts between the carbon particles and particle extracts. Aromatic additives labeled carbon particles more efficiently than aliphatic additives. Column chromatography of the particle extracts showed that, in most cases, the majority of the radioactivity eluted in fractions identical to the specific fuel additive employed, suggesting that a large amount of the particle-associated organic compounds consisted of uncombusted fuel constituents. Applying an electrical load to the engine-electrical generator increased carbon particle radioactivity, but had variable effects on the amount of radioactivity in the particle extracts. 67Ga-tetramethylheptanedione was also studied as a fuel additive to label carbon particles. 67Ga was incorporated into the exhaust particles and lung deposition of particles in rats was found to be approximately 10%. However, the 67Ga-radiolabel was found to separate from the particles in vivo, making it an unsuitable radiolabel for studying the long-term lung retention of diesel exhaust carbonaceous particles. PMID:6205579

Dutcher, J S; Sun, J D; Lopez, J A; Wolf, I; Wolff, R K; McClellan, R O

1984-07-01

356

Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.  

PubMed

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-? and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-?. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined. PMID:22633906

Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

2012-05-23

357

Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation.  

PubMed

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo. PMID:18044973

Wimalasena, D Shyamali; Cramer, John C; Janowiak, Blythe E; Juris, Stephen J; Melnyk, Roman A; Anderson, D Eric; Kirk, Kenneth L; Collier, R John; Bann, James G

2007-11-29

358

Endemic Gastrointestinal Anthrax in 1960s Lebanon: Clinical Manifestations and Surgical Findings  

PubMed Central

Anthrax is an ancient disease caused by the gram-positive Bacillus anthracis; recently, it has gained much attention because of its potential use in biologic warfare. Anthrax infection occurs in three forms: cutaneous, inhalational, and gastrointestinal. The last type results from ingestion of poorly cooked contaminated meat. Intestinal anthrax was widely known in Lebanon in the 1960s, when a series of >100 cases were observed in the Bekaa Valley. We describe some of these cases, introduce the concept of the surgical management of advanced intestinal anthrax, and describe some of the approaches for treatment.

Kanafani, Zeina A; Ghossain, Antoine; Sharara, Ala I; Hatem, Joseph M.

2003-01-01

359

Systemic but not mucosal immunity induced by AVA prevents inhalational anthrax.  

PubMed

Improved vaccines and adjuvants are being developed to reduce the threat posed by a terrorist attack involving aerosolized anthrax spores. Nevertheless, uncertainty persists concerning the relative benefits of inducing mucosal vs systemic immunity to host survival following inhalational exposure to anthrax spores. This work examines the effect of delivering the licensed human vaccine (anthrax vaccine adsorbed, AVA) combined with a CpG oligodeoxynucleotide (ODN) adjuvant intraperitoneally or intranasally to A/J mice. Results indicate that protection from inhalational anthrax correlates with the induction of a strong systemic rather than mucosal immune response, and demonstrate that protection is significantly improved and accelerated by the addition of CpG ODN. PMID:17913545

Klinman, Dennis M; Currie, Debra; Lee, Gloria; Grippe, Vanessa; Merkel, Tod

2007-08-10

360

Evaluation of the House Fly Musca domestica as a Mechanical Vector for an Anthrax  

PubMed Central

Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs.

Fasanella, Antonio; Scasciamacchia, Silvia; Garofolo, Giuliano; Giangaspero, Annunziata; Tarsitano, Elvira; Adone, Rosanna

2010-01-01

361

Botox (Botulinum Toxin)  

MedlinePLUS

... people when there are many effective and safe cosmetic procedures that can temporarily reduce a very prominent ... form of botulinum toxin is Type A (Botox® Cosmetic, Allergan, Inc). Botulinum toxin, what we will now ...

362

9 CFR 309.7 - Livestock affected with anthrax; cleaning and disinfection of infected livestock pens and driveways.  

Code of Federal Regulations, 2013 CFR

...2013-01-01 2013-01-01 false Livestock affected with anthrax; cleaning and disinfection of infected livestock pens and driveways. 309.7 Section...ANTE-MORTEM INSPECTION § 309.7 Livestock affected with anthrax; cleaning...

2013-01-01

363

Guidance for Protecting Responders' Health during the First Week Following a Wide-Area Aerosol Anthrax Attack.  

National Technical Information Service (NTIS)

This guidance provides recommendations for protecting personnel responding to a wide-area anthrax attack from developing anthrax as a result of exposure to aerosolized Bacillus anthracis (B. anthracis) spores and for minimizing the amount of exposure in t...

2012-01-01

364

Risk factors associated with anthrax in cattle on smallholdings.  

PubMed

SUMMARY Unprecedented high rates of anthrax outbreaks have been observed recently in cattle and humans in Bangladesh, with 607 human cases in 2010. By enrolling 15 case and 15 control cattle smallholdings in the spatial zone in July-September 2010, we conducted a case-control study, data of which were analysed by matched-pair analysis and multivariable conditional logistic regression. Feeding animals with uprooted and unwashed grass [odds ratio (OR) 41·2, 95% confidence interval (CI) 3·7-458·8, P=0·003], and feeding water hyacinth (Eichhornia crassipes) (OR 22·2, 95% CI 1·2-418·7, P=0·039) were independent risk factors for anthrax in cattle. PMID:22123521

Biswas, P K; Islam, M Z; Shil, S K; Chakraborty, R K; Ahmed, S S U; Christensen, J P

2011-11-29

365

[Anthrax in Chad: a zoonosis that still exists today].  

PubMed

An epidemic of human and animal anthrax raged in Chad mainly in the Department of Chari Baguirmi from September to December 1988, infesting more than 50% of donkeys and horses. 716 human cases have been reported, with 88 deaths. Thanks to a geographical distribution of animal and human prevalence, one sees immediately the interdependency between sanitary state of live-stock and public health. An unusual means of transmission from donkey to donkey by insects as the vector is suggested to explain the intensity of animal epidemics. Two strains of B. anthracis were isolated and described. Systematic annual prophylactic inoculation of the live-stock is recommended, and also resumption of research to create a polyvalent vaccine for cattle plague/peripneumonia and anthrax. PMID:2811650

Lamarque, D; Haessler, C; Champion, R; Granga, D; Bendina; Steinmetz, P; Guelina, A; Maurice, Y

366

Evaluation of mucoadhesive carrier adjuvant: Toward an oral anthrax vaccine.  

