PLoS ONE: Anthrax = Toxins Inhibit Neutrophil Signaling Pathways in Brain Endothelium and = Contribute to the Pathogenesis of Meningitis

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Anthrax Toxins Inhibit = Neutrophil=20 Signaling Pathways in Brain Endothelium and Contribute to the = Pathogenesis of=20 Meningitis

Nina M. van Sorge²,=20 Celia M. Ebrahimi¹,=20 Shauna M. McGillivray²,=20 Darin Quach¹,=20 Mojgan Sabet³,=20 Donald G. Guiney³,=20 Kelly S. Doran¹^,²^*

1 Department of Biology and Center for = Microbial=20 Sciences, San Diego State University, San Diego, California, United = States of=20 America, 2 Department of = Pediatrics,=20 University of California San Diego, La Jolla, California, United States = of=20 America, 3 Department of = Medicine,=20 University of California San Diego, La Jolla, California, United States = of=20 America

Abstract
Background Anthrax=20 meningitis is the main neurological complication of systemic infection = with=20 Bacillus anthracis approaching 100% mortality. The presence of = bacilli in=20 brain autopsies indicates that vegetative bacteria are able to breach = the=20 blood-brain barrier (BBB). The BBB represents not only a physical = barrier but=20 has been shown to play an active role in initiating a specific innate = immune=20 response that recruits neutrophils to the site of infection. Currently, = the=20 basic pathogenic mechanisms by which B. anthracis penetrates the = BBB and=20 causes anthrax meningitis are poorly understood.
Methodology/Principal=20 Findings Using=20 an in vitro BBB model, we show for the first time that B.=20 anthracis efficiently invades human brain microvascular endothelial = cells=20 (hBMEC), the single cell layer that comprises the BBB. Furthermore,=20 transcriptional profiling of hBMEC during infection with B. = anthracis=20 revealed downregulation of 270 (87%) genes, specifically key neutrophil=20 chemoattractants IL-8, CXCL1 (Gro=CE=B1) and CXCL2 (Gro=CE=B2), thereby = strongly=20 contrasting hBMEC responses observed with other meningeal pathogens. = Further=20 studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA = and=20 in vivo assays indicated that anthrax toxins actively suppress = chemokine=20 production and neutrophil recruitment during infection, allowing = unrestricted=20 proliferation and dissemination of the bacteria. Finally, mice = challenged with=20 B. anthracis Sterne, but not the toxin-deficient strain, = developed=20 meningitis.
Conclusions/Significance These=20 results suggest a significant role for anthrax toxins in thwarting the = BBB=20 innate defense response promoting penetration of bacteria into the = central=20 nervous system. Furthermore, establishment of a mouse model for anthrax=20 meningitis will aid in our understanding of disease pathogenesis and = development=20 of more effective treatment strategies.

Citation: van Sorge NM, Ebrahimi CM, McGillivray SM, = Quach=20 D, Sabet M, et al. (2008) Anthrax Toxins Inhibit Neutrophil Signaling = Pathways=20 in Brain Endothelium and Contribute to the Pathogenesis of Meningitis. = PLoS ONE=20 3(8): e2964. doi:10.1371/journal.pone.0002964

Editor: Debbie Fox, The Research Institute for = Children at=20 Children's Hospital New Orleans, United States of America

Received: May 16, 2008; Accepted: = July 22,=20 2008; Published: August 13, 2008

Copyright: =C2=A9 2008 van Sorge et al. This is an = open-access=20 article distributed under the terms of the Creative Commons Attribution = License,=20 which permits unrestricted use, distribution, and reproduction in any = medium,=20 provided the original author and source are credited.

Funding: This work was supported by grant = 1U01AI077989 from=20 the NIH to D.G.G. and R01NS051247 from the NINDS/NIH to K.S.D.

Competing interests: The authors have declared that = no=20 competing interests exist.

* E-mail: kdoran@sciences.sdsu.edu
=

Introduction Bacillus=20 anthracis is a Gram-positive spore-forming bacterium that causes = anthrax in=20 humans and animals [1].=20 The recent threat of B. anthracis as a potential bioterrorism = agent has=20 sparked renewed interest into disease pathogenesis and treatment = strategies.=20 Infection occurs upon entry of bacterial spores through the skin,=20 gastrointestinal mucosa or the lung [2].=20 Spores, initially taken up by resident macrophages [3]=20 and dendritic cells [4],=20 germinate to vegetative bacteria during phagocyte migration to the = regional=20 lymph nodes. Vegetative bacteria are then released from the phagocytes, = enter=20 the bloodstream [2]=20 and proliferate in long chains at preferred sites like the brain, = allowing entry=20 into the central nervous system (CNS) and development of anthrax = meningitis. The=20 incidence of anthrax meningitis after cutaneous infection is = approximately 5% [5],=20 however in an outbreak of inhalational anthrax, approximately 50% of = patients=20 displayed signs of hemorrhagic meningitis [6].=20 Additionally, experimental studies of inhalational anthrax in monkeys=20 demonstrated meningitis in 77% of cases examined [7].=20 In general, anthrax meningitis is associated with a fulminant and = rapidly=20 progressive deteriorating course approaching 100% mortality despite = intensive=20 antibiotic therapy [5].
The=20 major virulence factors of B. anthracis are encoded on two native = plasmids, pXO1 and pXO2 [2].=20 The pXO1 plasmid contains the toxin-gene complex comprised of protective = antigen=20 (PA), lethal factor (LF) and edema factor (EF) [1],=20 [2].=20 These three toxin components combine to form two binary toxins, lethal = toxin=20 (LT), a zinc metalloprotease that cleaves mitogen activated protein = kinases [8],=20 and edema toxin (ET), an adenylate cyclase that increases intracellular = cyclic=20 AMP concentrations [9].=20 The pXO2 plasmid encodes genes involved in the production of the = polyglutamyl=20 capsule [1],=20 [2].=20 Fully virulent strains of B. anthracis contain both plasmids, = whereas the=20 unencapsulated Sterne strain (pXO1⁺, = pXO2^=E2=88=92) is used for=20 vaccination purposes [10].=20 In addition, the Sterne strain has been widely used in both in = vitro and=20 in vivo studies of anthrax infection as it causes lethal disease = similar=20 to the encapsulated B. anthracis strain in mice [11].=20 Currently however, no small animal model of anthrax meningitis exists = that could=20 facilitate our understanding of disease pathogenesis and the = contribution of=20 specific virulence factors to penetration of the CNS.
Several=20 studies have demonstrated the presence of numerous Gram-positive bacilli = in the=20 cerebrospinal fluid and brain [5],=20 [6],=20 suggesting that B. anthracis is capable of breaching the = blood-brain=20 barrier (BBB). The human BBB, which is comprised principally of a single = layer=20 of specialized brain microvascular endothelial cells (BMEC), serves as a = critical barrier to protect the CNS against microbial invasion. In = addition to=20 providing barrier function, the BBB has also been shown to play an = active role=20 in initiating a specific innate immune response promoting neutrophil = recruitment=20 [12],=20 the clinical and diagnostic hallmark of acute bacterial meningitis. This = response is thought to be effective in clearing bacteria from the = cerebral=20 microvasculature in the majority of BBB encounters with bacteria. We = hypothesize=20 that penetration of the BBB by B. anthracis likely involves = bacterial=20 invasion and transcytosis across brain endothelium, direct damage by = bacterial=20 toxins and/or activation of host inflammatory pathways that compromise = BBB=20 integrity. A comprehensive study of the BBB response to B. = anthracis=20 infection could therefore aid in our understanding of disease = pathogenesis.
In=20 this study, we examine for the first time the interaction of B. = anthracis=20 with the human BBB using a well established hBMEC model [13],=20 specific pXO1 and isogenic toxin mutants and a newly-developed mouse = model for=20 anthrax meningitis. Our study demonstrates that B. anthracis = penetrates=20 brain endothelium directly and that anthrax toxins contribute to this = process.=20 Additionally, anthrax toxins suppress the BBB neutrophil recruitment = response=20 promoting unchecked bacterial replication within the CNS and = establishment of=20 meningitis in a newly developed model of hematogeneous anthrax=20 meningitis.

