American Journal of Respiratory and Critical Care Medicine

With emphysema, diaphragm length adaptation results in shortened fibers. We hypothesize that passive diaphragm stretch occurring acutely after lung volume reduction surgery (LVRS) results in fiber injury. Bilateral LVRS was performed in emphysematous hamsters. Studies were performed 1 (D1) and 4 (D4) days after LVRS, and compared with sham-treated groups. Sarcolemmal rupture was evident in 10.9% of fibers in LVRS-D1 and reduced to 1.6% in LVRS-D4. Ultrastructural analysis revealed focal abnormalities in both LVRS-D1 and LVRS-D4 animals in over one-third of fibers. Myofibrillar disruption was not observed in sham-treated animals. Diaphragm insulin-like growth factor-I (IGF-I) was increased in LVRS-D4 compared with other emphysematous groups. Increased IGF-I immunoreactivity was localized to types IIA and I fibers. The abundance of the splice variant of IGF-I mRNA sensitive to muscle stretch (IGF-IEb) increased 3.2-fold in LVRS D-4 diaphragms, compared with emphysema-sham animals. The main form of IGF-I mRNA was unchanged. Marked force deficit was observed in the LVRS-D1 diaphragm, compared with emphysema-sham and emphysema (no surgery) animals. These data highlight a markedly compromised ventilatory pump acutely after LVRS. Acute fiber stretch predisposes to muscle fiber injury and may also be a necessary mechanotransductive stimulus for fiber remodeling as the diaphragm adapts to reduced lung volume.

Lung volume reduction surgery (LVRS) is a new treatment option for emphysema (EMP) whereby 20–30% of the most diseased portions of each lung are removed (1, 2). The National Emphysema Treatment Trial recently reported results on 1,218 patients with severe EMP randomized to LVRS or maximal medical therapy (3). Improvements in survival and/or exercise capacity and health related quality of life were noted in distinct subgroups of patients (3). Whereas mechanisms for such improvement are not entirely known, reduced lung volume and improved elastic recoil may be important in restoring more normal diaphragm configuration, optimal length (Lo), and function (4, 5). Although this might be a positive change in the long term, the short-term influences of a rapid reduction in lung volume and its acute geometric impact on the foreshortened EMP diaphragm are unknown and best studied in an animal model of EMP. The latter is an excellent model of hyperinflation and diaphragm length adaptation (i.e., fiber shortening due to loss of sarcomeres in series) (68).

An abrupt increase in diaphragm fiber length might be expected after LVRS in the EMP animal due to the establishment of a more normal chest configuration and reduced lung volume with a resultant decrement in force generation (fiber stretch beyond Lo). We postulate that these abrupt changes in the acute phase after LVRS may provide the basis for acute diaphragm muscle injury due to the influences of passive stretch on the conditioned diaphragm (9, 10). Diaphragm fiber injury would be expected to further curtail diaphragm force generating capacity over and above functional geometric considerations, by reducing diaphragm muscle-specific force (i.e., force/muscle cross-sectional area). This would have important clinical implications for ventilatory pump function during the vulnerable perioperative period after LVRS. Furthermore, we suggest that diaphragm muscle injury and its subsequent regeneration may complement passive stretch of the diaphragm as a trigger for diaphragm fiber remodeling (i.e., length readaptation) and that insulin-like growth factor-I (IGF-I) is likely an important regulator for this regenerative process, as shown in other models of injury and healing (11).

Our study aims were, therefore, to examine the short-term impact of LVRS on diaphragm muscle with respect to (1) possible fiber injury, (2) specific force, and (3) IGF-I expression in EMP hamsters. Some of our results have been reported in abstract form (12, 13).

Induction and Verification of EMP

EMP was induced in male hamsters (n = 34; initial body weight approximately 100 g; age 4–6 weeks) under general anesthesia by the intratracheal instillation of pancreatic porcine elastase (35 IU/100 g body weight in 0.3 ml 0.9% saline) (Sigma, St. Louis, MO). Control animals (n = 7) received 0.3 ml 0.9% saline intratracheally. EMP was later verified by measuring static pressure volume relationships of the lungs, with the volume at 25 cm H2O being defined as maximum lung volume (MLV; 14).