PubMed

The aim of present study was to evaluate the potential of mucoadhesive alginate-coated chitosan microparticles (A-CHMp) for oral vaccine against anthrax. The zeta potential of A-CHMp was - 29.7 mV, and alginate coating could prevent the burst release of antigen in simulated gastric fluid. The results indicated that A-CHMp was mucoadhesive in nature and transported it to the peyer's patch upon oral delivery. The immunization studies indicated that A-CHMp resulted in the induction of potent systemic and mucosal immune responses, whereas alum-adjuvanted rPA could induce only systemic immune response. Thus, A-CHMp represents a promising acid carrier adjuvant for oral immunization against anthrax. PMID:23452384

Mangal, Sharad; Pawar, Dilip; Agrawal, Udita; Jain, Arvind K; Vyas, Suresh P

2013-03-01

367

Pharmacology of botulinum toxin  

Microsoft Academic Search

Background: Botulinum toxin has a well-defined role among dermatologists for the treatment of facial wrinkling, brow position, and palmar and axillary hyperhidrosis. Objective: The purpose of this study is to educate dermatologists on the pharmacology of botulinum toxin. Methods: A retrospective review of the literature on botulinum toxin from 1962 to the present was conducted. We examined the clinical applications

Wilber Huang; Jill A. Foster; Arlene S. Rogachefsky

2000-01-01

368

Detection of Protein Toxins  

Technology Transfer Automated Retrieval System (TEKTRAN)

We have focused on ricin, shiga-like toxin, botulinum neurotoxin (BoNT), and staphylococcal enterotoxin A (SEA), developing sensitive test methods for toxins and marker compounds in food matrices. Although animal models provide the best means for risk assessment, especially for crude toxins in compl...

369

Toxines botuliques : utilisation pratique  

Microsoft Academic Search

Botulinum toxins (A and B) are neurotoxins derived from Clostridium botulinum. Clostridium are anaerobic bacteria. C. botulinum produces exotoxins (A to G) with distinct antigenicities. The neurotoxins inhibit the release of the neurotransmitter acetylcholine from the axon terminals of motor neurons. Botulinum toxin is officially used in clinic for the treatment of muscular hyperactivity (strabismus, blepharospam, cervical dystonia). Botulinum toxins

A Durand; G Serment

2003-01-01

370

SPECT assay of radiolabeled monoclonal antibodies  

SciTech Connect

The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

Jaszczak, R.J.

1992-02-01

371

A semisynthesis platform for investigating structure-function relationships in the N-terminal domain of the anthrax lethal factor  

PubMed Central

Many bacterial toxins act by covalently altering molecular targets within the cytosol of mammalian cells and therefore must transport their catalytic moieties across a membrane. The Protective-Antigen (PA) moiety of anthrax toxin forms multimeric pores that transport the two enzymatic moieties, the Lethal Factor (LF) and the Edema Factor, across the endosomal membrane to the cytosol. The homologous PA-binding domains of these enzymes contain N-terminal segments of highly charged amino acids that are believed to enter the pore and initiate N- to C-terminal translocation. Here we describe a semisynthesis platform that allows chemical control of this segment in LFN, the PA-binding domain of LF. Semisynthetic LFN was prepared in milligram quantities by native chemical ligation of synthetic LFN14-28 ?thioester with recombinant N29C-LFN29-263 and compared with two variants containing alterations in residues 14-28 of the N-terminal region. The properties of the variants in blocking ion conductance through the PA pore and translocating across planar phospholipid bilayers in response to a pH gradient were consistent with current concepts of the mechanism of polypeptide translocation through the pore. The semisynthesis platform thus makes new analytical approaches available to investigate the interaction of the pore with its substrates.

Pentelute, Brad L.; Barker, Adam P.; Janowiak, Blythe E.; Kent, Stephen B. H.; Collier, R. John

2010-01-01

372

Total decontamination cost of the anthrax letter attacks.  

PubMed

All of the costs associated with decontamination following the 2001 anthrax letter attacks were summarized, estimated, and aggregated based on existing literature and news media reports. A comprehensive list of all affected structures was compiled. Costs were analyzed by building class and decontamination type. Sampling costs and costs of worker relocation were also included. Our analysis indicates that the total cost associated with decontamination was about $320 million. PMID:22313022

Schmitt, Ketra; Zacchia, Nicholas A

2012-02-07

373

Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC  

Microsoft Academic Search

A novel and sensitive HPLC method for the determination of benzethonium chloride (BZC) in anthrax vaccine was developed. Adjuvant Alhydrogel was removed by syringe filter after a simple sample pretreatment—acidification prior to injection. Chromatography was performed by isocratic reverse phase separation with methanol\\/262mM ammonium acetate (80\\/20, v\\/v) on an endcapped C18 column with diode array detector (DAD). The method showed

Hsiaoling Wang; Alfred V. Del Grosso; Joan C. May

2006-01-01

374

Economic Impacts of a Wide Area Release of Anthrax  

SciTech Connect

This analysis explores economic impacts that might result from a wide-area release of anthrax. The intent is not to provide a quantitative analysis of such a disaster, but to: 1. Define the general categories of economic impacts that the region should be concerned about; and, 2. Explore what types of private sector businesses or industries, if any, may have the greatest impact on speeding the economic recovery of the region.

Judd, Kathleen S.; Olson, Jarrod; Stein, Steven L.; Lesperance, Ann M.