Results B.=20 anthracis invades brain microvascular endothelial cells Analysis=20 of cerebral spinal fluid and brains from patients with anthrax = meningitis show=20 the presence of numerous Gram-positive bacilli [5],=20 [6],=20 indicating that B. anthracis is able breach the BBB. We = hypothesized that=20 B. anthracis, like other meningeal pathogens [14],=20 [15],=20 is able to invade human BMEC (hBMEC), a single-cell layer that comprises = the=20 BBB. We therefore examined B. anthracis Sterne interactions with = hBMEC=20 using transmission electron microscopy (TEM). After a 1 hour infection = period,=20 bacteria were observed in close association with the cell membrane (Fig.=20 1A) and in close proximity to cell surface microvillus = projections (Fig.=20 1B). After further exposure numerous bacteria were found either = entering=20 hBMEC or in membrane-bound intracellular vacuoles (Fig.=20 1C=E2=80=93F).

Figure=20 1. doi:10.1371/journal.pone.0002964.g001
To=20 quantify the number of adherent and invasive organisms, we optimized our = previously established quantitative hBMEC adherence and invasion assays = [12],=20 [16]=20 for B. anthracis. HBMEC were grown to confluency and infected = with=20 increasing concentrations (multiplicities of infection, MOI) of B.=20 anthracis Sterne (MOI 1 represents approximately = 1=C3=9710⁵ CFU); data=20 are expressed as the recovered total cell-associated or intracellular = colony=20 forming units (CFU). The number of adherent bacteria steadily increased = with=20 increasing MOI (Fig.=20 2A) and ranged from 25=E2=80=9340% of the initial inoculum. = Correspondingly,=20 more intracellular organisms were recovered after infection with a = higher MOI=20 and longer incubation time (Fig.=20 2B), representing between 2=E2=80=9310% of the initial = inoculum.

Figure=20 2. doi:10.1371/journal.pone.0002964.g002
As=20 the brain endothelium cells are responsible for maintaining biochemical=20 homeostasis within the central nervous system (CNS), entry of molecules = into the=20 CNS is a strictly regulated process [17],=20 [18].=20 However, to further demonstrate that the interaction of B. = anthracis=20 Sterne is not just due to random uptake, we incubated hBMEC with two = related=20 non-pathogenic Bacillus species, B. thuringensis and B. = subtilis.=20 Both of these strains were unable to invade hBMEC (Fig.=20 2C), demonstrating that the invasive ability is specific to = B.=20 anthracis Sterne.
The=20 EM studies suggested that B. anthracis Sterne alters the host=20 cytoskeleton to initiate its own uptake (Fig.=20 1). To confirm this observation experimentally, invasion = experiments=20 were performed in the presence of cytochalasin D, a potent inhibitor of=20 cytoskeletal rearrangements. This inhibitor has been shown previously to = effectively block invasion of hBMEC by other bacterial pathogens [16].=20 As shown in Fig.=20 2D, addition of cytochalasin D resulted in a dose-dependent = inhibition=20 of B. anthracis Sterne invasion into hBMEC. Together these = results=20 suggest that B. anthracis Sterne modulates the host cytoskeleton = to=20 induce it own uptake.
Expression=20 profile of hBMEC following B. anthracis Sterne infection Understanding=20 changes in gene expression that occur in response to B. anthracis = infection will facilitate further analysis of anthrax meningitis = pathogenesis.=20 We performed microarray analysis to assess the overall response of hBMEC = to=20 infection with B. anthracis Sterne. After 6 hours of infection, = 304 genes=20 exhibited a more than two-fold change in transcript abundance (Table=20 S1A, Fig=20 3C). Figure=20 3A depicts the mean differences in total gene expression in=20 Sterne-infected hBMEC cells compared to uninfected cells; down- and = upregulated=20 genes in response to B. anthracis Sterne are represented by blue = and red=20 dots, respectively. Interestingly, the majority of affected genes (270 = out-of=20 304, 87%) were downregulated in response to B. anthracis Sterne = infection=20 compared to the uninfected control (Fig=20 3A, C). However, transcription was not globally impaired as 34 = genes=20 showed a more than two fold increased expression upon infection with = B.=20 anthracis Sterne (Fig.=20 3A, C, Table=20 S1A).

Figure=20 3. doi:10.1371/journal.pone.0002964.g003
Differential=20 gene expression profile induced by B. anthracis lacking the pXO1 = plasmid Anthrax=20 toxins are considered to be the major virulence factors in B. = anthracis=20 infection. We therefore assessed the contribution of the pXO1 plasmid, = encoding=20 anthrax toxins, to the hBMEC transcriptional response. In parallel = microarray=20 studies, the B. anthracis Sterne =CE=94pXO1 strain affected 121 = hBMEC genes by=20 more than two-fold after 6 hours of infection compared to the uninfected = control=20 (Fig.=20 3C, Table=20 S1B). In contrast to infection with the Sterne bacteria, only 38 = out of=20 121 (31%) genes were downregulated upon infection with =CE=94pXO1 = bacteria (Fig.=20 3B, C). The microarray data also show that mRNA levels for = various=20 housekeeping genes like =CE=B2-actin and GAPDH were similar for samples = infected with=20 Sterne or the =CE=94pXO1 mutant (data not shown). To visualize how the = down- and=20 upregulated genes of Sterne-infected cells (colored blue and red, = respectively,=20 Fig.=20 3A) were affected in =CE=94pXO1-infected cells, the same genes were = followed in=20 the MA plot of =CE=94pXO1-infected hBMEC cells compared to media control = (Fig.=20 3B); 90% of genes were differentially affected in = response to=20 bacteria that lacked pXO1 (Fig.=20 3B). Also 40 additional genes were more than two fold induced in = the=20 absence of the pXO1 plasmid. Overall, this indicates that the hBMEC=20 transcriptional response is strongly influenced by the presence of the = pXO1=20 plasmid, whereas only 10% of the genes are regulated in a = pXO1-independent=20 manner (Fig.=20 3A=E2=80=93C).
Since=20 gene expression was strongly influenced by the pXO1 plasmid, we next = sought to=20 identify the most differentially regulated genes in response to = infection with=20 Bacillus Sterne or =CE=94pXO1 mutant strains. Figure=20 3D displays mean differences in gene expression comparing = =CE=94pXO1 versus=20 B. anthracis Sterne infected hBMEC; more than two fold = differentially=20 down- and upregulated genes are colored blue and red, respectively. The = identity=20 of these genes and their expression in hBMEC compared to media are = displayed in=20 Figure=20 4. Analysis of differentially expressed genes by gene ontology = (GO) of=20 molecular function indicated significant overrepresentation of genes = involved=20 with transcription (p =3D 1.3=C3=9710^=E2=88=9212), = growth (p =3D=20 7.9=C3=9710^=E2=88=926), RNA binding (p =3D = 1.7=C3=9710^=E2=88=925), protein=20 kinase inhibition (p =3D 2.8=C3=9710^=E2=88=925), and = cytokine activity=20 (p =3D 6.7=C3=9710^=E2=88=925). Of particular interest = however, was the=20 observation that some of the most strongly differentially affected genes = were=20 potent neutrophil chemoattractants IL-8 (CXCL8), CXCL1 (Gro=CE=B1) and = CXCL2 (Gro=CE=B2),=20 since we have previously shown that neutrophil recruitment is a major = part of=20 the innate host defense response against bacterial infection [12].=20 This was supported by GO analysis showing significant downregulation of = genes=20 with chemokine activity (p =3D 8.1=C3=9710^=E2=88=924) = and correspondingly=20 genes involved in induction of positive chemotaxis (p =3D=20 4.2=C3=9710^=E2=88=925) and leukocyte activation (p =3D = 9.1=C3=9710^=E2=88=924).=20 These microarray data suggest that factors on the pXO1 plasmid may = interfere=20 with the BBB innate immune defense, specifically neutrophil = recruitment.

