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* Unité Interactions Hôte-Pathogène, Département de Biologie des Agents Transmissibles, Centre de Recherches du Service de Santé des Armées, La Tronche, France;
Centre National de la Recherche Scientifique, Université Lyon 1, Unité Mixte de Recherche 5086, Institut de Biologie et Chimie des Protéines, Institut Fédératif de Recherche 128, Biosciences Gerland, Lyon, France; and
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-D-glutamate capsule (2) and 2) two toxins known as lethal toxin and edema toxin (3). B. anthracis is responsible for two very different clinical diseases: the cutaneous form, which is relatively benign, easily identified and treated with antibiotics, and the inhalational form, which is extremely severe, fulminating, and difficult to diagnose (4). The exact mechanisms leading to the severe clinical form remain unclear, but the contrast between skin and pulmonary infections may be explained by the architectural and functional singularities of the lung tissue (5). The lung architecture is devoted to respiratory exchanges through the alveolocapillary barrier. However, the mucosal surface of the pulmonary tract is exposed to diverse pathogens and the thin layer of exchange must be protected from damage by inflammatory processes. Alveolar macrophages (AMs)3 that participate in the elimination of particles and pathogens exhibit numerous anti-inflammatory properties (6, 7). Furthermore, two main subsets of lung dendritic cells (LDCs) have been described in the lung, participating in the control of the immune response: myeloid LDCs and plasmacytoid LDCs (reviewed in Refs. 8, 9, 10). Myeloid LDCs play the major role in Ag presentation in the lung (11, 12), whereas plasmacytoid LDCs efficiently clear virus and resolve inflammation (13, 14, 15). Although LDCs have been implicated in trafficking between the lung and thoracic lymph nodes (TLNs) in several models of allergen and particle inhalation (16, 17), the role of LDCs in inhalational anthrax is still unclear.
AMs from bronchoalveolar lavage (BAL) can phagocytose spores in several animal models of inhalational anthrax (18, 19, 20). Previous animal infection experiments reported that numerous infected phagocytes were found in TLNs in the few hours following infection, inducing lymphadenitis (21, 22, 23, 24). Thus, the TLNs are a main target for the proliferation and spread of the pathogen throughout the organism, leading ultimately to death. Clinical data and gross necropsy from the Fall 2001 cohorts have confirmed the same kinetics of infection in humans (25). Pulmonary anthrax does not correspond to a true pulmonary disease, and it can spread in the absence of lung injury. This activity signifies that B. anthracis spores may "hijack" phagocytic cells encountered in the lungs, inhibiting the production of inflammatory signals and forcing them to migrate to the TLNs. In accordance with this notion, we and others have shown in several models of macrophages and dendritic cells (DCs) in vitro that toxins secreted early after germination extinguish proinflammatory signals (26, 27, 28, 29, 30). The nature of the lung phagocytic cells that migrate has not yet been precisely defined, although it has been suggested that AMs play the role of the "Trojan horse" (20). LDCs continually sample the contents of the alveoli for particulate Ags, and unlike the AM population, then carry them to the TLNs (17); we have already shown that LDCs phagocytose anthrax spores more efficiently than AMs (31). We hypothesized that LDCs may represent a better target for anthrax spores. We examined the interactions between anthrax spores and the different lung phagocytic cell populations by using intranasal (i.n.) infection, which mimics an aerosol, and by following up the infection at the cellular level in mouse models.
We show that AMs immediately capture anthrax spores, whereas interstitial LDCs capture them within 30 min without transmigrating across the epithelial barrier of the alveoli. These results suggest a specific mechanism of alveolar spore sampling by transepithelial extensions. Then, LDCs rapidly carry the spores to the TLNs within 30 min, and this transport increases until 6 h after infection. Monocytes and LDCs are recruited 6 h later in the alveoli and constitute a new pool of alveolar cells. The timing of infection observed in this model is very rapid and is consistent with the fulminating form of inhalational anthrax.
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Materials and Methods |
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C57BL/6 (H2b) mice (Centre d’Elevage R. Janvier) were housed in clean standard conditions in our animal care facility (Centre de Recherches du Service de Santé des Armées, La Tronche, France), and CX3CR1gfp/+ mice on a C57BL/6 genetic background (32) were maintained under specific pathogen-free conditions at the Plateau de Biologie Expérimentale de la Souris (Ecole Normale Supérieure de Lyon, France). The local ethics committees approved our animal experiments according to international guidelines.