2009-05-29

375

SNR analysis: molecular investigation of an anthrax epidemic  

PubMed Central

Background In Italy, anthrax is endemic but occurs sporadically. During the summer of 2004, in the Pollino National Park, Basilicata, Southern Italy, an anthrax epidemic consisting of 41 outbreaks occurred; it claimed the lives of 124 animals belonging to different mammal species. This study is a retrospective molecular epidemiological investigation carried out on 53 isolates collected during the epidemic. A 25-loci Multiple Locus VNTR Analysis (MLVA) MLVA was initially performed to define genetic relationships, followed by an investigation of genetic diversity between epidemic strains through Single Nucleotide Repeat (SNR) analysis. Results 53 Bacillus anthracis strains were isolated. The 25-loci MLVA analysis identified all of them as belonging to a single genotype, while the SNR analysis was able to detect the existence of five subgenotypes (SGTs), allowing a detailed epidemic investigation. SGT-1 was the most frequent (46/53); SGTs 2 (4/53), 3 (1/53) 4 (1/53) and 5 (1/53) were detected in the remaining seven isolates. Conclusions The analysis revealed the prevalent spread, during this epidemic, of a single anthrax clone. SGT-1 - widely distributed across the epidemic area and present throughout the period in question - may, thus, be the ancestral form. SGTs 2, 3 and 4 differed from SGT-1 at only one locus, suggesting that they could have evolved directly from the latter during the course of this epidemic. SGT-5 differed from the other SGTs at 2-3 loci. This isolate, thus, appears to be more distantly related to SGT-1 and may not be a direct descendant of the lineage responsible for the majority of cases in this epidemic. These data confirm the importance of molecular typing and subtyping methods for in-depth epidemiological analyses of anthrax epidemics.

2010-01-01

376

Public response to an anthrax attack: a multiethnic perspective.  

PubMed

The 2001 anthrax attacks emphasized the need to develop outreach that would more effectively support racial/ethnic minority populations during a bioterrorism incident. Given the importance of antibiotic prophylaxis in a future anthrax attack, it should be a priority to better support racial/ethnic minorities in mass dispensing programs. To examine the needs and perspectives of racial/ethnic minorities, this study used a nationally representative poll of 1,852 adults, including 1,240 whites, 261 African Americans, and 282 Hispanics. The poll examined public reactions to a ''worst-case scenario'' in which cases of inhalation anthrax are discovered without an identified source and the entire population of a city or town is asked to receive antibiotic prophylaxis within 48 hours. Findings suggest willingness across all racial/ethnic groups to comply with recommendations to seek prophylaxis at dispensing sites. However, findings also indicate possible barriers for racial/ethnic minorities, including greater concern about pill safety and multiple attacks as well as lesser knowledge about inhalation anthrax. Across all racial/ethnic groups, roughly half would prefer to receive antibiotics at mass dispensing sites rather than through the US Postal Service. People in racial/ethnic minority groups were more likely to say this preference stems from a desire to speak with staff or to exchange medication formulation or type. Findings suggest the need for tailored outreach to racial/ethnic minorities through, for example, emphasis on key messages and enhanced understandability in communications, increased staff for answering questions in relevant dispensing sites, and long-term trust building with racial/ethnic minority communities. PMID:23244501

Steelfisher, Gillian K; Blendon, Robert J; Brulé, Amanda S; Ben-Porath, Eran N; Ross, Laura J; Atkins, Bret M

2012-12-01

377

Adverse medical events in British service personnel following anthrax vaccination  

Microsoft Academic Search

The safety of the UK anthrax vaccine in British service personnel was evaluated by a retrospective cohort study of randomly selected personnel from five Royal Air Force bases by investigating adverse medical events and consultation rates for a period before and after vaccination. Vaccination acceptance rate varied from 27 to 89% (P=0.0001). In the vaccinated cohort 11.1% (n=368) reported side-effects.

Joanne E Enstone; Martin C. J Wale; Jonathan S Nguyen-Van-Tam; James C. G Pearson

2003-01-01

378

Detection of anthrax vaccine virulence factors by polymerase chain reaction  

Microsoft Academic Search

In Italy, an attenuated Bacillus anthracis strain, named ‘Carbosap’, is used for immunization against ovine and bovine anthrax. Analysis on ‘Carbosap’, Sterne vaccine strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and primers specific for the chromosome. The results obtained from polymerase chain

Antonio Fasanella; Stefania Losito; Teresa Trotta; Rosanna Adone; Salvatore Massa; Franco Ciuchini; Doriano Chiocco

2001-01-01

379

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design*  

PubMed Central

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-?-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis ?-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

Wu, Sean J.; Eiben, Christopher B.; Carra, John H.; Huang, Ivan; Zong, David; Liu, Peixian; Wu, Cindy T.; Nivala, Jeff; Dunbar, Josef; Huber, Tomas; Senft, Jeffrey; Schokman, Rowena; Smith, Matthew D.; Mills, Jeremy H.; Friedlander, Arthur M.; Baker, David; Siegel, Justin B.

2011-01-01

380

Improvement of a potential anthrax therapeutic by computational protein design.  

PubMed

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-?-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis ?-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis. PMID:21768086

Wu, Sean J; Eiben, Christopher B; Carra, John H; Huang, Ivan; Zong, David; Liu, Peixian; Wu, Cindy T; Nivala, Jeff; Dunbar, Josef; Huber, Tomas; Senft, Jeffrey; Schokman, Rowena; Smith, Matthew D; Mills, Jeremy H; Friedlander, Arthur M; Baker, David; Siegel, Justin B

2011-07-18

381

Bacillus anthracis Edema Toxin Suppresses Human Macrophage Phagocytosis and Cytoskeletal Remodeling via the Protein Kinase A and Exchange Protein Activated by Cyclic AMP Pathways  

Microsoft Academic Search

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. It produces edema toxin (EdTx), a powerful adenylate cyclase that increases cyclic AMP (cAMP) levels in host cells. Because other cAMP-increasing agents inhibit key macrophage (M) functions, such as phagocytosis, it was hypothesized that EdTx would exhibit similar suppressive activities. Our previous GeneChip data showed that EdTx downregulated

Linsey A. Yeager; Ashok K. Chopra; Johnny W. Peterson

2009-01-01

382

Anthrax Lethal Toxin Impairs Innate Immune Functions of Alveolar Macrophages and Facilitates Bacillus anthracis Survival.  