Figure=20 4. Material=20 and Methods.
doi:10.1371/journal.pone.0002964.g004
Confirmation=20 of microarray expression data: quantitative RT-PCR and ELISA To=20 confirm our microarray results we used quantitative RT-PCR to analyze = the=20 relative transcript abundance in hBMEC of the following genes involved = in the=20 host immune response: IL-6, IL-8, CXCL1, CXCL2, and CCL20. Figure=20 5A depicts the relative fold change in hBMEC transcript levels = upon=20 infection with B. anthracis Sterne or =CE=94pXO1 mutant bacteria = compared to=20 the uninfected control. As was observed in our microarray studies, the=20 transcript levels of IL-8, CXCL1 and CXCL2 were significantly = downregulated in=20 cells infected with B. anthracis Sterne compared to uninfected = control or=20 hBMEC infected with the =CE=94pXO1 strain (Fig.=20 5A). In contrast, IL-6 and CCL20 transcripts were not = downregulated in=20 response to Sterne infection. This response was not significantly = different upon=20 infection with =CE=94pXO1 mutant bacteria, indicating that gene = regulation for these=20 genes is independent of the presence of the plasmid.

Figure=20 5. Material=20 and Methods. Data represent mean and standard deviation of three = independent=20 experiments performed in triplicate. (B) Protein expression of IL-6, = IL-8,=20 CXCL1, CXCL2 and CCL20 in hBMEC supernatants 6 h post infection with = B.=20 anthracis Sterne (B.a.) or =CE=94pXO1 bacteria using ELISA. = Experiments were=20 performed three times in triplicate. Bars represent mean and standard = deviation=20 of one representative experiment. * p<0.05, ** = p<0.005, ***=20 p<0.001.
doi:10.1371/journal.pone.0002964.g005
Effects=20 on gene transcription are not always paralleled by changes in protein = expression=20 [19].=20 Therefore, we analyzed hBMEC supernatants for the presence of IL-6, = IL-8, CXCL1,=20 CXCL2, and CCL20 protein 6 hours after B. anthracis Sterne = infection.=20 Induction of chemokines IL-8, CXCL1 and CXCL2 was markedly reduced when = cells=20 were infected with the Sterne bacteria compared to uninfected controls = (Fig.=20 5B). In contrast, IL-6 and CCL20 protein levels were unaffected = and=20 induced, respectively. Infection of hBMEC with the =CE=94pXO1 strain = restored=20 secretion of IL-8 and CXCL1 to levels secreted by uninfected cells, = while IL-6,=20 CXCL2, and CCL20 protein expression levels did not differ in the absence = of the=20 pXO1 plasmid compared to B. anthracis Sterne-infected hBMEC, = suggesting=20 that additional chromosomal factors may influence protein expression (Fig.=20 5B). Overall, these independent experiments generally confirmed = our=20 observations from the microarray experiment and suggest a role for = pXO1-encoded=20 factors in the downregulation of neutrophil chemokines in hBMEC.
Anthrax=20 toxins inhibit expression of IL-8 and suppress neutrophil recruitment in = vivo To=20 establish whether anthrax toxins were responsible for the downregulation = of=20 IL-8, the most potent and strongly affected neutrophil chemokine, we = utilized=20 isogenic mutants that specifically lacked LF (=CE=94LF), EF (=CE=94EF) = or both anthrax=20 toxins (=CE=94LF/EF). Infection of hBMEC with the =CE=94LF/EF bacterial = strain resulted in=20 a significant induction of IL-8 gene transcription (Fig.=20 6A) and restoration of IL-8 protein secretion (Fig.=20 6B) compared to B. anthracis Sterne infected cells. The = presence=20 of either LF or EF was still sufficient to suppress IL-8 transcript and = protein=20 expression (Fig.=20 6A, B), suggesting that both toxins are involved in = downregulation of=20 this neutrophil chemokine.