B. anthracis strains and i.n. infection
B. anthracis Sterne-p6gfp strain was provided by F. Tonello (University of Padova, Padova, Italy). The Sterne-p6gfp was constructed by electroporation of plasmid pAFp6gfp, a gift from T. Hoover (U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD) into the Sterne strain (pXO1+/pXO2–) (33).
B. anthracis Sterne 7702 strain (pXO1+/pXO2–) was provided by M. Mock (Institut Pasteur, Paris, France). The spores were chemically labeled with Alexa Fluor 647 (Invitrogen Life Technologies) as follows: 3 x 109 CFU of the Sterne strain of B. anthracis were stained with the reactive dye in 10 ml of H2O/0.1 M sodium bicarbonate for 1 h protected from light. Spores were washed five times in H2O/0.1 M sodium bicarbonate. The spores were maintained in H2O at 4°C to avoid germination (34).
Spores were diluted to a final concentration of 2 x 109 CFU/ml and heat activated for 10 min at 70°C. Mice were anesthetized and 50 µl of spores were i.n. administered at 108 CFU/mouse.
Analysis of infection in BALs, total lung cells, and TLNs
C57BL/6 mice were killed 10 min, 30 min, and 1, 6, 24, and 72 h after i.n. infection by the Sterne-p6gfp strain. BAL cells, lung cells, and draining TLNs were collected at each time point.
BAL cells and total lung cells were obtained as previously described (31). Briefly, the lungs were washed three times with 1 ml of ice-cold PBS/2 mM EDTA to collect BAL cells. Single-cell suspensions of lung cells were isolated by enzymatic digestion of lung tissue by collagenase (Worthington Biochemical) and DNase (Sigma-Aldrich).
Two mice were killed at each time point for measuring the bacterial load. Lungs were removed without previous BALs, and Potter-dissociated in 1 ml of ice-cold H2O. Serial dilutions were plated on TS agar containing kanamycin (Sigma-Aldrich) directly or after heating at 65°C for 30 min to kill any vegetative bacteria.
Cells were incubated with Fc receptor blocking Ab (anti-mouse CD16/CD32 Ab, clone 2.4G2; BD Biosciences) to reduce nonspecific binding. Cells were incubated for 30 min with anti-CD11b PE (clone M1/70), anti-I-A/I-E PE (clone M5/114.15.2), anti-CD11c biotin (clone HL3), or corresponding isotypes (all from BD Biosciences) at 4°C. Streptavidin-allophycocyanin was used after incubation with biotinylated mAb. Cells were then fixed with PBS, 1% BSA, 0.1% azide, and 4% paraformaldehyde. Cell acquisition was conducted on a FC500 (Beckman Coulter) and the data were analyzed with CXP software (Beckman Coulter).
At 24 h, BAL cells were stained for CD11c and CD11b and separated by FACS on a FACSVantage (BD Biosciences). Cytocentrifuge preparations of the different gates were colored with Giemsa. Cytospins were observed on an Axioimager Z1 microscope (Zeiss).
For confocal microscopy analysis, C57BL/6 BAL cells were collected 1 h after i.n. infection and stained with CD11b-unlabeled Ab (clone M1/70; Beckman Coulter) and CD11c-biotinylated Ab (clone HL3; BD Biosciences) for 30 min at 4°C. Cells were then washed and incubated with rabbit anti-rat IgG-Cy3 (Jackson ImmunoResearch Laboratories) and streptavidin-Cy5 (Beckman Coulter) for 30 min 4°C. Cells were washed twice, and fixed in PBS, 1% BSA, 0.1% azide, and 4% paraformaldehyde. Cytocentrifuge preparations were performed, fixed, and mounted with Vectashield DAPI medium (4',6'-diamidino-2-phenylindole; DakoCytomation). Cells were observed on a confocal Axioplan LSM510 microscope (Zeiss).
Analysis of spore-LDC contacts and transports
BAL cells and total lung cells of noninfected CX3CR1+/gfp mice were collected as described and analyzed by flow cytometry.
CX3CR1+/gfp mice infected by Alexa Fluor 647 Sterne spores were serially killed at 10 min, 30 min, and 1, 4, and 24 h after infection for analysis of the early steps of infection. Mice were intratracheally injected with 1% agarose.
