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Home医源资料库在线期刊美国呼吸和危急护理医学2006年第173卷第1期

Differential Roles of CD14 and Toll-like Receptors 4and 2 in Murine Acinetobacter Pneumonia

来源:美国呼吸和危急护理医学
摘要:Objective:TogainfirstinsightintotheroleofCD14andToll-likereceptors4and2inhostresponsetoA。MeasurementsandMainResults:BacterialcountswereincreasedinCD14andToll-likereceptor4gene–deficientmice,andonlytheseanimalsdevelopedbacteremia。Thepulmonarycytokine......

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    Laboratory of Experimental Internal Medicine, Department of Pathology, and Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam
    Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
    Department of Immunology, The Scripps Research Institute, La Jolla, California
    Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
    Department of Internal Medicine I, Medical University Vienna, Vienna, Austria

    ABSTRACT

    Rationale: Acinetobacter baumannii is an opportunistic bacterial pathogen that is increasingly associated with gram-negative nosocomial pneumonia, but the molecular mechanisms that play a role in innate defenses during A. baumannii infection have not been elucidated.

    Objective: To gain first insight into the role of CD14 and Toll-like receptors 4 and 2 in host response to A. baumannii pneumonia.

    Methods: Respective gene-deficient mice were intranasally infected with A. baumannii, and bacterial outgrowth, lung inflammation, and pulmonary cytokine/chemokine responses were determined. To study the importance of LPS in the inflammatory response, mice were also challenged with A. baumannii LPS.

    Measurements and Main Results: Bacterial counts were increased in CD14 and Toll-like receptor 4 gene–deficient mice, and only these animals developed bacteremia. The pulmonary cytokine/chemokine response was impaired in Toll-like receptor 4 knockout mice and the onset of lung inflammation was delayed. In contrast, Toll-like receptor 2–deficient animals displayed an earlier cell influx into lungs combined with increased macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 concentrations, which was associated with accelerated elimination of bacteria from the pulmonary compartment. Neither CD14 nor Toll-like receptor 4 gene–deficient mice responded to intranasal administration of LPS, whereas Toll-like receptor 2 knockout mice were indistinguishable from wild-type animals.

    Conclusions: Our results suggest that CD14 and Toll-like receptor 4 play a key role in innate sensing of A. baumannii via the LPS moiety, resulting in effective elimination of the bacteria from the lung, whereas Toll-like receptor 2 signaling seems to counteract the robustness of innate responses during acute A. baumannii pneumonia.

    Key Words: Acinetobacter  bacterial pneumonia  inflammation  lipopolysaccharide  macrophage  Toll-like receptor

    Members of the genus Acinetobacter have recently gained increased recognition as bacterial pathogens that have the potential to cause severe infections in critically ill patients in intensive care units (1, 2). One of the species within the genus, A. baumannii, has gained particular notoriety as one of the leading causes of opportunistic nosocomial infections worldwide (3–7). The predominant site of A. baumannii infection is the pulmonary compartment; 15 to 25% of ventilator-associated pneumonias are attributable to this pathogen (8, 9). The crude mortality of ventilator-associated pneumonias from A. baumannii has been shown to be as high as 75% (8, 10). There is additional evidence that A. baumannii induces community-acquired pneumonia, predominately among young alcoholics in tropical climates (11). Pneumonia induced by A. baumannii is frequently associated with a sudden and severe onset that, in most cases, requires mechanical ventilation, and systemic complications, including septic shock, have been repeatedly described (11). The high rate of antibiotic resistance and widespread colonization of skin, mucosal membranes, and medical equipment make A. baumannii a pathogen of high importance and concern (12–18). Given the increased clinical importance of A. baumannii and the lack of knowledge regarding host defense mechanisms against this opportunistic pathogen, we developed a murine model of A. baumannii pneumonia.