National Technical Information Service (NTIS)

Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contrib...

W. J. Ribot R. G. Panchal K. C. Brittingham G. Ruthel T. A. Kenny

2006-01-01

383

A three-dose intramuscular injection schedule of anthrax vaccine adsorbed generates sustained humoral and cellular immune responses to protective antigen and provides long-term protection against inhalation anthrax in rhesus macaques.  

PubMed

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10)-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-?) and interleukin-4 (IL-4) CD4(+) cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses. PMID:22933399

Quinn, Conrad P; Sabourin, Carol L; Niemuth, Nancy A; Li, Han; Semenova, Vera A; Rudge, Thomas L; Mayfield, Heather J; Schiffer, Jarad; Mittler, Robert S; Ibegbu, Chris C; Wrammert, Jens; Ahmed, Rafi; Brys, April M; Hunt, Robert E; Levesque, Denyse; Estep, James E; Barnewall, Roy E; Robinson, David M; Plikaytis, Brian D; Marano, Nina

2012-08-29

384

Novel inhibitors of Anthrax edema factor  

PubMed Central

Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D- pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF- active site in existing crystal structures and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design.

Chen, Deliang; Misra, Milind; Sower, Laurie; Peterson, Johnny W.; Kellogg, Glen E.; Schein, Catherine H.

2008-01-01

385

Relationship Between Prepregnancy Anthrax Vaccination and Pregnancy and Birth Outcomes Among US Army Women.  

National Technical Information Service (NTIS)

With the advent of the Persian Gulf War, the US Department of Defense determined there was a credible threat of anthrax exposure to its troops from biological weapons and a large-scale vaccination program with anthrax vaccine was started. Substantial conc...

A. R. Wiesen C. T. Littell

2002-01-01

386

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax.  

PubMed

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%-60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis. PMID:23108317

Tonry, Jessica H; Popov, Serguei G; Narayanan, Aarthi; Kashanchi, Fatah; Hakami, Ramin M; Carpenter, Calvin; Bailey, Charles; Chung, Myung-Chul

2012-10-26

387

Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax.  

PubMed

Prevention of inhalational anthrax after Bacillus anthracis spore exposure requires a prolonged course of antibiotic prophylaxis. In response to the 2001 anthrax attack in the United States, approximately 10,000 people were offered 60 days of antibiotic prophylaxis to prevent inhalational anthrax, but adherence to this regimen was poor. We sought to determine whether a short course of antibiotic prophylaxis after exposure could protect non-human primates from a high-dose spore challenge if vaccination was combined with antibiotics. Two groups of 10 rhesus macaques were exposed to approximately 1,600 LD50 of spores by aerosol. Both groups were given ciprofloxacin by orogastric tube twice daily for 14 days, beginning 1-2 h after exposure. One group also received three doses of the licensed human anthrax vaccine (anthrax vaccine adsorbed) after exposure. In the ciprofloxacin-only group, four of nine monkeys (44%) survived the challenge. In contrast, all 10 monkeys that received 14 days of antibiotic plus anthrax vaccine adsorbed survived (P = 0.011). Thus postexposure vaccination enhanced the protection afforded by 14 days of antibiotic prophylaxis alone and completely protected animals against inhalational anthrax. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax and may impact public health management of a bioterrorism event. PMID:16672361

Vietri, Nicholas J; Purcell, Bret K; Lawler, James V; Leffel, Elizabeth K; Rico, Pedro; Gamble, Christopher S; Twenhafel, Nancy A; Ivins, Bruce E; Heine, Henry S; Sheeler, Ryan; Wright, Mary E; Friedlander, Arthur M

2006-05-03

388

Validation of an anti-PA-ELISA for the potency testing of anthrax vaccine in mice  

Microsoft Academic Search

The potency test for the anthrax vaccine currently licensed for human use in the United States (Anthrax Vaccine Adsorbed) involves the protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis. Lethal challenge tests entail the use of specialized containment facilities for the safe and secure handling of the challenge strain. This potential

María Pombo; Inge Berthold; Elise Gingrich; María Jaramillo; Mary Leef; Lev Sirota; Henry Hsu; Juan Arciniega

2004-01-01

389

Medical counterbioterrorism: The response to provide anthrax prophylaxis to New York city US postal service employees  

Microsoft Academic Search

Study objective: We describe and analyze a recent rapid deployment of disaster medical assistance teams and other government agencies to provide medical screening and anthrax prophylaxis to New York City US Postal Service employees potentially exposed to letters contaminated with anthrax spores. Methods: A description of the response effort is presented. Data were collected on standardized forms and included the

Robert Partridge; John Alexander; Tom Lawrence; Selim Suner

2003-01-01

390

The role of HLA–DR–DQ haplotypes in variable antibody responses to Anthrax Vaccine Adsorbed  

Microsoft Academic Search

Host genetic variation, particularly within the human leukocyte antigen (HLA) loci, reportedly mediates heterogeneity in immune response to certain vaccines; however, no large study of genetic determinants of anthrax vaccine response has been described. We searched for associations between the immunoglobulin G antibody to protective antigen (AbPA) response to Anthrax Vaccine Adsorbed (AVA) in humans, and polymorphisms at HLA class

N M Pajewski; S D Parker; G A Poland; I G Ovsyannikova; W Song; K Zhang; B A McKinney; V S Pankratz; J C Edberg; R P Kimberly; R M Jacobson; J Tang; R A Kaslow

2011-01-01

391

The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor  

Microsoft Academic Search

Recent events have created an urgent need for new therapeutic strategies to treat anthrax. We have applied a mixture-based peptide library approach to rapidly determine the optimal peptide substrate for the anthrax lethal factor (LF), a metalloproteinase with an important role in the pathogenesis of the disease. Using this approach we have identified peptide analogs that inhibit the enzyme in

Benjamin E Turk; Thiang Yian Wong; Robert Schwarzenbacher; Emily T Jarrell; Stephen H Leppla; R John Collier; Robert C Liddington; Lewis C Cantley

2003-01-01

392

Review of the Scientific Approaches Used During the FBI's Investigation of the 2001 Anthrax Letters.  

