scholarly journals Anthrax Lethal Toxin Induces Ketotifen-Sensitive Intradermal Vascular Leakage in Certain Inbred Mice

2006 ◽  
Vol 74 (2) ◽  
pp. 1266-1272 ◽  
Author(s):  
Yehoshua Gozes ◽  
Mahtab Moayeri ◽  
Jason F. Wiggins ◽  
Stephen H. Leppla
Keyword(s):  
Mast Cell ◽  
Protective Antigen ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Lethal Factor ◽  
Clinical Signs ◽  
Lethal Toxin ◽  
Vascular Leakage ◽  
Mouse Strains

ABSTRACT Bacillus anthracis lethal toxin (LT) is a bipartite toxin composed of protective antigen (PA) and lethal factor (LF). Injection of LT produces clinical signs characteristic of anthrax infection, including pleural edema and vascular collapse in various animal models. We utilized the classic Miles leakage assay to quantify vascular leakage in mice. LT injected intradermally induced leakage as early as 15 to 25 min in some inbred mouse strains, but not in others, whereas PA or LF individually did not induce leakage. A third component of anthrax toxin, edema factor, did not induce leakage alone or with PA. Leakage was quantified in eight mouse strains, and no correlation was found between sensitivity to intradermal leakage and sensitivity to the lethality of systemically administered LT. The leakage could be inhibited by ketotifen, an inhibitor of mast cell degranulation, but not by azelastine, a histamine receptor 1 antagonist, or by ketanserin, a serotonin 5-HT2A receptor antagonist. LT was cytotoxic to MC/9 mast cells (in vitro) by 7 h after toxin treatment but did not induce histamine release from these cells. Mast cell-deficient mice exhibited the leakage event and had no increased resistance to systemic LT. Human umbilical vein endothelial cells were resistant to LT over 12 h, with only 20% of cells succumbing by 24 h, suggesting that endothelial cell killing is not the cause of the rapid LT-mediated leakage event. We describe here a ketotifen-sensitive vascular leakage event induced by LT which is the most rapid in vivo or in vitro LT-mediated effect reported to date.

scholarly journals Association of Bacillus anthracis Capsule with Lethal Toxin during Experimental Infection

2008 ◽  
Vol 77 (2) ◽  
pp. 749-755 ◽  
Author(s):  
J. W. Ezzell ◽  
T. G. Abshire ◽  
R. Panchal ◽  
D. Chabot ◽  
S. Bavari ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Size Exclusion ◽  
Terminal Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Animal Blood

ABSTRACT Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA63) and the residual 20-kDa fragment (PA20) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA63 was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-γ-d-glutamic acid (γ-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/γ-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including γ-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for γ-DPGA in anthrax pathogenesis.

scholarly journals Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

2016 ◽  
Vol 23 (3) ◽  
pp. 213-218 ◽  
Author(s):  
Mahtab Moayeri ◽  
Jacqueline M. Tremblay ◽  
Michelle Debatis ◽  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Inbred Mouse ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Serum Levels ◽  
Mouse Strains ◽  
Common Component ◽  
Content Type ◽  
Neutralizing Agent

ABSTRACTBacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated theirin vivoefficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.

scholarly journals Enhanced VWF biosynthesis and elevated plasma VWF due to a natural variant in the murine Vwf gene

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 3061-3067 ◽  
Author(s):  
Heidi L. Lemmerhirt ◽  
Jordan A. Shavit ◽  
Gallia G. Levy ◽  
Suzanne M. Cole ◽  
Jeffrey C. Long ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Genetic Modifiers ◽  
Wide Range ◽  
Natural Variant ◽  
Vitro Analysis

Abstract Both genetic and environmental influences contribute to the wide variation in plasma von Willebrand factor (VWF) levels observed in humans. Inbred mouse strains also have highly variable plasma VWF levels, providing a convenient model in which to study genetic modifiers of VWF. Previously, we identified a major modifier of VWF levels in the mouse (Mvwf1) as a regulatory mutation in murine Galgt2. We now report the identification of an additional murine VWF modifier (Mvwf2). Mvwf2 accounts for approximately 16% of the 8-fold plasma VWF variation (or ∼ 25% of the genetic variation) observed between the A/J and CASA/RkJ strains and maps to the murine Vwf gene itself. Twenty SNPs were identified within the coding regions of the A/J and CASA/RkJ Vwf alleles, and in vitro analysis of recombinant VWF demonstrated that a single SNP (+7970G>A) and the associated nonsynonymous amino acid change (R2657Q) confers a significant increase in VWF biosynthesis from the CASA/RkJ Vwf allele. This change appears to represent a unique gain of function that likely explains the mechanism of Mvwf2 in vivo. The identification of a natural Vwf gene variant among inbred mice affecting biosynthesis suggests that similar genetic variation may contribute to the wide range of VWF levels observed in humans.

scholarly journals Animal models of Graves' disease

2000 ◽  
pp. 1-8 ◽  
Author(s):  
M Ludgate
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Nod Mice ◽  
Autoimmune Response ◽  
Mouse Strains ◽  
Graves Disease ◽  
Genetic Immunisation