    The first line of defense against invading bacteria is provided by the innate immune system, which recognizes pathogen-associated molecular patterns, conserved microbial patterns shared by large groups of pathogens, but not found in higher eukaryotes (19–21). In recent years, it has become evident that both the recognition and the subsequent response to pathogens are mainly transferred by members of the Toll-like receptor (TLR) family (22–25). Of the 11 described TLRs, TLR4 and TLR2 are the key receptors signaling the presence of bacteria. TLR4 signaling is triggered by the interaction with LPS, the major cell wall component of gram-negative bacteria (26). CD14, a glycosylphosphatidylinositol-anchored molecule, is an important player in the LPS-signaling process, because it enhances LPS binding to MD-2 (27). This process, in turn, enables LPS binding to TLR4. In the absence of either CD14 or TLR4, the LPS-induced inflammatory responses are greatly reduced (28, 29). TLR2, in contrast to TLR4, has received attention primarily as an important pattern recognition receptor for gram-positive bacteria, although it might also contribute to the host innate immune defense against gram-negative pathogens (30–34). TLR2 recognizes peptidoglycan and lipoproteins, which are major constituents of the cell wall of gram-positive bacteria but, to a lesser degree, are also present in gram-negative microorganisms. Here, we provide first insight into the role of these important signaling receptors in A. baumannii pneumonia.

    METHODS

    Mice

    Pathogen-free 7- to 9-wk-old C57/BL6 mice were obtained from Harlan Sprague-Dawley (Horst, The Netherlands), as were CD14 gene–deficient (CD14–/–) mice from Jackson Laboratories (Bar Harbor, ME) (35). TLR4–/– and TLR2–/– mice were generated as described (29, 32). All mice were bred in the animal facility of the Academic Medical Center in Amsterdam and backcrossed six times to C57/BL6 background. Age- and sex-matched C57/BL6 wild-type mice were used as controls. The institutional animal care and use committee approved all experiments.

    Induction of Pneumonia

    A. baumannii (strain RUH 2037, allocated to the European clone I) (36) was isolated during an Acinetobacter outbreak in 1986 from sputum of a patient suffering from pneumonia. Detailed information regarding the bacterial strain is provided in the online supplement. Bacteria were grown to midlogarithmic phase at 37°C using Luria Bertani broth (Difco, Detroit, MI), then washed and resuspended in sterile isotonic saline (106–108 cfu/50 μl). Mice were anesthetized by inhalation of isoflurane (Upjohn, Ede, The Netherlands) and 50 μl were inoculated intranasally. At indicated time points, mice were killed and bacterial counts were determined as described (34, 37). In some experiments, a bronchoalveolar lavage (BAL) was performed; total cell numbers were counted using a Coulter counter (Beckmann Coulter, Fullerton, CA) and differential cell counts were done on cytospin preparations stained with Giemsa. For details, see the online supplement.

    Cytokine/Chemokine and Myeloperoxidase Measurements

    Lungs were homogenized as described (34, 37, 38). Tumor necrosis factor (TNF)-, interleukin (IL)-6, IL-10, and monocyte chemoattractant protein-1 (MCP-1) were measured using the cytometric bead array (BD Bioscience, San Jose, CA). IL-1, keratinocyte-derived chemokine, and macrophage inflammatory protein (MIP)-2 were measured using ELISA (R&D Systems, Minneapolis, MN), as was myeloperoxidase (HyCult Biotechnology, Uden, The Netherlands). Further details are described in the online supplement.

    Histologic Examination

    Lungs were harvested at indicated time points, fixed in 10% formalin, and embedded in paraffin. Four-micrometer sections were stained with hematoxylin and eosin and analyzed by a pathologist blinded for groups. To score lung inflammation and damage, the entire lung surface was analyzed regarding the presence of the following: interstitial inflammation, edema, endothelialitis, bronchitis, and pleuritis as described (34, 38). Granulocyte staining was done as described previously (34, 37). See online supplement for details.

    LPS Pneumonitis

    LPS (100 ng) purified from A. baumannii strain 24 (RUH 872, allocated to the European clone I [additional information on this LPS preparation is provided in the online supplement]) (39, 40) was administered (in 50 μl saline) intranasally to mice that were anesthetized by inhalation of isoflurane (Upjohn). Six hours later, a BAL was performed, total cell numbers were counted using a hemocytometer (Türk chamber), and differential cell counts were done on cytospin preparations stained with Giemsa (41).

    Statistical Analysis

    Differences between groups were calculated by Mann-Whitney U test or one-way analysis of variance where appropriate using GraphPad Prism software (GraphPad Software, San Diego, CA). Values are expressed as mean ± SEM. A p value of less than 0.05 was considered statistically significant.