Lungs from CX3CR1+/gfp mice were cut into 0.1- to 0.5-mm thick sections and mounted on slides for microscopic examination. TLNs were also removed and mounted on slides before analysis.
All observations were performed on a confocal Axioplan LSM510 microscope (Zeiss).
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Results |
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Kinetics of BAL infection
We discriminated between different cells by using the CD11c/CD11b expression pattern as previously described (12, 17, 31, 35). Inhaled spores first encounter resident cells of the alveoli; therefore we analyzed BALs that are mostly composed of AMs (
90%, CD11chigh/CD11b–) in uninfected controls (Fig. 1A, R1, noninfected panel). As early as 10 min after infection with Sterne-p6gfp spores, 65% of AMs were positive for GFP, suggesting that they engulfed spores very rapidly. Subsequent confocal analysis of BAL cells confirmed that GFP-spores were inside the cells (Fig. 1B). The percentage of AMs phagocytosing Sterne-p6gfp spores was relatively stable for the first 24 h and progressively decreased thereafter (Fig. 1C). In the 6 h following instillation, two cell populations were recruited de novo in the alveoli: a population of CD11b+/CD11c– cells (Fig. 1, R3), which represented 40% of the total cells, and a cell population expressing intermediate levels of CD11b and CD11c markers (Fig. 1, R2), representing 4% of the total cells and increasing to 6% in the next 24 h. With respect to their appearance after Giemsa staining, cells were clearly LDCs (Fig. 1, R2), whereas other shown corresponded to a monocyte population (Fig. 1D, R3). In accordance with our previous results (31), 80% of the LDCs recruited in the alveoli were Sterne-p6gfp-positive (mean fluorescence intensity (MFI) of 20.5) at 6 h, whereas AMs (50% of the total cell population with an MFI of 16.0) and monocytes (38% of the total cell population with an MFI of 6.9) were less positive for Sterne-p6gfp spores. At 24 and 72 h, monocytes still represented a large proportion of BAL cells (36% at 24 h and 30% at 72 h) but were only weakly positive for Sterne-p6gfp spores (MFI of 6.0 at 24 h, and MFI of 4.4 at 72 h); this response was presumably a consequence of either lower phagocytic capabilities or the monocytes capacity to kill intracellular spores that may have germinated. In contrast, the population of LDCs increased to represent 20% of total cells at 72 h postinfection. However, these LDCs were less efficient at phagocytosing Sterne-p6gfp spores (55% and 30% of GFP+ cells, and with MFI of 19.2 and 4.3, respectively), or spores were less available for LDC phagocytosis.
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20% of total BAL cells and increased up to 30% at 72 h after infection. These cells had an intermediate phagocytosis capacity (MFI of 14.3 at 6 h after infection, and MFI of 10.2 at 24 h after infection) compared with LDCs. This cell population was very similar to AMs, and it appeared at the time the CD11c+/CD11blow population percentage decreased, so it was probably an activated AM population. Cell sorting of CD11c+/CD11blow followed by Giemsa staining clearly confirmed that this population corresponded to an AM population (Fig. 1D, R'). Kinetics of lung cell infection
Next, we analyzed the presence and phagocytosis ability of several lung cell populations, to differentiate which lung population captured spores.
As previously shown, we identified three main populations corresponding to macrophages (CD11chigh/CD11b– including interstitial macrophages (IM) and AMs still present in the alveoli after lavages) (Fig. 2A, R1), LDCs (CD11cint/CD11bint) (Fig. 2A, R2), and monocytes (CD11c–/CD11bhigh) (Fig. 2A, R3). Ten minutes after infection we found a Sterne-p6gfp-positive population representing 10% of the macrophage population (Fig. 2B). This population might have been AMs not collected by lavages, suggesting that AMs were the first sentinels to encounter spores in the earliest step of infection. LDCs (12% of GFP-positive cells) captured spores secondarily at 30 min, whereas monocytes did not. Notably, LDCs were absent from the alveoli at this early time of infection (Fig. 1A, 30 min total cells). The percentage of GFP-positive macrophages and LDCs did not change significantly over time, and LDCs were more efficient than macrophages at phagocytosing spores, as observed in BALs (Fig. 2C). The percentage of the macrophage population did not change following infection, whereas monocytes were recruited 30 min after infection and then decreased over time. Recruitment of LDCs was transiently observed 24 h after infection.