Graves' disease (GD) is an autoimmune condition in which goitre and hyperthyroidism are induced by thyroid stimulating antibodies (TSAB) which mimic the action of thyrotrophin (TSH). The target of the autoimmune response is the thyrotrophin receptor (TSHR) and, since its cloning, a number of differing approaches have been adopted in an attempt to develop an animal model of GD. Methods in which synthetic peptides or fragments of the receptor produced in bacteria or insect cells have been injected into animals together with immunological adjuvants have had only limited success in inducing some of the signs and symptoms of GD. Genetic immunisation resulted in thyroiditis in the majority, but TSAB formation in only a minority, of treated inbred mice. Transfer of receptor in vitro primed T cells to syngeneic naive recipients, with priming either using a bacterial fusion protein or genetic immunisation, induced destructive thyroiditis in non-obese diabetic (NOD) mice but lymphocytic thyroiditis in BALBc mice. Furthermore, the orbits of 17/22 of the BALBc animals, but not the NOD animals, with thyroiditis had orbital changes similar to those seen in thyroid eye disease. TSAB and elevated thyroxine levels were induced in AKR/N mice injected with fibroblasts expressing the full length human TSHR and murine major histocompatibility complex (MHC) class II homologous to the recipient mice. No thyroiditis was induced but preliminary results from a different group using the same protocol suggest that receptor autoantibodies and thyroid dysfunction could be transferred using T cells primed in vitro with the receptor and MHC-II expressing cells. The majority of the studies described above have studied inbred mouse strains. In a novel departure, the NMR outbred strain has been treated by genetic immunisation with very promising results, including the induction of increased thyroxine levels in 4/30 female mice, accompanied by TSAB in addition to thyroiditis, and with signs of hyperactivity and orbital pathology. This review discusses the various protocols together with the information regarding the pathogenesis of GD which each has contributed, and concludes with an evaluation of how close we are to mimicking this polygenic, multifactorial disease.

scholarly journals Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

2006 ◽  
Vol 281 (43) ◽  
pp. 32755-32764 ◽  
Author(s):  
Wei Wang ◽  
Chandrika Mulakala ◽  
Sabrina C. Ward ◽  
Grace Jung ◽  
Hai Luong ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Raw 264.7 Cells ◽  
Protective Effects ◽  
Anthrax Lethal Toxin ◽  
Arginine Residues

θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

scholarly journals Chemically-induced formation of an inhibitor of hepatic uroporphyrinogen decarboxylase in inbred mice with iron overload

1987 ◽  
Vol 246 (1) ◽  
pp. 221-226 ◽  
Author(s):  
A G Smith ◽  
J E Francis
Keyword(s):  
Iron Overload ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Decarboxylase Activity ◽  
Uroporphyrinogen Decarboxylase ◽  
Chemically Induced ◽  
Heat Treated ◽  
Inhibitory Potential

An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2′,3′,4′-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase.

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

2007 ◽  
Vol 75 (11) ◽  
pp. 5443-5452 ◽  
Author(s):  
Herman F. Staats ◽  
S. Munir Alam ◽  
Richard M. Scearce ◽  
Shaun M. Kirwan ◽  
Julia Xianzhi Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Neutralization Assay ◽  
Passive Transfer ◽  
Lethal Toxin ◽  
Significant Protection

ABSTRACT 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 immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.

scholarly journals Cisplatin Inhibition of Anthrax Lethal Toxin

2006 ◽  
Vol 50 (8) ◽  
pp. 2658-2665 ◽  
Author(s):  
Mahtab Moayeri ◽  
Jason F. Wiggins ◽  
Robin E. Lindeman ◽  
Stephen H. Leppla
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Inbred Strains ◽  
Lethal Toxin ◽  
Anthrax Lethal Toxin ◽  
Biologically Relevant ◽  
Mouse Macrophages ◽  
Mitogen Activated Protein

ABSTRACT Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages derived from certain inbred strains. LT is comprised of a receptor binding component, protective antigen (PA), which delivers the enzymatic component, lethal factor (LF), into cells. We found that mouse macrophages were protected from toxin by the antitumor drug cis-diammineplatinum (II) dichloride (cisplatin). Cisplatin was shown to inhibit LT-mediated cleavage of cellular mitogen-activated protein kinases (MEKs) without inhibiting LF's in vitro proteolytic activity. Cisplatin-treated PA lost 100% of its ability to function in toxicity assays when paired with untreated LF, despite maintaining the ability to bind to cells. Cisplatin-treated PA was unable to form heptameric oligomers required for LF binding and translocation. The drug was shown to modify PA in a reversible noncovalent manner. Not surprisingly, cisplatin also blocked the actions of anthrax edema toxin and of LF-Pseudomonas aeruginosa exotoxin A fusion peptide (FP59), both of which require PA for translocation. Treatment of BALB/cJ mice or Fischer F344 rats with cisplatin at biologically relevant concentrations completely protected the animals from a coadministered lethal dose of LT. However, treatment with cisplatin 2 hours before or after animals received a lethal bolus of toxin did not protect them.

scholarly journals CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of Bacillus anthracis Lethal Toxin-Neutralizing Antibodies

2010 ◽  
Vol 78 (4) ◽  
pp. 1610-1617 ◽  
Author(s):  
T. Scott Devera ◽  
Lindsay M. Aye ◽  
Gillian A. Lang ◽  
Sunil K. Joshi ◽  
Jimmy D. Ballard ◽  
...  
Keyword(s):  
T Cells ◽  
Bacillus Anthracis ◽  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Nkt Cells ◽  
Lethal Toxin ◽  
Type I ◽  
Lethal Challenge

ABSTRACT The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

scholarly journals A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

2010 ◽  
Vol 54 (11) ◽  
pp. 4750-4757 ◽  
Author(s):  
Gaobing Wu ◽  
Yuzhi Hong ◽  
Aizhen Guo ◽  
Chunfang Feng ◽  
Sha Cao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Vaccine Candidate ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Lethal Challenge ◽  
Edema Factor ◽  
Trivalent Vaccine

ABSTRACT 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., PAF427D. 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.

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