    RESULTS

    A. baumannii Pneumonia Model

    To enable the investigation of host defense mechanisms in A. baumannii pneumonia in vivo, we first developed a suitable mouse model. A well documented strain of A. baumannii isolated from a clinical case of Acinetobacter pneumonia was selected for this purpose, and mice (n = 5/group) were infected intranasally with inocula ranging from 106 to 108 cfu. Mice were killed after 1, 4, 24, 48, or 72 h to follow the inflammatory response over time. As depicted in Figure 1, lung bacterial counts temporarily increased until 4 h after infection and gradually declined thereafter (Figure 1). However, bacteria were still detectable in lung tissue 72 h after inoculation. Likewise, pulmonary TNF- production reached peak levels at t = 4 h and quickly decreased thereafter, whereas the number of infiltrating polymorphonuclear cells (PMNs; determined as myeloperoxidase concentration) remained at a constant level between 4 and 48 h after infection (Figure 1). Histologic examination of lung tissue illustrated the early onset of pneumonia as reflected by dense pulmonary infiltrates (Figure 2). Of the mice challenged with 108 cfu of A. baumannii, two died after 24 h. On the basis of these pilot experiments, subsequent studies were performed with a bacterial inoculum of 107 cfu and mice were killed at 4 h (peak of cytokine response and bacterial counts) and 24 h (peak of PMN influx and onset of bacterial clearance) after challenge.

    CD14 and TLR4 Contribute to Clearance of A. baumannii

    Having established a murine model of A. baumannii pneumonia, we next studied host innate defense pathways possibly involved in the observed inflammatory responses. CD14 and TLRs are important pattern recognition receptors that contribute to the initiation of an adequate inflammatory response during infections and, hence, to an effective host immune defense. Because CD14 and TLR4 are known to recognize LPS from gram- negative bacteria, we first investigated their respective roles during A. baumannii pneumonia in vivo. Wild-type and gene-deficient mice were inoculated with 107 cfu of A. baumannii and killed after 4 and 24 h. At 4 h after inoculation, significantly higher lung bacterial counts were found in CD14–/– and TLR4–/– mice as compared with wild-type animals (Figure 3). At 24 h, wild-type mice had lower bacterial counts than either group of gene-deficient mice, though this difference was only statistically significant for TLR4–/– animals (Figure 3). To investigate systemic bacterial dissemination, blood samples were cultured for the presence of A. baumannii. Whereas blood cultures were sterile at 4 h in all mice, approximately 50% of mice lacking either CD14 or TLR4 had positive blood cultures at t = 24 h (6/10 and 4/9 mice, respectively) as compared with wild-type animals (0/8 mice).

    We next examined whether TLR2 might also play a role in host innate defense against A. baumannii. TLR2–/– and wild-type mice were infected and lung bacterial counts enumerated. No difference was found at t = 4 h after infection but, somewhat surprisingly, a significantly lower number of bacteria was observed in lungs of TLR2–/– mice 24 h after induction of pneumonia (Figure 4). The observed difference in bacterial clearance between wild-type mice and mice lacking TLR2 was still observed at 44 h after inoculation (Figure 4).

    Taken together, these results suggest that CD14 and TLR4 contribute to lung bacterial clearance and prevent systemic dissemination of A. baumannii, whereas TLR2 signaling counteracts elimination of the bacteria from the lungs.