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Kinetics of spore transport to the TLNs
We gated our analysis of TLN populations on CD11c-positive cells representing a population strongly expressing MHC class II (Fig. 3A), previously described as TLN DCs (11). We analyzed the percentage of Sterne-p6gfp-positive cells gated on CD11c+ cells reaching the TLNs after i.n. infection to assess DC spore carriage to regional lymphoid tissue (Fig. 3B). We observed a significant increase of GFP-positive cells from 27% at 30 min to a 75% peak 6 h after infection and a progressive decrease to 46% at 72 h (Fig. 3B). The MFI of Sterne-p6gfp-positive cells gated on CD11c+ cells showed similar kinetics increasing up to 6 h and decreasing thereafter to 72 h (8.9 and 6.0 at 6 and 72 h, respectively). The progressive decrease observed after 6 h could have been the consequence of 1) an influx of new LDC with fewer intracellular spores, 2) the killing of spores and bacilli already present within resident TLN DCs, or 3) a greater efflux than influx of spore- bearing DCs.
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To address the role of LDCs in spore transport from alveoli to TLNs through the intact alveoloepithelial barrier, we used transgenic CX3CR1+/gfp mice that specifically expressed GFP in DCs. Mice were infected with Alexa Fluor 647-labeled Sterne spores (32). First, we ascertained that GFP was specifically expressed in LDCs but not in BAL AMs (Fig. 4A) and total lung cells (Fig. 4B). As previously shown in the digestive tract (36, 37) and in the lung (38), GFP was specifically expressed in CX3CR1-LDCs (CD11cint/CD11bint) present in pulmonary parenchyma (Fig. 4, gate R2), but not in AMs (CD11c+/CD11b–) from BAL fluids (Fig. 4, gate R1). Confocal analysis of lungs and TLNs from uninfected CX3CR1+/gfp mice showed a thin web of green fluorescent cells harboring long dendrites in alveolar walls and TLNs (Fig. 4C). Ten minutes after i.n. infection, some Alexa Fluor 647-labeled Sterne spore-DC contacts were observed in the lung, but LDC-spore colocalizations were more evident at 30 min and 1 h and spores were principally observed in dendrite extensions (Fig. 5A). At 30 min, several LDCs colocalized with Alexa Fluor 647-labeled Sterne spores in the TLNs, demonstrating rapid kinetics of sampling and transport that were more pronounced after 1 h. These results confirmed our flow cytometry analysis, indicating that LDCs efficiently transport anthrax spores to the TLNs rapidly after infection. The subsequent triggering of massive LDC migration by the infection was confirmed by the diminution of LDC web density in the lung after 4 h, associated with spores colocalizing in the TLNs. Nevertheless, the detection of Alexa Fluor 647 spores at this 4-h time point was more difficult. This result could have been due to the killing of B. anthracis spores by DCs or alternatively the loss of the Alexa dye from within the phagolysosomes. Lung analysis at 24 h showed a recolonization by LDCs (data not shown). In accordance with our previous model, TLN analysis showed DCs capturing Alexa Fluor 647-labeled Sterne spores in TLNs at 30 min increasing up to 1 h (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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These data present an original scenario for B. anthracis entry by the pulmonary route. AMs are the first scavengers of spores, while LDCs capture spores from the outside of the alveoli within the first half hour of infection. The passage across the epithelium, or transepithelial crossing, constitutes the first step of pathogen diffusion, and confocal microscopy data clearly showed spores inside LDC dendrites 30 min after infection, although they were not observed in the BALs. LDCs can thus be proposed as the major effectors of the transepithelial crossing. AMs may first encounter aerosol anthrax spores, may phagocytose them, and as recently shown, may recognize sporal pathogen-associated molecular pattern through a MyD88-dependent mechanism (39). The activation of AMs may lead to the release of chemokine signaling for LDC recruitment, activation and spore uptake by a mechanism yet to be determined. A rat model recently showed that LDCs are able to send cell extensions between epithelial cells in the tracheal lumen to capture Ags, with an acceleration of Ag sampling within 30 min after bacterial stimulus (40). Another report on mouse lungs suggests that a subpopulation of LDCs display CD103 