Figure=20 6. doi:10.1371/journal.pone.0002964.g006
As=20 the anthrax toxins decreased neutrophil chemokine transcription and = expression,=20 we hypothesized that neutrophil recruitment might be suppressed after = infection=20 with B. anthracis Sterne compared to the =CE=94LF/EF mutant = strain. To examine=20 the effects of anthrax toxins on neutrophil chemotaxis in vivo, = we=20 analyzed neutrophil recruitment to the site of infection using two = independent=20 assays. First, neutrophil recruitment was assessed upon subcutaneous = injection=20 of B. anthracis Sterne or the =CE=94LF/EF mutant strain into the = right or left=20 flank of mice, respectively, After 4 hours, mice were euthanized and the = site of=20 subcutaneous injection was excised, homogenized and analyzed for the = neutrophil=20 enzyme myeloperoxidase (MPO), which serves as an effective indicator of=20 neutrophil infiltration [20].=20 MPO levels and therefore accumulating neutrophils were significantly = lower upon=20 infection with B. anthracis Sterne compared to the =CE=94LF/EF = mutant strain=20 (Fig.=20 6C). Using a second independent measurement, we quantified the = amount of=20 neutrophils entering the peritoneal cavity upon i.p. injection of B.=20 anthracis Sterne or =CE=94LF/EF mutant bacteria. PBS and a 3% = thioglycolate=20 solution were included as negative and positive controls, respectively. = After 4=20 hours, cells were extracted from the peritoneal cavity and the amount of = accumulated neutrophils was quantified by flow cytometry. Although = neutrophils=20 were recruited upon infection by B. anthracis Sterne compared to = the PBS=20 control, neutrophil accumulation was significantly reduced compared to = the=20 toxin-deficient isogenic mutant (Fig.=20 6D). In general, neutrophil accumulation by the =CE=94LF/EF = mutant was=20 comparable to the positive 3% thioglycolate control (Fig.=20 6D). Overall, these results suggest that B. anthracis = anthrax=20 toxins interfere with transcription and secretion of neutrophil = chemokines, as=20 well as neutrophil recruitment during active infection.
Anthrax=20 meningitis mouse model Our=20 data suggest that B. anthracis is capable of penetrating the BBB. = In=20 addition, anthrax toxins suppress the brain endothelial host response = which=20 could promote unrestricted proliferation and further dissemination of = B.=20 anthracis in the CNS. To test the contribution of anthrax toxins to = the=20 pathogenesis of CNS infection, we developed a mouse model of anthrax = meningitis.=20 Mice were injected intravenously with B. anthracis Sterne or = =CE=94LF/EF=20 bacteria (n =3D 12 per group). Mice were euthanized when they became = moribund with=20 severely labored breathing (between days two and twelve for B. = anthracis=20 Sterne-infected mice) after which brain and blood were collected. All of = the=20 =CE=94LF/EF-infected mice and approximately 10% of the Sterne-infected = mice survived=20 until the experimental endpoint of three weeks (Fig.=20 7A). Five out-of eight Sterne-infected mice (63%) had high = bacterial=20 counts in the brain (Fig.=20 7B), while no bacteria were recovered from the brains of = =CE=94LF/EF-infected=20 mice (Fig.=20 7B). Microscopic examination of brain tissue from mice infected = with the=20 Sterne strain showed thickening of the meninges, an influx of = inflammatory cells=20 and substantial hemorrhaging (Fig.=20 7D=E2=80=93F). In addition, Gram stain revealed the presence of = numerous bacilli=20 in both the meninges and the parenchyma (Fig.=20 7G, H). The brains of mice that were infected with the = =CE=94LF/EF strain did=20 not show any signs of infection over the course of the experiment and = exhibited=20 normal brain architecture (Fig.=20 7C).

Figure=20 7. doi:10.1371/journal.pone.0002964.g007
The=20 absence of clinical symptoms in =CE=94LF/EF-infected mice could = partially be due to=20 reduced virulence of this strain in vivo [21].=20 Therefore, we performed additional in vitro experiments to assess = whether=20 anthrax toxins contribute directly to the penetration of brain = endothelium.=20 Compared to the parent strain, the toxin deficient strain exhibited a = 70=E2=80=9380%=20 reduction in hBMEC invasion (Fig.=20 7I) and was less able to penetrate hBMEC monolayers in a = transmigration=20 assay (Fig.=20 7J). Together these data indicate that B. anthracis = Sterne is=20 indeed capable of crossing the BBB in vivo, establishing the = classic=20 signs of meningitis and meningoencephalitis, and that the expression of = anthrax=20 toxins may directly contribute to this process.

Discussion Infection=20 with B. anthracis resulting in systemic disease is associated = with high=20 morality characterized by septicemia, toxemia, and meningitis [6],=20 [22]=E2=80=93[24].=20 The presence of bacilli in brain autopsies indicates that vegetative = bacteria=20 are able to disseminate from the bloodstream to the CNS, however, the = basic=20 pathogenic mechanisms by which B. anthracis penetrates the BBB = have not=20 been described. Using electron microscopy and an established in = vitro=20 model of the BBB, we demonstrate here for the first time that B.=20 anthracis is capable of invading hBMEC, the single cell layer that = comprises=20 the BBB. Our observations extend recent studies reporting invasion of = B.=20 anthracis into non-phagocytic fibroblasts and epithelial cell lines = [25].=20 Furthermore, our results suggest that uptake of B. anthracis = Sterne in=20 hBMEC is specific and requires actin cytoskeleton rearrangements. = Interestingly,=20 a very recent report identified a pXO1-encoded adhesin, BslA important = for=20 adherence to keratinocytes and lung epithelial cells [26].=20 Studies to identify and characterize additional factors involved in = hBMEC=20 adherence and invasion, including the BslA adhesin, are in progress.
