Stephen H Leppla

National Institute of Allergy and Infectious Disease, 베서스다, Maryland, United States

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Publications (281)1830.72 Total impact

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    ABSTRACT: Immune inhibitor A(InhA)-type metallopeptidases are potential virulence factors secreted by members of the Bacillus cereus group. Two paralogs from anthrax-causing Bacillus anthracis (BaInhA1 and BaInhA2) were shown to degrade host tissue proteins with broad substrate specificity. Analysis of their activation mechanism and the crystal structure of a zymogenic BaInhA2 variant revealed a ∼750-residue four-domain structure featuring a pro-peptide, a catalytic domain, a domain reminiscent of viral envelope glycoproteins, and a MAM domain grafted into the latter. This domain, previously found only in eukaryotes, is required for proper protein expression in B. anthracis and evinces certain flexibility. Latency is uniquely modulated by the N-terminal segment of the pro-peptide, which binds the catalytic zinc through its α-amino group and occupies the primed side of the active-site cleft. The present results further our understanding of the modus operandi of an anthrax secretome regulator.
    Full-text · Article · Jan 2016 · Structure
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    ABSTRACT: Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides which form the bipartite lethal and edema toxins (LT, ET). 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 their in vivo efficacy. In this work, gene therapy with an adenoviral vector (Ad/VNA2-PA) promoting 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. Levels were 10-100 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 >0.1 μg/ml survived LT challenge and 9 of 10 C57BL/6J mice with serum levels >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.
    No preview · Article · Jan 2016 · Clinical and vaccine Immunology: CVI
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    ABSTRACT: Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation.
    Full-text · Article · Nov 2015 · Scientific Reports
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    Allison J. Greaney · Stephen H. Leppla · Mahtab Moayeri
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    ABSTRACT: The inflammasomes are intracellular protein complexes that play an important role in innate immune sensing. Activation of inflammasomes leads to activation of caspase-1 and maturation and secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. In certain myeloid cells, this activation can also lead to an inflammatory cell death (pyroptosis). Inflammasome sensor proteins have evolved to detect a range of microbial ligands and bacterial exotoxins either through direct interaction or by detection of host cell changes elicited by these effectors. Bacterial exotoxins activate the inflammasomes through diverse processes, including direct sensor cleavage, modulation of ion fluxes through plasma membrane pore formation, and perturbation of various host cell functions. In this review, we summarize the findings on some of the bacterial exotoxins that activate the inflammasomes.
    Preview · Article · Nov 2015 · Frontiers in Immunology
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    ABSTRACT: In this study, we attempt to target both the urokinase plasminogen activator and the mitogen-activated protein kinase pathway in acute myeloid leukemia (AML) cell lines and primary AML blasts using PrAgU2/LF, a urokinase-activated anthrax lethal toxin. PrAgU2/LF was cytotoxic to five out of nine AML cell lines. Cytotoxicity of PrAgU2/LF appeared to be nonapoptotic and was associated with MAPK activation and urokinase activity because all the PrAgU2/LF-sensitive cell lines showed both uPAR expression and high levels of MEK1/2 phosphorylation. Inhibition of uPAR or desensitization of cells to MEK1/2 inhibition blocked toxicity of PrAgU2/LF, indicating requirement for both uPAR expression and MAPK activation for activity. PrAgU2/LF was also cytotoxic to primary blasts from AML patients, with blasts from four out of five patients showing a cytotoxic response to PrAgU2/LF. Cytotoxicity of primary AML blasts was also dependent on uPAR expression and phos-MEK1/2 levels. CD34(+) bone marrow blasts and peripheral blood mononuclear cells lacked uPAR expression and were resistant to PrAgU2/LF, demonstrating the lack of toxicity to normal hematological cells and, therefore, the tumor selectivity of this approach. Dose escalation in mice revealed that the maximal tolerated dose of PrAgU2/LF is at least 5.7-fold higher than that of the wild-type anthrax lethal toxin, PrAg/LF, further demonstrating the increased safety of this molecule. We have shown, in this study, that PrAgU2/LF is a novel, dual-specific molecule for the selective targeting of AML.
    Full-text · Article · Oct 2015 · Translational oncology
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    ABSTRACT: The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.
    Full-text · Article · Sep 2015 · Oncotarget