National Technical Information Service (NTIS)

Less than a month after the September 11, 2001, attacks, letters containing spores of anthrax bacteria (Bacillus anthracis, or B. anthracis) were sent through the U.S. mail. Between October 4 and November 20, 2001, 22 individuals developed anthrax; 5 of t...

2011-01-01

393

Anthrax Vaccination and Risk of Optic Neuritis in the United States Military, 1998-2003  

Microsoft Academic Search

Background: Numerous case reports have suggested a possible association between optic neuritis and receipt of several different vaccines. The most frequently iden- tified vaccines associated with optic neuritis in the lit- erature are influenza and hepatitis B, and a report de- scribing 2 US military cases suggests an association with the currently used anthrax vaccine (anthrax vac- cine adsorbed). Objective:Totestthehypothesisthatopticneuritismay

Daniel C. Payne; Charles E. Rose; John Kerrison; Aaron Aranas; Susan Duderstadt; Michael M. McNeil

2006-01-01

394

Anthrax Detection: Agencies Need to Validate Sampling Activities in Order to Increase Confidence in Negative Results.  

National Technical Information Service (NTIS)

In September and October 2001, contaminated letters laced with Bacillus anthracis, or anthrax spores, were sent through the mail to two senators, Thomas Daschle and Patrick Leahy, and members of the media. The letters led to the first cases of anthrax dis...

2005-01-01

395

Anthracimycin, a potent anthrax antibiotic from a marine-derived actinomycete.  

PubMed

Licensed to kill: A new antibiotic, anthracimycin (see scheme), produced by a marine-derived actinomycete in saline culture, shows significant activity toward Bacillus anthracis, the bacterial pathogen responsible for anthrax infections. Chlorination of anthracimycin gives a dichloro derivative that retains activity against Gram-positive bacteria, such as anthrax, but also shows activity against selected Gram-negative bacteria. PMID:23776159

Jang, Kyoung Hwa; Nam, Sang-Jip; Locke, Jeffrey B; Kauffman, Christopher A; Beatty, Deanna S; Paul, Lauren A; Fenical, William

2013-06-17

396

Use of medical simulation to teach bioterrorism preparedness: the anthrax example.  

PubMed

The 2001 anthrax bioterrorism attacks demonstrated vulnerability for future similar attacks. This article describes mechanisms that can be used to prepare the medical community and healthcare facilities for the diagnosis and management of a subsequent bioterrorism attack should such an event occur and the fundamentals of medical simulation and its use in teaching learners about the diagnosis of management of anthrax exposure. PMID:23263314

Olsen, Martin E

2013-01-01

397

Development of an in vitro-based potency assay for anthrax vaccine  

Microsoft Academic Search

The potency assay currently used to evaluate consistency of manufacture for the anthrax vaccine is contingent upon meeting specified parameters after statistical analysis of the percent survival and time to death of vaccinated guinea pigs after challenge with spores of a virulent strain of Bacillus anthracis. During the development of a new anthrax vaccine based upon recombinant protective antigen (rPA)

S. F. Little; W. M. Webster; B. E. Ivins; P. F. Fellows; S. L. Norris; G. P. Andrews

2004-01-01

398

UK armed forces responses to an informed consent policy for anthrax vaccination: A paradoxical effect?  

Microsoft Academic Search

BackgroundIn recognition of concerns that anthrax vaccination might be a trigger for “Gulf war syndrome”, anthrax vaccinations were offered to UK armed forces in the 2003 Iraq conflict using explicit as opposed to implicit consent, as is the policy for all other vaccinations. This paper examines responses of personnel to this policy.

Dominic Murphy; Christopher Dandeker; Oded Horn; Matthew Hotopf; Lisa Hull; Margaret Jones; Theresa Marteau; Roberto Rona; Simon Wessely

2006-01-01

399

News Note: A new dual vaccine for protection against both smallpox and anthrax  

Cancer.gov

Scientists have developed and tested a new protective vaccine against smallpox and anthrax, two agents of bioterrorism, in animal models. Liyanage P. Perera, Ph.D., NCI, and colleagues made the enhanced dual vaccine by inserting the genes for protective parts of anthrax and the immune-boosting chemical, interleukin-15, into the backbone of the licensed smallpox vaccine, ACAM2000.

400

Antibody Responses to a Spore Carbohydrate Antigen as a Marker of Nonfatal Inhalation Anthrax in Rhesus Macaques ?  

PubMed Central

The Bacillus anthracis exosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (J. M. Daubenspeck et al., J. Biol. Chem. 279:30945–30953, 2004). We hypothesized that serologic responses to anthrose may have diagnostic value in confirming exposure to aerosolized B. anthracis. We evaluated the serologic responses to a synthetic anthrose-containing trisaccharide (ATS) in a group of five rhesus macaques that survived inhalation anthrax following exposure to B. anthracis Ames spores. Two of five animals (RM2 and RM3) were treated with ciprofloxacin starting at 48 hours postexposure and two (RM4 and RM5) at 72 h postexposure; one animal (RM1) was untreated. Infection was confirmed by blood culture and detection of anthrax toxin lethal factor (LF) in plasma. Anti-ATS IgG responses were determined at 14, 21, 28, and 35 days postexposure, with preexposure serum as a control. All animals, irrespective of ciprofloxacin treatment, mounted a specific, measurable anti-ATS IgG response. The earliest detectable responses were on days 14 (RM1, RM2, and RM5), 21 (RM4), and 28 (RM3). Specificity of the anti-ATS responses was demonstrated by competitive-inhibition enzyme immunoassay (CIEIA), in which a 2-fold (wt/wt) excess of carbohydrate in a bovine serum albumin (BSA) conjugate of the oligosaccharide (ATS-BSA) effected >94% inhibition, whereas a structural analog lacking the 3-hydroxy-3-methyl-butyryl moiety at the C-4" of the anthrosyl residue had no inhibition activity. These data suggest that anti-ATS antibody responses may be used to identify aerosol exposure to B. anthracis spores. The anti-ATS antibody responses were detectable during administration of ciprofloxacin.