We=20 have used microarray analysis to examine the acute response of brain = endothelium=20 to infection with vegetative B. anthracis Sterne. We have shown=20 previously that the BBB plays an active role in initiating a very = specific=20 innate immune response to bacterial infection by inducing gene = expression of=20 factors promoting neutrophil recruitment [12].=20 Most strikingly, B. anthracis infection reduced steady-state = expression=20 of 270 genes by more than two-fold corresponding to 87% of all affected = gene=20 transcripts. This contrasts typical host cellular responses to microbial = pathogens where the number of host genes induced by infection is = significantly=20 higher than the number of down-regulated genes [12],=20 [27].=20 The majority of downregulated genes were related to transcription, = signal=20 transduction, stress, host immune response, and proliferation. As = anthrax toxins=20 are the major secreted B. anthracis virulence factors, we also = analyzed=20 the gene expression profile of hBMEC upon infection with a strain = lacking the=20 pXO1 plasmid, =CE=94pXO1, which encodes both anthrax toxins. Ninety = percent of=20 affected genes upon B. anthracis Sterne infection were = differentially=20 affected upon infection with =CE=94pXO1 bacteria, and in total only 31% = of genes in=20 =CE=94pXO1-infected cells were downregulated. Additionally, = approximately 10% of=20 genes were regulated independently of pXO1, suggesting possible = involvement of=20 B. anthracis chromosomal factors to host response. Overall, these = results=20 suggest a major role for plasmid encoded factors and toxins in = regulating the=20 brain endothelial host response.
Of=20 particular interest was the unambiguous effect on the expression levels = of genes=20 belonging to the CXC chemokine family, particularly the neutrophil = chemotactic=20 factors IL-8, CXCL1 and CXCL2 in response B. anthracis Sterne = infection.=20 Notably the expression levels of other major pro-inflammatory mediators = such as=20 TNF=CE=B1 and IL-1 were not affected by B. anthracis Sterne or = =CE=94pXO1=20 infection. Neutrophil recruitment is thought to be part of the very = first line=20 of CNS defense against bacterial infection [12]=20 as many Gram-positive and Gram-negative meningeal pathogens induce = expression of=20 these genes in hBMEC [12],=20 [28],=20 van Sorge et al. unpublished data). Active impairment of neutrophil = recruitment=20 could therefore benefit survival and proliferation of B. = anthracis, as=20 both spores and vegetative bacteria are efficiently killed by human = neutrophils=20 [29].=20 Our results clearly demonstrate that the suppression of CXCL1 and IL-8=20 expression is pXO1- and toxin-dependent, respectively. These data = complement=20 observations in recent studies where systemic infection with the = encapsulated=20 strain impaired production of cytokines in a toxin-dependent manner [30]=20 and purified LT reduced IL-8 production by the destabilization of IL-8 = mRNA in=20 HUVEC in vitro [31].
We=20 hypothesized that altered chemokine expression would result in impaired=20 neutrophil recruitment upon active infection with B. anthracis = Sterne.=20 Using two independent in vivo assays, we demonstrated that = neutrophil=20 chemotaxis was indeed reduced to the site of infection with the Sterne = strain as=20 compared to infection with the =CE=94LF/EF mutant. Similar observations = were recently=20 published in a systemic infection model using encapsulated WT B.=20 anthracis (pXO1⁺, pXO2⁺); host neutrophil = recruitment=20 in spleen and liver was significantly increased in the absence anthrax = toxins=20 compared to infection with the parent strain [32].=20 In addition, purified LT has been shown to directly impair neutrophil = motility=20 [33],=20 [34].=20 Toxin-mediated subversion of the innate immune system, specifically = targeting=20 neutrophils, may therefore contribute to unchecked bacterial replication = and a=20 more fulminent disease course.
Establishment=20 of an anthrax meningitis model is critical to better understand disease=20 pathogenesis. The current rabbit and rhesus monkey models of inhalation = anthrax=20 [7],=20 [35]=20 both report signs of meningitis in a subgroup of animals, however, a = mouse model=20 would be preferable due to availability, lower costs and = well-characterized=20 genetic systems. We found that intravenous injection of immunocompetent = outbred=20 CD-1 mice with B. anthracis Sterne resulted in penetration of = bacilli=20 into the CNS. Microscopic analysis of brain sections confirmed the = development=20 of meningitis, showing inflammatory cell infiltration, hemorrhaging, = thrombosis,=20 edema and areas full of bacilli. While we did observe neutrophil = infiltration in=20 the brains of B. anthracis Sterne infected mice at the time of = death, we=20 speculate that an initial reduction or delay in host neutrophilic = response may=20 promote acute unrestricted bacterial proliferation and further CNS = dissemination=20 ultimately responsible for the rapidly progressive deteriorating course=20 associated with anthrax meningitis. These observations reflect autopsy = findings=20 in patients [5]=20 validating the utility of this newly developed mouse model of = hematogenous=20 anthrax meningitis. Finally, development of anthrax meningitis requires=20 expression of anthrax toxins as no signs of disease developed in mice = infected=20 with the =CE=94LF/EF mutant strain. Additional in vitro studies = suggested that=20 this could be due to a direct contribution of the toxins to penetration = of brain=20 endothelium; however, we cannot exclude the possibility that the lack of = clinical symptoms observed during infection with the toxin-deficient = mutant may=20 partially reflect a generalized reduction in virulence.
In=20 summary, our studies provide the first evidence that B. anthracis = is=20 capable of invading the human BBB. We have also demonstrated that = diverse=20 functional classes of genes, including chemokines involved in neutrophil = recruitment and signaling, were downregulated in brain endothelium upon = B.=20 anthracis infection suggesting that the pathogen actively suppresses = the BBB=20 innate immune response. This signaling appears to be mediated largely by = the=20 bacterial pXO1-encoded toxins. Our in vivo studies indicate that = the=20 anthrax toxins contribute to impaired neutrophil recruitment and the = development=20 of anthrax meningitis. Additional studies aimed at further understanding = the=20 mechanisms governing the pathogenesis of anthrax meningitis should aid = in the=20 development of preventative therapies for this serious CNS = infection.