    Humoral and Cellular Factors

    To gain further insight into the impaired bacterial clearance in mice lacking CD14 or TLR4, we next investigated the pulmonary cytokine and chemokine responses to A. baumannii. Early after infection (4 h), TLR4–/– mice displayed a reduced ability to produce IL-6, TNF-, keratinocyte-derived chemokine, and MIP-2 as compared with wild-type animals (p < 0.05 in all cases), whereas IL-1, IL-10, and MCP-1 levels were comparable to wild-type mice (Table 1). Cytokine concentrations did not differ between TLR4–/– and wild-type mice at 24 h, whereas keratinocyte- derived chemokine levels remained lower in the TLR4-deficient animals than in the wild-type animals (Table 1). Pulmonary cytokine and chemokine concentrations in CD14–/– and wild-type mice were largely similar (Table E1 in the online supplement). The onset of pulmonary inflammation, as assessed by histologic examination of lungs at t = 4 h, was delayed in the absence of TLR4 (inflammation score at t = 4 h: 9.3 ± 0.6 for wild-type and 7.1 ± 0.6 for TLR4–/– mice; p < 0.05), whereas no difference was observed between CD14–/– mice and their wild-type counterparts (inflammation score at t = 4 h: 10.5 ± 0.9 in wild-type and 11.5 ± 0.9 in CD14–/– mice; not significant). At this early time point in particular, TLR4–/– mice demonstrated a reduced influx of PMNs into BAL fluid (BALF), whereas CD14–/– mice displayed moderately, albeit insignificantly, reduced PMN numbers in BALF (Table 2; p < 0.05, TLR4–/– vs. wild-type mice). In line with increased bacterial counts, mice lacking CD14 had significantly higher pulmonary inflammation scores than their wild-type counterparts 24 h after infection (10.0 ± 0.6 for wild-type and 12.4 ± 0.4 for CD14–/– mice; p < 0.05). Likewise, the proportion of mice with confluent pneumonia was higher in TLR4–/– animals (14 and 50% of wild-type and TLR4–/– mice, respectively, displayed areas of confluent pneumonia), although no difference between the lung inflammation score of wild-type or TLR4–/– animals was found at t = 24 h (12.1 ± 0.7 in wild-type vs. 12.0 ± 0.7 in TLR4–/– mice).

    In TLR2–/– mice, the most striking finding was a significant increase in pulmonary MIP-2 and MCP-1 concentrations 4 h after infection, whereas TNF-, IL-1, IL-6, or IL-10 levels did not differ when compared with wild-type mice (Figure 5; see also Table E2). Keratinocyte-derived chemokine concentrations were decreased in lungs of TLR2–/– mice at this early time point (see Table E2). Moreover, TLR2–/– mice displayed an earlier and more pronounced inflammatory cell influx into the lungs (Figure E1 and Figure 6). The early recruitment of PMNs into the pulmonary compartment was confirmed by Ly-6 immunohistochemical stainings and the detection of higher pulmonary myeloperoxidase concentrations in TLR2–/– (Figures 5 and 6, insets). Of note, TLR2–/– mice did not have an increased influx of PMNs into BALF (Table 2).

    The Role of A. baumannii LPS in Pulmonary Infection

    So far, our studies were performed using whole bacteria. Given the involvement of LPS-signaling receptors CD14 and TLR4 in the innate response within the pulmonary compartment, we next inoculated mice intranasally with 100 ng of purified A. baumannii LPS, strain 24. The LPS of this strain has been shown previously to possess the same immunochemical properties as that of strain RUH 2037 (40), which was used in the studies described previously. Bronchoalveolar PMN influx and cytokine/chemokine concentrations were assessed 6 h after LPS administration. As expected, CD14 and TLR4 were crucial for the induction of the inflammatory response; mice lacking either signaling receptor did not mount a substantial PMN influx or TNF- or IL-6 release (Figure 7; IL-6 data not shown). These results confirm that A. baumannii LPS is a major immunostimulatory component that leads to a proinflammatory response during pneumonia with whole A. baumannii bacteria. Accordingly, we did not find a role for TLR2 in LPS-induced pneumonitis. Neither PMN influx nor alveolar TNF- or IL-6 concentrations differed between TLR2–/– and wild-type mice (Figure 7; IL-6 data not shown).

    DISCUSSION

    A. baumannii pneumonia poses an increased threat to hospitalized patients, as reflected in the rising number of nosocomial pneumonia cases caused by this bacterial species and the relatively high incidence of mortality among infected individuals (4, 8). In light of the high antibiotic resistance of A. baumannii, knowledge about host defense mechanisms is highly warranted. We hereby introduce an acute A. baumannii pneumonia model that allows the in vivo investigation of these molecular mechanisms during infection with this bacterium. We observed that the release of proinflammatory cytokines mediated by CD14 and TLR4 signaling is crucial to bacterial clearance within the lungs and to the prevention of systemic bacterial spread in vivo. In contrast, TLR2-related pathways delayed early MIP-2 and MCP-1 release as well as pulmonary inflammation, which were accompanied by an impaired elimination of A. baumannii from the lungs.