Saile, Elke; Boons, Geert-Jan; Buskas, Therese; Carlson, Russell W.; Kannenberg, Elmar L.; Barr, John R.; Boyer, Anne E.; Gallegos-Candela, Maribel; Quinn, Conrad P.

2011-01-01

401

Antibody responses to a spore carbohydrate antigen as a marker of nonfatal inhalation anthrax in rhesus macaques.  

PubMed

The Bacillus anthracis exosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (J. M. Daubenspeck et al., J. Biol. Chem. 279:30945-30953, 2004). We hypothesized that serologic responses to anthrose may have diagnostic value in confirming exposure to aerosolized B. anthracis. We evaluated the serologic responses to a synthetic anthrose-containing trisaccharide (ATS) in a group of five rhesus macaques that survived inhalation anthrax following exposure to B. anthracis Ames spores. Two of five animals (RM2 and RM3) were treated with ciprofloxacin starting at 48 hours postexposure and two (RM4 and RM5) at 72 h postexposure; one animal (RM1) was untreated. Infection was confirmed by blood culture and detection of anthrax toxin lethal factor (LF) in plasma. Anti-ATS IgG responses were determined at 14, 21, 28, and 35 days postexposure, with preexposure serum as a control. All animals, irrespective of ciprofloxacin treatment, mounted a specific, measurable anti-ATS IgG response. The earliest detectable responses were on days 14 (RM1, RM2, and RM5), 21 (RM4), and 28 (RM3). Specificity of the anti-ATS responses was demonstrated by competitive-inhibition enzyme immunoassay (CIEIA), in which a 2-fold (wt/wt) excess of carbohydrate in a bovine serum albumin (BSA) conjugate of the oligosaccharide (ATS-BSA) effected >94% inhibition, whereas a structural analog lacking the 3-hydroxy-3-methyl-butyryl moiety at the C-4" of the anthrosyl residue had no inhibition activity. These data suggest that anti-ATS antibody responses may be used to identify aerosol exposure to B. anthracis spores. The anti-ATS antibody responses were detectable during administration of ciprofloxacin. PMID:21389148

Saile, Elke; Boons, Geert-Jan; Buskas, Therese; Carlson, Russell W; Kannenberg, Elmar L; Barr, John R; Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P

2011-03-09

402

Recombinant vaccine displaying the loop-neutralizing determinant from protective antigen completely protects rabbits from experimental inhalation anthrax.  

PubMed

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2?2-2?3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD(50)) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines. PMID:23283638

Oscherwitz, Jon; Yu, Fen; Jacobs, Jana L; Cease, Kemp B

2013-01-02

403

Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies  

PubMed Central

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-?-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin-neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.

Ramirez, Karina; Ditamo, Yanina; Galen, James E.; Baillie, Les W. J.; Pasetti, Marcela F.

2010-01-01

404

Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch.  

PubMed Central

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed.

Martin, B M; Ramirez, A N; Gurrola, G B; Nobile, M; Prestipino, G; Possani, L D

1994-01-01

405

Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch.  

PubMed

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed. PMID:7998956

Martin, B M; Ramirez, A N; Gurrola, G B; Nobile, M; Prestipino, G; Possani, L D

1994-11-15

406

Structure-function relationship of lapemis toxin: a synthetic approach.  

PubMed

The synthetic approach to the structure-function relationship of lapemis toxin has been very useful in clarifying the important binding regions. To identify the neurotoxic binding domain(s) of lapemis toxin, several peptides were synthesized using the 9-fluorenylmethoxycarbonyl protocols. These peptides were based on the sequence of lapemis toxin, a 60-amino-acid, short-chain postsynaptic neurotoxin found in sea snake (Lapemis hardwickii) venom. The peptides were purified using high-performance liquid chromatography and sequenced to verify the correct synthesis, isolation, and purity. The synthetic peptide names and single letter sequences were Peptide A1 (15 mer) CCNQQSSQPKTTTNC Peptide B1 (18 mer) CYKKTWSDHRGTRIERGC Peptide B2 (16 mer) YKKTWSDHRGTRIERG Peptide C1 (12 mer) CPQVKPGIKLEC Peptide NS (20 mer) EACDFGHIKLMNPQRSTVWY. The peptide NS (nonsense peptide) sequence was arbitrarily determined and used as a control peptide. Biological activities of the synthetic peptides were determined by in vivo as well as by in vitro assay methods. For the in vivo assay, lethality was determined by intravenous injection in mice (Swiss Webster). For the in vitro assay, peptide binding to the Torpedo californica nicotinic acetylcholine receptor was determined. The peptides were found to be nontoxic at approximately 114 times the known LD50 of lapemis toxin. Binding studies with 125I-radiolabeled lapemis toxin and tyrosine-containing peptides indicated that lapemis toxin and peptide B1 bound the receptor, while the other peptides had no detectable binding. The central loop domain of lapemis toxin (peptide B1) plays a dominate role in the toxin's binding ability to the receptor. These results and the hydrophilicity analysis predict peptide B1 may serve as an antagonist or antigen to neutralize the neurotoxin effects in vivo. PMID:1929436

Miller, R A; Tu, A T

1991-11-15

407

Isolation and characterization of a novel toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch.  