Materials=20 and Methods Bacterial=20 strains and endothelial cell culture Bacillus=20 anthracis Sterne (pXO1⁺, pXO2^=E2=88=92) and = mutant derivatives=20 were grown in Brain-Heart infusion broth (BHI; Sigma) as shaking = cultures under=20 aerobic conditions at 37=C2=B0C. B. anthracis Sterne was cured of = the pXO1=20 plasmid by passage at 43=C2=B0C. Specific LF, EF and LF/EF deletion = mutants were=20 generously provided by Scott Stibitz (Center for Biologics Evaluation = and=20 Research, Bethesda, Maryland) and described previously [36].=20 For log-phase cultures of B. anthracis, fresh BHI was inoculated = with the=20 overnight culture at a 1:20 dilution and grown to OD₆₀₀ =3D = 0.4=20 (1=C3=9710⁷ CFU/ml). Growth kinetics of all strains was = similar under the=20 experimental conditions used in our assays.
The=20 human brain microvascular endothelial cell line hBMEC, obtained from = Kwang Sik=20 Kim (Johns Hopkins University, Baltimore, Maryland, USA), were = originally=20 isolated as previously described [13],=20 [37],=20 and maintain the morphologic and functional characteristics of primary = brain=20 endothelium [13],=20 [15].=20 HBMEC were cultured using RPMI 1640 (Gibco), supplemented with 10% fetal = calf=20 serum (FBS; Gibco), 10% Nuserum (BD Biosciences, San Jose, California, = USA), and=20 modified Eagle's medium nonessential amino acids (Gibco) without = addition of=20 antibiotics. All experiments used cells at passage 8=E2=80=9314.
HBMEC=20 infection and transmigration assays For=20 hBMEC invasion assays, cells were seeded in collagen-coated 24 well = tissue=20 culture plates until they reached 90=E2=80=93100% confluency. B. = anthracis=20 cultures were grown to log-phase as described above. Log-phase bacteria = were=20 pelleted, washed in PBS and resuspended in RPMI 1640 10% FBS to the = appropriate=20 concentration. HBMEC monolayers, washed twice with PBS before the = addition of=20 bacterial cultures, were infected with different multiplicity of = infection (MOI;=20 MOI of 1 is approximately 1=C3=9710⁵ CFU) in a final volume = of 500 =C2=B5l of=20 RPMI 10% FBS. Plates were centrifuged at 800=C3=97 g for 5 min to = synchronize=20 the infection, and subsequently incubated at 37=C2=B0C with 5% = CO₂. After=20 2=E2=80=934 h, monolayers were washed three times with PBS before the = addition of 1 ml=20 of RPMI 10% FBS containing 50 =C2=B5g of gentamicin for 15 min to kill = extracellular=20 bacteria. Control experiments confirmed that B. anthracis was = killed by=20 this concentration of gentamicin within 15 minutes (data not shown). The = monolayers were washed three times with PBS before the addition of 0.1 = ml of=20 0.25% trypsin/EDTA solution (5 min 37=C2=B0C) followed by 0.4 ml of = 0.025% Triton=20 X-100 to liberate intracellular bacteria. The number of invasive = bacteria was=20 quantified by plating serial dilutions of the lysate on THB or BHI agar = plates.=20 To assess the effect of host cytoskeleton on B. anthracis = invasion, hBMEC=20 cells were incubated for 30 min with the indicated concentration of = cytochalasin=20 D (Sigma) before addition of bacteria. To assess the level of = surface-adherent=20 (total cell-associated) bacteria, bacteria were quantified from hBMEC = monolayers=20 prior to addition of extracellular antibiotics after 45 min of = incubation as=20 described above only washing six times with PBS prior to bacterial = enumeration.=20 All cellular adherence and invasion assays were performed at least in = triplicate=20 and repeated at least three times.
For=20 transmigration assays, polar hBMEC monolayers were established on=20 collagen-coated Transwell plates, 3 =C2=B5m pore size (Transwell-COL; = Corning-Costar=20 Corp., MA, USA) as described previously [16].=20 Monolayers were incubated with 2=C3=9710⁵ CFU of log-phase = grown bacteria.=20 After 4 hours, the number of bacteria in the lower chamber was = quantified by=20 serial dilution plating on THA plates. The experiment was performed at = least=20 three times in triplicate.
Transmission=20 electron microscopy Infection=20 experiments were performed similar to the adherence assay described = above with=20 B. anthracis Sterne for 1 hour or 4 hours. After washing, samples = were=20 immersed in modified Karnovsky's fixative (1.5% glutaraldehyde, 3%=20 paraformaldehyde and 5% sucrose in 0.1 M cacodylate buffer, pH 7.4) for = at least=20 8 hours, post fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer = for 1 hour=20 and stained en loc in 1% uranyl acetate for 1 hour. Samples were = dehydrated in=20 ethanol, embedded in epoxy resin, sectioned at 60 to 70 nm, and picked = up on=20 carbon-coated formvar grids. Grids were stained with uranyl acetate and = lead=20 nitrate, viewed using a JEOL 1200EX II (JEOL, Peabody, MA) or Philips = CM-10=20 (FEI, Hilsboro, OR) transmission electron microscope and photographed = using a=20 Gatan digital camera (Gatan, Peabody, CA).
Microarray=20 analysis Microarray=20 experiments were performed using Sentrix Human-8 Expression BeadChips, = which=20 analyzed 25,440 transcripts (Illumina, San Diego, CA) according to=20 manufacturer's instructions. In brief, a 250 ng aliquote of total RNA, = isolated=20 as described above, from each sample was amplified to cDNA, transcribed = to cRNA=20 and biotin labelled using Ambion's TotalPrep kit (Austin, TX), according = to the=20 instructions. cRNA concentrations were checked with the Agilent = Bioanalyzer, and=20 cRNA quality was controlled by BioRad's Experion Automated = Electrophoresis=20 System and RNA Std Sens Analysis Kit (BioRad Laboratories, Hercules, = CA). Each=20 sample cRNA (750 ng) was hybridized to Illumina's Sentrix Human-8 = Expression=20 BeadChip arrays at 58=C2=B0C overnight (18 h, shaking) following the = Illumina=20 Whole-Genome Gene Expression Protocol for BeadStation. Hybridized = biotinylated=20 cRNA was detected with 1 mg/ml streptavidin-Cy3 (Amersham Biosciences,=20 Piscataway, NJ). BeadChips were scanned with Illumina BeadArray Reader. = Data was=20 analyzed using a statistical algorithm developed for high-density=20 oligonucleotide arrays [38].