    A. baumannii exhibits some resemblance to Pseudomonas aeruginosa: both are gram-negative, nonglucose fermenters that are strongly associated with nosocomial pneumonia. Although many reports have described host defense mechanisms against P. aeruginosa, very little is known about A. baumannii. We have taken first steps to fill this gap by establishing a murine model of A. baumannii infection and thereby encountered some similarities to the P. aeruginosa model we frequently study in our laboratory (42, 43). Thus, both groups of bacteria rapidly induce a robust inflammatory response within the lungs, and bacteria are eventually cleared by the host unless high infection doses are administered. Until recently, the role of TLRs during this type of acute pneumonia was not entirely clear; we therefore decided to investigate the respective roles of CD14, TLR4, and TLR2 during A. baumannii pneumonia. While this work was in progress, two other groups reported an important role for myeloid differentiation factor (MyD) 88, the main adaptor protein involved in TLR and IL-1 signaling, during P. aeruginosa pneumonia (44, 45). Both reports describe a severely impaired PMN influx and cytokine/chemokine response within the lungs of MyD88–/– mice that was associated with higher bacterial counts in this organ. Because IL-1 and IL-18 pathways (which also use MyD88 as adaptor) have been shown to play no supportive role during P. aeruginosa pneumonia (42, 43), Power and colleagues then focused on TLRs and investigated the contribution of TLR2 and TLR4 to host defense (45). Power and colleagues made use of C3H/HeJ mice that harbor a mutation in TLR4 that renders this receptor dysfunctional, whereas in the study reported here TLR4–/– animals backcrossed to a C57/BL6 background were used. Though in most instances the differences are minor, C3H/HeJ mice do not precisely mirror the situation found in TLR4–/– mice that were used here. We also performed experiments in C3H/HeJ mice (data not shown) and revealed a less prominent role for TLR4 using these mice when compared with TLR4–/– animals. However, just like Power and colleagues in P. aeruginosa pneumonia (45), we found an impaired early (4 h) cytokine and PMN response in TLR4 gene–deficient mice in A. baumannii pneumonia. In addition, we also observed an increased bacterial load in TLR4–/– mice after 4 h, whereas the difference in C3H/HeJ mice did not reach significance at this early time point (data not shown). Because LPS is considered the main ligand for TLR4 and CD14, we expanded our studies and used purified LPS form A. baumannii to demonstrate that both TLR4 and CD14 are indeed the two crucial receptors involved in the signaling cascade during A. baumannii infection in vivo.

    To investigate the receptor that counts responsible for the inflammatory response to A. baumannii in the absence of TLR4 or CD14, we moved on and examined the role of TLR2, the receptor for bacterial lipoproteins and peptidoglycan. Much to our surprise, we found that the absence of TLR2 accelerated PMN influx into lung tissue (as assessed by myeloperoxidase levels in whole-lung homogenates and histology), although PMN counts in BALF of TLR2–/– mice did not differ from BALF PMN counts in wild-type mice early after infection. Nonetheless, TLR2 deficiency was associated with an improved bacterial clearance from this organ. When Power and colleagues investigated P. aeruginosa pneumonia in TLR2–/– mice, they found a very moderately decreased PMN influx in lung tissue (as indicated by reduced myeloperoxidase levels) after 4 h but no impairment of the cytokine/chemokine responses (45). However, the authors did not report on lung cfu nor did they investigate later time points when improved bacterial clearance might have become apparent. We found an early onset of pulmonary inflammation together with increased MIP-2 and MCP-1 concentrations in TLR2–/– mice 4 h after infection.

    Alveolar macrophages have been reported to be the main source of MCP-1 during pulmonary infection (46) and LPS, via TLR4/CD14 and direct as well as indirect nuclear factor-B activation, is the major trigger for the secretion of MCP-1 (47, 48). MCP-1 has also been described in respiratory epithelial cells 24 h after infection with P. aeruginosa (49). Moreover, two reports described the highly beneficial role of MCP-1 during gram-negative infection: early MCP-1 administration contributed to a faster elimination of bacteria, whereas late MCP-1 administration (at t = 24 h) reduced lung injury and improved the resolution of P. aeruginosa pneumonia via enhanced uptake of potentially harmful apoptotic PMNs (49, 50). Likewise, increased MIP-2 levels have been described to improve the PMN influx and phagocytosis of bacteria during gram-negative pneumonia (51). Accordingly, we found an improved pulmonary clearance of A. baumannii in the presence of elevated MCP-1 and MIP-2 concentrations. The robust and early (4 h) onset of pulmonary inflammation in the absence of TLR2–/– might therefore explain the improved bacterial clearance observed at later time points (24 and 44 h).