E
7 integrin, potentially enabling these cells to migrate through the epithelium (41). Our results fit well with the possibility that LDCs may send extensions into the alveoli because we did not observe any recruitment of LDCs in the BALs at 30 min, whereas at the same time 12% of LDCs in the lung were already infected. These LDCs that have phagocytosed spores may have sampled the alveoli through cell extensions without transmigration, which would have taken a longer time. The signal that triggers LDC rapid sampling is still unknown, but CX3CL1, the CX3CR1 receptor ligand, could play a role, as exemplified by pathogen uptake in the gut (36). Moreover, we show in our experiments with CX3CR1+/gfp mice that CX3CR1-positive LDCs participate in spore uptake and transport. The mechanisms involved in LDC migration to TLNs are not yet determined. Spores have been shown to trigger the reprogramming of chemokine expression by human DCs due to a loss of transcription in tissues retaining chemokine receptors (CCR2, CCR5) and due to the induction of lymph node homing receptor CCR7 gene transcription (42). It is likely that the recognition of sporal pathogen-associated molecular pattern by a MyD88-dependent mechanism (39) may also facilitate the reprogramming of chemokine receptors and increase the physiological migration of LDCs toward the draining TLNs. In this early phase of infection, AMs and LDCs may cooperate closely in their respective roles: sampling and transport of Ag for LDCs, and capture and destruction of pathogens for AMs.
After 6 h of infection, LDCs and monocytes move to the alveoli. These two populations may have been recruited by AM signals, even though it is not clear whether monocytes differentiate into LDCs in the alveoli or whether differentiated LDCs come up directly from interstitial tissues. Furthermore, some spores may have germinated inside the cells en route and started to secrete their toxins to impair LDC functions (28, 29, 31).
Although the role of pathogens in the migration of LDCs has previously been reported in several models of viral (43, 44, 45) or Mycobacterium tuberculosis (46, 47) infection, we describe for the first time the very rapid kinetics of pathogen migration using LDCs.
We propose that LDCs are the real galloping Trojan horse of anthrax spores in inhalational anthrax because they are able to sample alveoli and to migrate to the TLNs in the first half hour of infection. In contrast, AMs phagocytose spores without transporting them. In the next 72 h, LDCs efficiently transport spores, facilitating pathogen diffusion throughout the body. The mechanisms of spread after spore germination may involve the capsule. If this scenario is the case, the use of a capsulated and fully virulent strain of B. anthracis may be required for studies to elucidate these processes.
One of the most striking features of our results is the extreme rapidity of spore transport, starting 30 min after infection, which is very consistent with the fulminating form of inhalational anthrax. This result signifies that exposure to an anthrax aerosol must be considered as a profound infection as early as 30 min following exposure, and the patient should be medically treated as soon as possible. These results should be taken into account for medical management after exposure to an anthrax aerosol.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Grant CO 010808 from the Délégation Générale pour l’Armement and by 135OP3B loi de finances rectificative Etat-Major des Armées from the Service de Santé des Armées. S.J. is the incumbent of the Pauline Recanati Career Development Chair and a Scholar of the Benoziyo Center for Molecular Medicine. 
2 Address correspondence and reprint requests to Dr. J.-N. Tournier, Unité Interactions Hôte-Pathogène, Département de Biologie des Agents Transmissibles, Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Grésivaudan, 38702 La Tronche, France. E-mail address: jntournier{at}crssa.net
3 Abbreviations used in this paper: AM, alveolar macrophage; BAL, bronchoalveolar lavage; DC, dendritic cell; LDC, lung DC; MFI, mean fluorescence intensity; TLN, thoracic lymph node.