RNA=20 isolation, cDNA preparation and qPCR HBMEC=20 monolayers were infected with B. anthracis Sterne, or isogenic = mutants=20 (=CE=94pXO1, =CE=94LF, =CE=94EF, or =CE=94LF/EF) for 6 hour. Total RNA = was extracted using the=20 RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's = instruction,=20 and 1 =C2=B5g of RNA reverse transcribed to cDNA (Superscript = First-strand synthesis=20 kit, Invitrogen). Quantitative PCR (qPCR) was performed using the = following=20 primer sets: IL-6 forward primer 5=E2=80=B2- GGA GAC = TTG CCT GGT GAA AA=20 -3=E2=80=B2 and IL-6 reverse primer 5=E2=80=B2- CAG GGG TGG TTA TTG CAT=20 CT -3=E2=80=B2, IL-8 forward primer 5=E2=80=B2- AGC TCT GTG TGA AGG TGC=20 AG - 3=E2=80=B2 and IL-8 reverse primer 5=E2=80=B2- AAT TTC TGT GTT GGC=20 GCA GT - 3=E2=80=B2, CXCL1 forward primer 5=E2=80=B2 - CTC TTC CGC TCC=20 TCT CAC AG - 3=E2=80=B2, and CXCL1 reverse primer 5=E2=80=B2 - GGG GAC=20 TTC ACG TTC ACA CT -3, CXCL2 forward primer 5=E2=80=B2- CTC AAG=20 AAT GGG CAG AAA GC -3=E2=80=B2, and CXCL2 reverse primer 5=E2=80=B2- AAA=20 CAC ATT AGG CGC AAT CC -3=E2=80=B2, CCL20 forward primer 5=E2=80=B2- GCG=20 CAA ATC CAA AAC AGA CT -3=E2=80=B2 and CCL20 reverse primer 5=E2=80=B2-=20 CAA GTC CAG TGA GGC ACA AA -3=E2=80=B2, and GAPDH forward primer = 5=E2=80=B2- GAA GGT GAA GGT CGG AGT CAA CG = -3=E2=80=B2 and GAPDH reverse=20 primer5=E2=80=B2- TCC TGG AAG ATG GTG ATG GGA T = -3=E2=80=B2. PCR=20 reaction mixtures contained primers at a concentration 10 =C2=B5M and = PCR mix (SYBR=20 GreenER qPCR Supermix for iCycler, Invitrogen) in a volume of 25 = =C2=B5l. qPCR=20 cycling was as follows for all genes: 50=C2=B0C for 2 min, 95=C2=B0C for = 7 min, followed=20 by 40 cycles of 95=C2=B0C for 15=E2=80=B3 and 61=C2=B0C for 1 min. = Melting curve analysis was=20 performed according to the manufacturer's instructions; PCR primer = efficiencies=20 were as follows: 1.92 for IL-6, 1.8 for IL-8, 1.83 for CXCL1, 1.99 for = CXCL2,=20 1.94 for CCL20 and 1.88 for GAPDH. Calculation of relative gene = expression=20 included adjustments for PCR efficiencies and using the following = equation:=20 Relative gene expression =3D target gene efficiency=C3=97(C_T = control -=20 C_T sample)/1.88=C3=97(C_T control - C_T = sample).
Chemokine=20 secretion in hBMEC supernatants HBMEC=20 supernatants were collected after infection with B. anthracis = Sterne,=20 =CE=94pXO1, =CE=94LF, =CE=94EF, or =CE=94LF/EF deletion mutants after 6 = hours. Concentrations of=20 IL-8 (R&D systems, Minneapolis, MN, USA), CXCL1 (R&D systems), = CXCL2=20 (BioSupplyUK) and CCL20 (R&D systems) were measured using = enzyme-linked=20 immunosorbent assays (ELISA) according to the manufacturer's = instructions. IL-6=20 and IL-8 concentrations were measured using the cytometric bead array = system=20 according to the manufacturer's instructions (BD Biosciences, Human = inflammation=20 kit).
Mouse=20 infection studies All=20 animal experiments were approved by the Committee on the Use and Care of = Animals, and performed using accepted veterinary standards. For the = meningitis=20 model, bacteria were grown to early log phase, washed in PBS and = resuspended to=20 an optical density of 0.4 in PBS. Vegetative bacteria were diluted in = PBS to=20 2=E2=80=933=C3=9710⁵ CFU/ml and 0.1 ml was injected = intravenously into 8 weeks old=20 out bred immunocompetent female CD-1 mice (Charles River Laboratories,=20 Wilmington, MA, USA). Mice were monitored for signs of infection at = least twice=20 a day for up to three weeks and euthanized when they became moribund. = Blood and=20 brain were collected and plated to determine bacterial counts. Half of = the brain=20 was stored in 10% formalin for further histology analysis performed at = the UCSD=20 Histopathology Core Facility (N. Varki, Director).
To=20 determine neutrophil recruitment in vivo, B. anthracis = Sterne and=20 =CE=94LF/EF mutant bacteria were grown to early log phase, washed and = resuspended in=20 PBS to and OD₆₀₀ =3D 0.4. Eight week old CD-1 female mice = were injected=20 with 1=C3=9710⁶ CFU of B. anthracis Sterne on the = right shaved=20 flank and with 1=C3=9710⁶ CFU of =CE=94LF/EF mutant bacteria = on the left shaved=20 flank in a volume of 0.1 ml. After 4 hours, mice were euthanized and the = site of=20 subcutaneous injection was excised for further analysis of = myeloperoxidase=20 activity (see below). Neutrophil recruitment was also assessed using an=20 intraperitoneal infection model. Eight week old CD-1 female mice were = injected=20 i.p. with 2=C3=9710⁶ CFU in 200 =C2=B5l PBS. PBS alone and a = 3% thioglycolate=20 solution were used as negative and positive control for neutrophil = recruitment,=20 respectively. After 4 hours, cells were harvested from the peritoneal = cavity in=20 PBS 0.2% BSA. One-hundred =C2=B5l of cell suspension was directly = stained with=20 FITC-labeled rat anti-mouse Gr-1 monoclonal antibody or the appropriate = isotype=20 control (both BD Pharmingen) for 30 min at 4=C2=B0C and analyzed by flow = cytometry.=20 The flow cytometer was set to count events during a fixed time (60 s) = thus=20 permitting quantification of the absolute number of recovered Gr-1 = positive=20 cells in each mouse [39].=20 A quality check was performed on the flow cytometer (Dual Laser = FACSCalibur Flow=20 Cytometer) before use to assure a constant flow rate.
Myeloperoxidase=20 assay Skin=20 samples of mice were homogenized in 500 =C2=B5l 0.05% = hexadecyltrimethylammonium=20 bromide (HTAB in 0.05 M phosphate buffer, pH 6; Sigma) solution. = Homogenates=20 were centrifuged for at 18,000=C3=97 g for 30 min at 4=C2=B0C. = Supernatants were=20 transferred to a clean microcentrifuge tube and stored at = =E2=88=9280=C2=B0C until further=20 analysis. Next, 10 mg of o-dianisidine dihydrochloride (DCC; = Sigma) was=20 added to 60 ml of freshly-prepared HTAB solution to yield DCC solution. = In=20 addition, activated substrate was prepared by adding one =C2=B5l of = 0.05% hydrogen=20 peroxide solution for every 99 =C2=B5l of DCC solution. Finally, the = reaction was=20 started by adding 90 ul of DCC solution in HTAB solution and 100 ml of = activated=20 solution to 10 =C2=B5l of skin supernatants 96 well flat-bottom plates. = The=20 absorbance was read every minute for 10 minutes at 450 nm using a=20 spectrophotometer. All samples were analyzed in triplicate. For = quantification=20 purposes, a calibration curve of horseradish peroxidase (Calbiochem) = ranging=20 from 100 mU/ml to 3.13 mU/ml was run in parallel with the samples in = triplicate=20 with every experiment.
Statistical=20 analysis Graphpad=20 Prism version 4.03 was used for statistical analysis. Differences in=20 adherence/invasion, mRNA expression, chemokine secretion in hBMEC = supernatants=20 were evaluated with a one-way ANOVA followed by Tukey's post hoc test.=20 Differences in neutrophil recruitment were determined using a paired=20 t-test for the MPO assay and an unpaired t-test for the=20 intraperitoneal infection model. Kaplan-Meier survival plots were = evaluated with=20 the log-rank test. Statistical significance was accepted at=20 p<0.05.