Animal Groups

Nine months after induction of EMP (9, 10, 15), the animals were divided into several groups: (1) EMP subjected to LVRS studied at either 1 or 4 days after surgery (LVRS-D1 [n = 10] or LVRS-D4 [n = 8]); (2) EMP subjected to bilateral thoracotomies but no LVRS, i.e., sham surgery (EMP-sham; n = 8); and (3) control hamsters subjected to sham surgery (CTL-sham; n = 7). Sham operated groups (CTL-sham and EMP-sham) were comprised of animals studied at either Day 1 (D1) or Day 4 (D4). Data from animals at these two time points were pooled, as they were not different, thus, yielding two final sham groups (i.e., CTL-sham and EMP-sham). In contractility studies, EMP animals not subjected to surgery (EMP; n = 8) were included to control for influences on muscle of anesthesia, bilateral thoracotomy, mechanical ventilation during surgery, long-acting opiate analgesia postsurgery, and the recovery period from anesthesia (reduced food intake and activity). EMP (no surgery) animals would not be subjected to the above major stressors. The experimental protocol was approved by the Cedars-Sinai Medical Center Animal Use and Care Committee.

LVRS

The animal was intubated and mechanically ventilated. LVRS was performed using sequential bilateral thoracotomy approaches (at the sixth-intercostal space) with the aim of removing approximately 30% of each lung, simulating lung volume reduction in patients. Sham surgery in EMP and CTL animals included bilateral thoracotomies as above but without lung resection.

Muscle Fiber Type Identification

This was determined using histochemical and immunohistochemical methods.

Histochemical procedures.

One or 4 days after LVRS, the diaphragm was excised under deep anesthesia and a segment of the midcostal region was mounted at its resting length and frozen in isopentane. Serial diaphragm cryosections were cut at 10 μm, and muscle fibers classified based on differences in staining intensity for myofibrillar ATPase (15, 16). These procedures allow classification of fibers into types I, IIA, IIB, and IIX (15).

Immunohistochemical procedures.

To identify the various myosin heavy chain isoforms in the hamster diaphragm, serial cryosections were incubated in the presence of mouse anti- myosin heavy chain monoclonal antibodies (17). Based on differences in immunoreactivity for myosin heavy chain, muscle fibers were classified as previously described (15).

Muscle Injury: Sarcolemmal Injury

Under general anesthesia, the right internal jugular vein was cannulated. A low molecular weight tracer was used to identify sarcolemmal rupture due to rapid diffusion within the fiber (18, 19). Procion orange, a fluorescent dye tracer (Sigma) was infused over a 30-minute period. Forty minutes after dye infusion, midcostal diaphragm samples were obtained, sectioned across the muscle length, and observed under fluorescent microscopy. Identified procion orange–containing fibers were expressed as a percent of total number of fibers for a given area.

Muscle Injury: Electron Microscopy

Electron microscopy (EM) was used to examine ultrastructural evidence of injury, including sarcomeric disruption, disorganization of myofilaments, misalignment of adjacent sarcomeres, and distortion or absence of Z-lines. Standard muscle processing for EM was employed. Results were expressed quantitatively (percentage of abnormal myocytes) and qualitatively for myofibrillar and mitochondrial status and other miscellaneous abnormalities.

IGF-I Studies
Diaphragm muscle IGF-I.

Diaphragm IGF-I was extracted (20) and assayed using a double-antibody system kit (DSL-2900; Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer's protocol. The rat IGF-I antibody used in the radioimmunoassay (RIA) kit has a high level cross-reactivity with hamster, mouse, and rat antigens (21). We have previously used this method successfully (2123).

Muscle protein concentrations.

Soluble muscle protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA) based on the Bradford method (24).

Immunohistochemical studies: IGF-I.

This has been previously described (21, 22). Sections were incubated in rabbit polyclonal antiserum AFP4892898 specific for IGF-I (provided by the National Hormone and Pituitary Program, Torrance, CA). Cross-reactivity with IGF-II has been reported to be less than 1%. The relative expression of IGF-I in different fiber types was obtained by microdensitometric measurements of IGF-I immunoreactivity. To determine specific fiber types expressing high levels of IGF-I after LVRS, further analysis was restricted to fibers in which IGF-I gray levels exceeded the mean gray levels for all fibers in sham animals.

mRNA studies: IGF-I.

Total RNA was extracted from samples of the costal diaphragm with TRIZOL reagent (Life Technologies, Rockville, MD) according to the manufacturer's protocol.

Oligonucleotides. The primers for IGF-I and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were designed based on published rat cDNA sequences because hamster sequences are not available. See online supplement for primers sequences and GeneBank accession numbers for IGF-I (25) and glyceraldehyde-3-phosphate dehydrogenase (26).

Semiquantitative reverse transcription polymerase chain reaction. Standard methods used for reverse transcription, and reverse transcription–generated cDNA for both IGF-I and glyceraldehyde-3-phosphate dehydrogenase were amplified using polymerase chain reaction. The relative amounts of the polymerase chain reaction products were measured by densitometry.