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    ABSTRACT: Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, we characterize the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in E. coli. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. We examine the impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs, and identify a sequence region of the Hfq proteins that is involved in hexamer formation. We found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation. This article is protected by copyright. All rights reserved. © 2015 The Protein Society.
    No preview · Article · Aug 2015 · Protein Science
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    ABSTRACT: Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients. Copyright © 2015 by The American Association of Immunologists, Inc.
    Full-text · Article · Aug 2015 · The Journal of Immunology
  • Allison J Greaney · Nolan K Maier · Stephen H Leppla · Mahtab Moayeri
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    ABSTRACT: The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1β maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1β secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to our understanding of the beneficial effects of sulforaphane. © Society for Leukocyte Biology.
    No preview · Article · Aug 2015 · Journal of leukocyte biology
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    ABSTRACT: Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed. Expected final online publication date for the Annual Review of Microbiology Volume 69 is September 08, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    Full-text · Article · Jul 2015 · Annual review of microbiology
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    ABSTRACT: The immunogenicity of Bacillus anthracis capsule (poly-γ-d-glutamic acid, PGA) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naïve juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (mAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1-2 weeks following the 1(st) immunization, with no booster effects following 2(nd) and 3(rd) immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1(st) immunization were ≤300, with the antibodies peaking above 10(4) following the 2(nd) immunization. The polyclonal anti-PGA shared the mAb 11D epitope and, similar to the mAbs, exerted opsonophagocytic killing of B. anthracis. Most importantly, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as second generation anthrax vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Full-text · Article · Jun 2015 · Clinical and vaccine Immunology: CVI
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    ABSTRACT: Background Lethal and edema toxin contribute to shock and lethality with Bacillus anthracis. We showed previously in a 96-h sedated canine model that raxibacumab, a monoclonal antibody against protective antigen, augmented hemodynamic support (HS) and improved survival with lethal toxin challenge. Here we study raxibacumab further. Using this model, we have now studied raxibacumab with 24 h edema toxin challenges (Study 1), and lethal and edema toxin challenges together (Study 2). Methods Using our canine model, we have now studied raxibacumab with 24h edema toxin challenges (Study-1), and lethal and edema toxin challenges together (Study-2). Results In Study 1, compared to no treatment, HS (titrated fluid and norepinephrine) increased mean arterial blood pressure (MAP, p ≤ 0.05) but not survival [0 of 10 (0/10) animals survived in each group] or median survival time [43.8 h (range 16.8 to 80.3) vs. 45.2 h (21.0 to 57.1)]. Compared to HS, HS with raxibacumab treatment at or 6 h after the beginning of edema toxin increased MAP and survival rate (6/7 and 7/8, respectively) and time [96.0 h (39.5 to 96.0) and 96.0 h (89.5 to 96.0), respectively]; (p ≤ 0.05). HS with raxibacumab at 12 h increased MAP (p ≤ 0.05) but not survival [1/5; 55.3 h (12.6 to 96.0)]. In Study-2, survival rate and time increased with HS and raxibacumab at 0 h (4/4) or 6 h after (3/3) beginning lethal and edema toxin compared to HS [0/5; 71.5 h (65 to 93)] (p = 0.01 averaged over raxibacumab groups). Conclusions Raxibacumab augments HS and improves survival during shock with lethal and edema toxin.
    No preview · Article · Feb 2015
  • Nolan K Maier · Stephen H Leppla · Mahtab Moayeri
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    ABSTRACT: Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of NF erythroid 2-related factor 2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1β release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome.
    No preview · Article · Feb 2015 · The Journal of Immunology
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    Full-text · Article · Jan 2015 · Toxicon
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    ABSTRACT: Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). Antibodies (Abs) against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only Abs (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well-characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from 'pre-pore' to its SDS and heat-resistant 'pore' conformation while not preventing cleavage of full length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface generated PA63 subunit but not pre-formed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin, and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Jan 2015 · Journal of Biological Chemistry
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    Shihui Liu · Mahtab Moayeri · Andrei P. Pomerantsev · Stephen H. Leppla