PubMed

A novel peptide, toxic to mice, was purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, by means of gel filtration and ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The complete amino acid sequence was determined by automatic Edman degradation of reduced and alkylated toxin, and by overlapping sequences of fragments of the toxin, generated by cleavage with proteinase V8 separated by HPLC. This toxin is composed of 66 amino acid residues, contains eight half-cystine residues, and is highly similar (91%) to the amino acid sequence deduced for toxin 1 of C. limpidus tecomanus and toxin 4 from C. noxius venom (89%). This peptide displaces the binding of radiolabeled toxin 2 of C. noxius from synaptosomal membranes of rat brain with superimposable kinetics, supporting the conclusion that it belongs to the beta-scorpion toxin class. Further characterization of C. l. limpidus toxin 1, as we have named it, was performed by means of competition experiments with monoclonal antibodies and various purified scorpion toxins, using an ELISA assay. A panel of six distinct monoclonal antibodies (mAB) against toxin 2 and 3 of C. noxius was used. From these, only three clones, originally named BCF1, BCF8 and BCF9, were able to recognize toxin 1 from C. l. limpidus. PMID:8053002

Ramírez, A N; Martin, B M; Gurrola, G B; Possani, L D

1994-04-01

408

[Hemolysis of Scolopendra toxins].  

PubMed

The hemolysis of toxins from alive Scolopendra subspinipes mutilans, medicinal material of Scolopendra subspimipes mutilans and S. multidens have been compared. The result shows that all the toxins have hemolytic activity. The hemolytic activity of the toxin from the medicinal materials of S. subspinipes mutilans is obviously lower than that from alive ones, and that from fresh medicinal materials are twice as high that from old ones, and that from S. multidens is higher than that from S. subspinipes multilans. PMID:12572496

Deng, F; Fang, H; Wang, K

1997-01-01

409

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax  

PubMed Central

The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ?7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions.

Marston, Chung K.; Bhullar, Vinod; Baker, Daniel; Rahman, Mahmudur; Hossain, M. Jahangir; Chakraborty, Apurba; Khan, Salah Uddin; Hoffmaster, Alex R.

2012-01-01

410

Exogenous Interferon-? and Interferon-? Increase Lethality of Murine Inhalational Anthrax  

PubMed Central

Background Bacillus anthracis, the etiologic agent of inhalational anthrax, is a facultative intracellular pathogen. Despite appropriate antimicrobial therapy, the mortality from inhalational anthrax approaches 45%, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Type I and Type II Interferons (IFN) are prominent members of the host innate immune response and are required for control of intracellular pathogens. We have previously described a protective role for exogenous Type I and Type II IFNs in attenuating intracellular B.anthracis germination and macrophage cell death in vitro. Methodology and Principal Findings We sought to extend these findings in an in vivo model of inhalational anthrax, utilizing the Sterne strain (34F2) of B.anthracis. Mice devoid of STAT1, a component of IFN-? and IFN-? signaling, had a trend towards increased mortality, bacterial germination and extrapulmonary spread of B.anthracis at 24 hrs. This was associated with impaired IL-6, IL-10 and IL-12 production. However, administration of exogenous IFN-?, and to a lesser extent IFN-?, at the time of infection, markedly increased lethality. While IFNs were able to reduce the fraction of germinated spores within the lung, they increased both the local and systemic inflammatory response manifest by increases in IL-12 and reductions in IL-10. This was associated with an increase in extrapulmonary dissemination. The mechanism of IFN mediated inflammation appears to be in part due to STAT1 independent signaling. Conclusions In conclusion, while endogenous IFNs are essential for control of B.anthracis germination and lethality, administration of exogenous IFNs appear to increase the local inflammatory response, thereby increasing mortality.

Gold, Jeffrey A.; Hoshino, Yoshihiko; Jones, Marcus B.; Hoshino, Satomi; Nolan, Anna; Weiden, Michael D.

2007-01-01

411

Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants.  

PubMed

The only US-licensed anthrax vaccine for human use, as well as several experimental vaccines containing solely purified recombinant protective antigen (rPA), are formulated using aluminum hydroxide (Al(OH)3) as an adjuvant. It has been suggested that effective adjuvanticity of aluminum salts for protein antigens depends, at least partially, on the degree of adsorption of the antigen to the adjuvant. On the other hand, the ease of antigen desorption from the adjuvant in a quantitative fashion may facilitate the assessment of vaccine characteristics in the laboratory. In this regard, aluminum phosphate (AlPO4), although deemed a "weaker" adjuvant than Al(OH)3, appears superior to the latter. To investigate the possibility of formulating rPA vaccines with AlPO4, as well as the significance of the adsorption of this antigen to the aluminum salt for adjuvanticity, we studied the effect of AlPO4 and Al(OH)3 on the induction of anti-rPA antibodies in mice. In a first immunization experiment the adjuvanticity of AlPO4 combined with rPA was examined. Antibodies against rPA were measured using an ELISA. Results indicated that AlPO4 is able to significantly increase the antibody response to rPA, irrespective of its degree of adsorption to the adjuvant. Based on these results, in a second experiment mice were immunized twice, with different formulations of rPA containing either AlPO4 or Al(OH)3, and rPA-antibodies were measured using ELISA and an in vitro toxin neutralization assay. Comparable immune responses to rPA were obtained with both aluminum salts. Additionally, results with AlPO4 as adjuvant confirmed that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response induced. PMID:15734073

Berthold, Inge; Pombo, Maria-Luz; Wagner, Leslie; Arciniega, Juan L

2005-03-14

412

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: Chemical synthesis, radiolabeling, and receptor characterization  

SciTech Connect

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, /sup 125/I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.

Cruz, L.J.; Kupryszewski, G.; LeCheminant, G.W.; Gray, W.R.; Olivera, B.M.; Rivier, J.

1989-04-18

413

Detection of the sentinel anthrax case in the United States.  

PubMed

First-hand knowledge of the detection of the first bioweapon in modern United States history is described in this article. The method by which the presumptive diagnosis of anthrax meningitis was made within 13 hours of the patient presenting to the emergency department is described using pre-analytic, analytic, and post-analytic phases. The lessons learned from this process are briefly presented so that other laboratories may learn from our experience: how to prepare; how to quickly analyze a potential bioweapon; how to communicate with staff and local, regional, and national authorities; and how to deal with disruptive media attention. PMID:14531222

Beall, Anne; Cooke, William; Trout, Joanne; Robb, James A

414

Decontamination of Anthrax spores in critical infrastructure and critical assets.  