    Why and how TLR2 precisely prevents the rise in MCP-1 and MIP-2 levels remains unclear. The possibility exists that TLR2 mediates antiinflammatory pathways that downregulate MCP-1 production. IL-10 has been shown to reduce MIP-2 and MCP-1 secretion by activated monocytes/macrophages (48, 52, 53), but we did not find any differences in IL-10 concentrations that could explain our findings. Increased MIP-2 levels might even be a consequence of elevated MCP-1 concentrations, as illustrated by the finding of synergistically enhanced MIP-2 release in the presence of MCP-1 and LPS (54). Alternatively, the lack of TLR2 signaling in gene-deficient mice could have been associated with an upregulation of other receptors with mainly proinflammatory properties, such as TLR4. This phenomenon has been described in TLR2–/– mice infected with P. aeruginosa that lack pilus expression (55). Another potential explanation could be that differences in the cellular expression profile of TLRs within the respiratory tract are associated with distinct responses. It seems quite well established that LPS induced pulmonary inflammation relies primarily on TLR4 expressing macrophages (56, 57). LPS directly activates alveolar macrophages to secrete proinflammatory cytokines such as TNF- and IL-1; these, in turn, stimulate respiratory epithelial cells to produce, for example, chemokines (56). Respiratory epithelial cells have been repeatedly shown to be unresponsive when stimulated with LPS alone, although TLR4 mRNA is present in these cells (56, 58–60). In contrast, less is known about pulmonary TLR2, but this signaling receptor is expressed on alveolar macrophages and has gained much attention recently as part of a lipid raft receptor assembly at the apical side of airway epithelial cells (61). This finding strongly indicates the direct involvement of TLR2 in the sampling of and response to pathogens within the lungs. Given the high rate of colonization with A. baumannii in critically ill patients, it is tempting to speculate that the reduced inflammatory response we observed to occur in the presence of TLR2 might even prove beneficial for the host by providing a delicate balance between situations requiring robust response for the rapid clearance of bacteria in individuals with high pulmonary bacterial counts and the risk of systemic dissemination (such as the model of acute infection described herein) and cases in which the low degree of colonization does not warrant such a vigorous response.

    A more recent study by Benjamim and colleagues investigated the immunosuppression and higher susceptibility to nosocomial pulmonary infections after cecal ligation puncture. Among other findings, the authors described an increased expression of TLR2 within the lungs after cecal ligation puncture (62). Considering our observation of a weakened immune response to A. baumannii in the presence of TLR2, these data suggest that preceding insults such as cecal ligation puncture lead to molecular alterations that include the upregulation of TLR2 and are associated with an impaired ability to combat nosocomial bacteria such as A. baumannii.

    In conclusion, we demonstrate that CD14 and TLR4 are indispensable for A. baumannii LPS-mediated signaling, resulting in the effective elimination of A. baumannii from the lungs in vivo, whereas TLR2 reduces the pulmonary inflammatory response and so delays elimination of bacteria from the lungs. Our model of A. baumannii pneumonia will be useful in further studies aimed at elucidating mechanisms involved in innate and adaptive immune responses to this increasingly important nosocomial pathogen.

    Acknowledgments

    The authors thank J. Daalhuisen, I. Kop, and V. Susott for expert technical assistance, and A. Tuip de Boer for the Ly-6 immunohistochemical stainings.

    FOOTNOTES

    Supported by grants from the Austrian Fonds zur Frderung der wissenschaftlichen Forschung in sterreich to S.K. and from the Mr Willem Bakhuis Roozeboom Foundation to C.W.W.

    This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

    Originally Published in Press as DOI: 10.1164/rccm.200505-730OC on October 6, 2005

    Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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作者: Sylvia Knapp, Catharina W. Wieland, Sandrine Florq 2007-5-14
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