    Full-text · Chapter · Jan 2015
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    ABSTRACT: Unlabelled: Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Importance: Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain "sensor" proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.
    Full-text · Article · Dec 2014 · mBio
  • A. Bekdash · S. H. Liu · S. H. Leppla · A.E. Frankel · R. Abi-Habib

    No preview · Article · Nov 2014 · European Journal of Cancer
  • Zonglin Hu · Stephen H Leppla · Baoguang Li · Christopher A Elkins
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    ABSTRACT: Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies.
    No preview · Article · Jul 2014 · Expert Review of Molecular Diagnostics
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    ABSTRACT: We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; Mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA- activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32%-87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers.
    No preview · Article · Jun 2014 · Toxicology and Applied Pharmacology

Publication Stats

15k Citations
1,830.72 Total Impact Points

Institutions

  • 2003-2015
    • National Institute of Allergy and Infectious Disease
      베서스다, Maryland, United States
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Parasitic Diseases (LPD)
      베서스다, Maryland, United States
  • 1991-2015
    • National Institutes of Health
      • • Laboratory of Parasitic Diseases
      • • Branch of Oral and Pharyngeal Cancer
      • • Branch of Oral Infection and Immunity
      • • Laboratory of Molecular Microbiology
      • • Laboratory of Molecular Biology
      베서스다, Maryland, United States
  • 2012
    • Medical College of Wisconsin
      Milwaukee, Wisconsin, United States
  • 2009-2012
    • The Catholic University of America
      • Department of Biology
      Washington, Washington, D.C., United States
  • 2010
    • University of California, San Francisco
      • Department of Pharmaceutical Chemistry
      San Francisco, California, United States
    • The Ohio State University
      • Center for Microbial Interface Biology
      Columbus, Ohio, United States
  • 2007
    • University of Chicago
      • Ben May Department for Cancer Research
      Chicago, Illinois, United States
  • 2006-2007
    • Scott & White
      Temple, Texas, United States
    • University of Texas at Austin
      • Department of Biomedical Engineering
      Austin, Texas, United States
  • 2005-2007
    • Van Andel Research Institute
      Grand Rapids, Michigan, United States
  • 2004
    • Naval Medical Research Center
      Silver Spring, Maryland, United States
  • 2003-2004
    • Wake Forest University
      • School of Medicine
      Winston-Salem, North Carolina, United States
  • 1996-2003
    • Northern Inyo Hospital
      BIH, California, United States
  • 1997-2001
    • University of Leicester
      • Department of Biochemistry
      Leicester, ENG, United Kingdom
    • University of Michigan
      Ann Arbor, Michigan, United States
  • 1999
    • Emory University
      Atlanta, Georgia, United States
  • 1998
    • NCI-Frederick
      Фредерик, Maryland, United States
  • 1979-1994
    • U.S. Army Medical Research Institute of Infectious Diseases
      Фредерик, Maryland, United States
    • Brooke Army Medical Center
      Houston, Texas, United States
  • 1989
    • University of Virginia
      • Department of Pharmacology
      Charlottesville, Virginia, United States
  • 1986-1989
    • United States Army Medical Research Institute for Infectious Diseases
      Maryland, United States
  • 1984
    • University of Cincinnati
      • Department of Molecular Genetics, Biochemistry, and Microbiology
      Cincinnati, Ohio, United States