SciTech Connect

Decontamination of anthrax spores in critical infrastructure (e.g., subway systems, major airports) and critical assets (e.g., the interior of aircraft) can be challenging because effective decontaminants can damage materials. Current decontamination methods require the use of highly toxic and/or highly corrosive chemical solutions because bacterial spores are very difficult to kill. Bacterial spores such as Bacillus anthracis, the infectious agent of anthrax, are one of the most resistant forms of life and are several orders of magnitude more difficult to kill than their associated vegetative cells. Remediation of facilities and other spaces (e.g., subways, airports, and the interior of aircraft) contaminated with anthrax spores currently requires highly toxic and corrosive chemicals such as chlorine dioxide gas, vapor- phase hydrogen peroxide, or high-strength bleach, typically requiring complex deployment methods. We have developed a non-toxic, non-corrosive decontamination method to kill highly resistant bacterial spores in critical infrastructure and critical assets. A chemical solution that triggers the germination process in bacterial spores and causes those spores to rapidly and completely change to much less-resistant vegetative cells that can be easily killed. Vegetative cells are then exposed to mild chemicals (e.g., low concentrations of hydrogen peroxide, quaternary ammonium compounds, alcohols, aldehydes, etc.) or natural elements (e.g., heat, humidity, ultraviolet light, etc.) for complete and rapid kill. Our process employs a novel germination solution consisting of low-cost, non-toxic and non-corrosive chemicals. We are testing both direct surface application and aerosol delivery of the solutions. A key Homeland Security need is to develop the capability to rapidly recover from an attack utilizing biological warfare agents. This project will provide the capability to rapidly and safely decontaminate critical facilities and assets to return them to normal operations as quickly as possible, sparing significant economic damage by re-opening critical facilities more rapidly and safely. Facilities and assets contaminated with Bacillus anthracis (i.e., anthrax) spores can be decontaminated with mild chemicals as compared to the harsh chemicals currently needed. Both the 'germination' solution and the 'kill' solution are constructed of 'off-the-shelf,' inexpensive chemicals. The method can be utilized by directly spraying the solutions onto exposed surfaces or by application of the solutions as aerosols (i.e., small droplets), which can also reach hidden surfaces.

Boucher, Raymond M.; Crown, Kevin K.; Tucker, Mark David; Hankins, Matthew Granholm

2010-05-01

415

The effect of seasonal variation on anthrax epidemiology in the upper Zambezi floodplain of western Zambia  

PubMed Central

Anthrax has become endemic throughout the upper Zambezi floodplain located in the Western Province of Zambia over the recent years. To date, no comprehensive study has been carried out to determine whether recurrence of anthrax outbreaks may be linked to differences in precipitation and human activities. Retrospective data for the period 1999 to 2007 showed that a total of 1,216 bovine cases of anthrax were reported. During the same period, 1,790 human anthrax cases and a corresponding case fatality rate of 4.63% (83/1,790) was documented in the upper Zambezi floodplain. Occurrence of human cases was highly correlated with cattle outbreaks (r = 0.94, p < 0.001). Differences in precipitation were significantly associated with the occurrence of anthrax outbreaks (?2 = 4.75, p < 0.03), indicating that the likelihood of outbreaks occurring was higher during the dry months when human occupancy of the floodplain was greater compared to the flooding months when people and livestock moved out of this region. Human dependency on the floodplain was shown to significantly influence the epidemiology of anthrax in the upper Zambezi floodplain of western Zambia. Methods for mitigating anthrax outbreaks by disrupting the cycle of transmission are herein highlighted.

Banda, Fredrick; Siamudaala, Victor Mukulule; Munyeme, Musso; Kasanga, Christopher Jacob; Hamududu, Byman

2012-01-01

416

Surveillance and control of anthrax and rabies in wild herbivores and carnivores in Namibia.  

PubMed

Anthrax has been studied intensively in Etosha National Park, Namibia since 1966; in addition, since 1975, mortality due to rabies and all other causes has been recorded, totalling 6,190 deaths. Standard diagnostic procedures demonstrated that at least 811 deaths (13%) were due to anthrax and 115 deaths (2%) were caused by rabies. Of the total number of deaths due to anthrax, 97% occurred in zebra (Equus burchelli), elephant (Loxodonta africana), wildebeest (Connochaetes taurinus) and springbok (Antidorcas marsupialis) while 96% of rabies deaths occurred in kudu (Tragelaphus strepsiceros), jackal (Canis mesomelas), bat-eared fox (Otocyon megalotis) and lion (Panthera leo). Anthrax deaths were highest in the rainy season for zebra, wildebeest and springbok, while elephant mortality peaked during dry seasons. No statistical relationship existed between seasonal rainfall and overall incidence of either anthrax or rabies. Control of anthrax is limited to prophylactic inoculation when rare or endangered species are threatened. Incineration of anthrax carcasses and chemical disinfection of drinking water are not feasible at Etosha. Rabies control consists of the destruction of rabid animals and incineration of their carcasses when possible. PMID:8518440

Berry, H H

1993-03-01

417

Methods for radiolabelling of monoclonal antibodies.  

PubMed

The use of radionuclide labels allows to study the pharmacokinetics of monoclonal antibodies, to control the specificity of their targeting and to monitor the response to an antibody treatment with high accuracy. Selection of label depends on the processing of an antibody after binding to an antigen, and on the character of information to be derived from the study (distribution of antibody in the extracellular space, target occupancy or determination of sites of metabolism). This chapter provides protocols for labelling of antibodies with iodine-125 (suitable also for other radioisotopes of iodine) and with indium-111. Since radiolabelling might damage and reduce the immunoreactive fraction and/or affinity of an antibody, the methods for assessment of these characteristics of an antibody are provided for control. PMID:24037848

Tolmachev, Vladimir; Orlova, Anna; Andersson, Karl

2014-01-01

418

Autodecomposition of radiolabeled human growth hormone  

SciTech Connect

Human growth hormone (hGH) was radiolabeled with /sup