Supporting=20 Information
Table=20 S1. (0.07 MB XLS)
A. Genes affected in hBMEC >2 fold by infection with B. anthracis = Sterne=20 B. Genes affected in hBMEC >2 fold by infection with pXO1 deficient=20 mutant

Acknowledgments
The=20 authors are grateful to Monique Stins and Kwang Sik Kim for providing = hBMEC,=20 Scott Stibitz for the isogenic =CE=94LF, =CE=94EF and =CE=94LF/EF B. = anthracis strains,=20 Marilyn Farquhar and Timo Meerloo for assistance with electron = microscopy and=20 Roman Sasik for assistance with microarray data analysis. The microarray = analysis was performed at the Biogem Core Facility of the University of=20 California San Diego, director Gary Hardiman, and histopathologic = analysis=20 performed by Nissi Varki.

Author Contributions

Conceived and = designed the=20 experiments: NMvS KD. Performed the experiments: NMvS CME SMM DQ. = Analyzed the=20 data: NMvS CME KD. Contributed reagents/materials/analysis tools: MS DG. = Wrote=20 the paper: NMvS KD.

References

Mock M, Fouet = A (2001)=20 Anthrax. Annu Rev Microbiol 55: 647=E2=80=93671. Find=20 this article online
Dixon TC, = Meselson M,=20 Guillemin J, Hanna PC (1999) Anthrax. N Engl J Med 341: = 815=E2=80=93826. Find=20 this article online
Guidi-Rontani C=20 (2002) The alveolar macrophage: the Trojan horse of Bacillus = anthracis.=20 Trends Microbiol 10: 405=E2=80=93409. Find=20 this article online
Cleret A, = Quesnel-Hellmann=20 A, Vallon-Eberhard A, Verrier B, Jung S, et al. (2007) Lung = dendritic=20 cells rapidly mediate anthrax spore entry through the pulmonary route. = J=20 Immunol 178: 7994=E2=80=938001. Find=20 this article online
Lanska DJ = (2002)=20 Anthrax meningoencephalitis. Neurology 59: 327=E2=80=93334. Find=20 this article online
Abramova FA, = Grinberg LM,=20 Yampolskaya OV, Walker DH (1993) Pathology of inhalational = anthrax in=20 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci U S = A 90:=20 2291=E2=80=932294. Find=20 this article online
Fritz DL, Jaax = NK, Lawrence=20 WB, Davis KJ, Pitt ML, et al. (1995) Pathology of experimental=20 inhalation anthrax in the rhesus monkey. Lab Invest 73: = 691=E2=80=93702. Find=20 this article online
Duesbery NS, = Webb CP,=20 Leppla SH, Gordon VM, Klimpel KR, et al. (1998) Proteolytic=20 inactivation of MAP-kinase-kinase by anthrax lethal factor. Science = 280:=20 734=E2=80=93737. Find=20 this article online
Leppla SH = (1982)=20 Anthrax toxin edema factor: a bacterial adenylate cyclase that = increases=20 cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S = A 79:=20 3162=E2=80=933166. Find=20 this article online
Turnbull = PC (1991)=20 Anthrax vaccines: past, present and future. Vaccine 9: = 533=E2=80=93539. Find=20 this article online
Welkos SL, = Friedlander=20 AM (1988) Pathogenesis and genetic control of resistance to the = Sterne=20 strain of Bacillus anthracis. Microb Pathog 4: 53=E2=80=9369. = Find=20 this article online
Doran KS, Liu GY, = Nizet=20 V (2003) Group B streptococcal beta-hemolysin/cytolysin = activates=20 neutrophil signaling pathways in brain endothelium and contributes to=20 development of meningitis. J Clin Invest 112: 736=E2=80=93744. Find=20 this article online
Stins MF, = Prasadarao NV, Zhou=20 J, Arditi M, Kim KS (1997) Bovine brain microvascular = endothelial cells=20 transfected with SV40-large T antigen: development of an immortalized = cell=20 line to study pathophysiology of CNS disease. In Vitro Cell Dev Biol = Anim 33:=20 243=E2=80=93247. Find=20 this article online
Doran KS, = Engelson EJ,=20 Khosravi A, Maisey HC, Fedtke I, et al. (2005) Blood-brain = barrier=20 invasion by group B Streptococcus depends upon proper = cell-surface=20 anchoring of lipoteichoic acid. J Clin Invest 115: 2499=E2=80=932507. = Find=20 this article online
Kim KS = (2001)=20 Escherichia coli translocation at the blood-brain barrier. = Infect Immun=20 69: 5217=E2=80=935222. Find=20 this article online
Nizet V, Kim KS, = Stins M,=20 Jonas M, Chi EY, et al. (1997) Invasion of brain microvascular=20 endothelial cells by group B streptococci. Infect Immun 65: = 5074=E2=80=935081.=20 Find=20 this article online
Betz AL = (1985)=20 Epithelial properties of brain capillary endothelium. Fed Proc 44: = 2614=E2=80=932615.=20 Find=20 this article online
Betz AL = (1992) An=20 overview of the multiple functions of the blood-brain barrier. NIDA = Res Monogr=20 120: 54=E2=80=9372. Find=20 this article online
Unwin RD, Whetton = AD=20 (2006) Systematic proteome and transcriptome analysis of stem cell=20 populations. Cell Cycle 5: 1587=E2=80=931591. Find=20 this article online
Bradley PP, = Priebat DA,=20 Christensen RD, Rothstein G (1982) Measurement of cutaneous=20 inflammation: estimation of neutrophil content with an enzyme marker. = J Invest=20 Dermatol 78: 206=E2=80=93209. Find=20 this article online
Pezard C, Berche = P, Mock=20 M (1991) Contribution of individual toxin components to = virulence of=20 Bacillus anthracis. Infect Immun 59: 3472=E2=80=933477. Find=20 this article online
Gleiser = CA (1967)=20 Pathology of anthrax infection in animal hosts. Fed Proc 26: = 1518=E2=80=931521. Find=20 this article online
Jernigan JA, = Stephens DS,=20 Ashford DA, Omenaca C, Topiel MS, et al. (2001) = Bioterrorism-related=20 inhalational anthrax: the first 10 cases reported in the United = States. Emerg=20 Infect Dis 7: 933=E2=80=93944. Find=20 this article online
Popov SG, Popova = TG, Grene E,=20 Klotz F, Cardwell J, et al. (2004) Systemic cytokine response = in murine=20 anthrax. Cell Microbiol 6: 225=E2=80=93233. Find=20 this article online
Russell BH, = Vasan R, Keene=20 DR, Xu Y (2007) Bacillus anthracis internalization by = human=20 fibroblasts and epithelial cells. Cell Microbiol 9: 1262=E2=80=931274. = Find=20 this article online
Kern JW, = Schneewind O=20 (2008) BslA, a pXO1-encoded adhesin of Bacillus anthracis. Mol=20 Microbiol 68: 504=E2=80=93515. Find=20 this article online
Schubert-Unkmeir A,=20 Sokolova O, Panzner U, Eigenthaler M, Frosch M (2007) Gene = expression=20 pattern in human brain endothelial cells in response to Neisseria=20 meningitidis. Infect Immun 75: 899=E2=80=93914. Find=20 this article online
Sokolova O, = Heppel N,=20 Jagerhuber R, Kim KS, Frosch M, et al. (2004) Interaction of=20 Neisseria meningitidis with human brain microvascular = endothelial=20 cells: role of MAP- and tyrosine kinases in invasion and inflammatory = cytokine=20 release. Cell Microbiol 6: 1153=E2=80=931166. Find=20 this article online
Mayer-Scholl A, Hurwitz=20 R, Brinkmann V, Schmid M, Jungblut P, et al. (2005) Human = neutrophils=20 kill Bacillus anthracis. PLoS Pathog 1: e23. Find=20 this article online
Drysdale M, = Olson G,=20 Koehler TM, Lipscomb MF, Lyons CR (2007) Murine innate immune = response=20 to virulent toxigenic and nontoxigenic Bacillus anthracis = strains.=20 Infect Immun 75: 1757=E2=80=931764. Find=20 this article online
Batty S, Chow EM, = Kassam A,=20 Der SD, Mogridge J (2006) Inhibition of mitogen-activated = protein=20 kinase signalling by Bacillus anthracis lethal toxin causes=20 destabilization of interleukin-8 mRNA. Cell Microbiol 8: = 130=E2=80=93138. Find=20 this article online
Heninger S, = Drysdale M,=20 Lovchik J, Hutt J, Lipscomb MF, et al. (2006) Toxin-deficient = mutants=20 of Bacillus anthracis are lethal in a murine model for = pulmonary=20 anthrax. Infect Immun 74: 6067=E2=80=936074. Find=20 this article online
During RL, Li W, = Hao B,=20 Koenig JM, Stephens DS, et al. (2005) Anthrax lethal toxin = paralyzes=20 neutrophil actin-based motility. J Infect Dis 192: 837=E2=80=93845. Find=20 this article online
During RL, = Gibson BG, Li W,=20 Bishai EA, Sidhu GS, et al. (2007) Anthrax lethal toxin = paralyzes=20 actin-based motility by blocking Hsp27 phosphorylation. Embo J 26: = 2240=E2=80=932250.=20 Find=20 this article online
Zaucha GM, Pitt = LM, Estep J,=20 Ivins BE, Friedlander AM (1998) The pathology of experimental = anthrax=20 in rabbits exposed by inhalation and subcutaneous inoculation. Arch = Pathol Lab=20 Med 122: 982=E2=80=93992. Find=20 this article online
Janes BK, Stibitz = S=20 (2006) Routine markerless gene replacement in Bacillus = anthracis.=20 Infect Immun 74: 1949=E2=80=931953. Find=20 this article online
Stins MF, = Prasadarao NV,=20 Ibric L, Wass CA, Luckett P, et al. (1994) Binding = characteristics of S=20 fimbriated Escherichia coli to isolated brain microvascular = endothelial=20 cells. Am J Pathol 145: 1228=E2=80=931236. Find=20 this article online
Sasik R, Calvo E, = Corbeil=20 J (2002) Statistical analysis of high-density oligonucleotide = arrays: a=20 multiplicative noise model. Bioinformatics 18: 1633=E2=80=931640. Find=20 this article online
Storie I, Sawle = A,=20 Goodfellow K, Whitby L, Granger V, et al. (2003) Flow rate = calibration=20 I: a novel approach for performing absolute cell counts. Cytometry B = Clin=20 Cytom 55: 1=E2=80=937. Find=20 this article online

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