Received for publication February 6, 2007. Accepted for publication April 11, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-glutamate in bacteria. Mol. Microbiol. 60: 1091-1098. [Medline]-induced release of NO and TNF. FEBS Lett. 462: 199-204. [Medline]E)7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J. Immunol. 176: 2161-2172. This article has been cited by other articles:
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  W. Wu, H. Mehta, K. Chakrabarty, J. L. Booth, E. S. Duggan, K. B. Patel, J. D. Ballard, K. M. Coggeshall, and J. P. Metcalf Resistance of Human Alveolar Macrophages to Bacillus anthracis Lethal Toxin J. Immunol., November 1, 2009; 183(9): 5799 - 5806. [Abstract] [Full Text] [PDF]
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 C. L. Loving, M. Osorio, Y.-G. Kim, G. Nunez, M. A. Hughes, and T. J. Merkel Nod1/Nod2-Mediated Recognition Plays a Critical Role in Induction of Adaptive Immunity to Anthrax after Aerosol Exposure Infect. Immun., October 1, 2009; 77(10): 4529 - 4537. [Abstract] [Full Text] [PDF]
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 C. K. Cote, J. Bozue, N. Twenhafel, and S. L. Welkos Effects of altering the germination potential of Bacillus anthracis spores by exogenous means in a mouse model J. Med. Microbiol., June 1, 2009; 58(6): 816 - 825. [Abstract] [Full Text] [PDF]
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F. J. Maldonado-Arocho and K. A. Bradley Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3{beta} Infect. Immun., May 1, 2009; 77(5): 2036 - 2042. [Abstract] [Full Text] [PDF]
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M. A. Crawford, Y. Zhu, C. S. Green, M. D. Burdick, P. Sanz, F. Alem, A. D. O'Brien, B. Mehrad, R. M. Strieter, and M. A. Hughes Antimicrobial Effects of Interferon-Inducible CXC Chemokines against Bacillus anthracis Spores and Bacilli Infect. Immun., April 1, 2009; 77(4): 1664 - 1678. [Abstract] [Full Text] [PDF]
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Y. P. Gauthier, J.-N. Tournier, J.-C. Paucod, J.-P. Corre, M. Mock, P. L. Goossens, and D. R. Vidal Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax Infect. Immun., March 1, 2009; 77(3): 1197 - 1207. [Abstract] [Full Text] [PDF]
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 O. N. Chesnokova, S. A. McPherson, C. T. Steichen, and C. L. Turnbough Jr. The Spore-Specific Alanine Racemase of Bacillus anthracis and Its Role in Suppressing Germination during Spore Development J. Bacteriol., February 15, 2009; 191(4): 1303 - 1310. [Abstract] [Full Text] [PDF]
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J.-N. Tournier, A. Cleret, A. Quesnel-Hellmann, Y. Xu, and B. H. Russell What Is the Relevance of Lung Epithelial Cells during the Dissemination of Spores in Inhalational Anthrax? Infect. Immun., January 1, 2009; 77(1): 565 - 566. [Full Text] [PDF]
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M. W. Lisanby, M. K. Swiecki, B. L. P. Dizon, K. J. Pflughoeft, T. M. Koehler, and J. F. Kearney Cathelicidin Administration Protects Mice from Bacillus anthracis Spore Challenge J. Immunol., October 1, 2008; 181(7): 4989 - 5000. [Abstract] [Full Text] [PDF]
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B. H. Russell, Q. Liu, S. A. Jenkins, M. J. Tuvim, B. F. Dickey, and Y. Xu In Vivo Demonstration and Quantification of Intracellular Bacillus anthracis in Lung Epithelial Cells Infect. Immun., September 1, 2008; 76(9): 3975 - 3983. [Abstract] [Full Text] [PDF]
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 C. Kim, S. Wilcox-Adelman, Y. Sano, W.-J. Tang, R. J. Collier, and J. M. Park From the Cover: Antiinflammatory cAMP signaling and cell migration genes co-opted by the anthrax bacillus PNAS, April 22, 2008; 105(16): 6150 - 6155. [Abstract] [Full Text] [PDF]
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P. Sanz, L. D. Teel, F. Alem, H. M. Carvalho, S. C. Darnell, and A. D. O'Brien Detection of Bacillus anthracis Spore Germination In Vivo by Bioluminescence Imaging Infect. Immun., March 1, 2008; 76(3): 1036 - 1047. [Abstract] [Full Text] [PDF]
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) and the percentage of GFP-positive cells for each population (
) for AM gate (R1), LDC gate (R2), and monocyte gate (R3). Data show cell percentages (±SD) representative of three independent experiments with similar results. C, MFI is represented at each time point for each population. Data show the cell percentages (±SD) representative of three independent experiments. D, CFU of total lungs after heat treatment at 65°C for 30 min (
) at each time point. Data show CFU (±SD) representative of three independent experiments with similar results.