Contractile Studies

Methods for determining in vitro diaphragm contractile properties have been previously described (9, 10, 15, 27). Briefly, a narrow strip of costal diaphragm was vertically mounted in a tissue bath containing a mammalian Ringer's aerated with 95% O2 and 5% CO2 kept at 26°C. Supramaximal muscle stimulation at Lo was performed using platinum plate electrodes placed on each side of the muscle. Peak twitch force, contraction, and half-relaxation times were determined from a series of single pulses. Force/frequency relationships were measured at stimulus frequencies ranging from 5–100 pulses/second. Diaphragm forces were normalized for physiologic cross-sectional areas (CSA) of the muscle segment and expressed in Newtons /cm2. Diaphragm fatigue resistance was determined after repetitive stimulations over a 2-minute period. A fatigue index was calculated as the ratio of the force after 2 minutes of stimulation to the initial force.

Statistical Analysis

The distribution of data was tested for normality. Statistical analysis was performed by using an analysis of variance (SigmaStat v. 2.0, Jandel Scientific, Erkrath, Germany). If a significant interaction was found, post hoc analysis (Newman-Keuls test) was used to compare differences in independent groups. An α-level of 0.05 was used to determine significance. Values presented are means ± SEM.

Additional methodologic details for all experimental procedures are provided in the online supplement.

Maximal Lung Volumes and Body Weights

Induction of EMP resulted in a 1.6-fold increase in maximum lung volume at 25 cm H2O pressure compared with saline-treated CTL animals (EMP-sham: 18.9 ± 0.8 ml vs. CTL-sham: 12.1 ± 0.4 ml; p < 0.0001). LVRS performed in EMP animals significantly reduced lung volumes by 26% (LVRS: 13.9 ± 0.3 ml; p = 0.0018). However, these lung volumes after LVRS were still significantly greater (15%) than those in CTL-sham hamsters (p = 0.0045). In addition, based on wet weight of resected lung specimens and remaining lung tissue at experiment termination, it was estimated that ∼ 28 ± 3% of total lung wet weight was removed during the LVRS procedure.

After LVRS, body weights fell by 4.8 ± 0.4% at Day 1 and by 12.2 ± 0.6% at Day 4 (see Table E1 in the online supplement for data on all groups).

Sarcolemmal Injury

Diffuse fluorescent uptake of procion orange in the cytoplasm of diaphragm muscle fibers identifies sarcolemmal rupture of those individual fibers. Evidence of sarcolemmal injury was most prominent in EMP animals 1 day after LVRS where 10.9 ± 1.3% of fibers showed diffuse uptake of the fluorescent dye (Figure 1C)

. Significantly less evidence of sarcolemmal rupture was noted 4 days after LVRS in EMP hamsters where only 1.6 ± 1.1% of fibers were positive for procion orange uptake compared with LVRS-D1 animals (Figure 1D; p < 0.001). By contrast, no evidence of sarcolemmal injury was evident in either CTL (0.4 ± 0.1%) or EMP (1.0 ± 0.2%) animals undergoing sham surgery (Figure 1A and B). The percentages of injured diaphragm fibers in the four experimental groups are depicted in Figure 2. Analysis of the diaphragm fiber types exhibiting injury in LVRS-D1 revealed a disproportionate percentage of injured type I and IIA fibers compared with type IIX diaphragm fibers (type I: 34.0 ± 2.4%, type IIA: 46.0 ± 1.9%, and type IIX: 20.0 ± 1.5%; p ⩽ 0.01).

EM Studies

In control sham operated animals, the diaphragm revealed ultrastructurally normal architecture with well-preserved Z bands, normal mitochondria, and glycogen normally distributed (Figure 3A)

. In EMP-sham animals, 13.7 ± 2.8% of myofibers showed ultrastructural abnormalities limited to mild degrees of mitochondrial swelling and fat deposition. By contrast, in animals subjected to LVRS, ultrastructural studies revealed prominent focal abnormalities. One day after LVRS in EMP animals, focal sarcomeric injury was demonstrated in 40.4 ± 2.1% of myocytes. This was evidenced by mild-to-moderate disorganization of myofilaments with wavy Z lines and A bands barely perceptible. Further, mild-to-moderate mitochondrial swelling was evident (Figure 3B). In some instances, complete rupture and separation of sarcomeric structures was noted (Figure 3C) with or without fat deposition or vacuolization. Four days after LVRS in EMP animals, 33.3 ± 9.0% of myocytes were abnormal. Further evidence of ultrastructural damage by Day 4 was characterized by extensive focal areas of vacuolization and severe mitochondrial swelling and sarcomeric disintegration together with lipid droplets and occasional irregular clusters of glycogen often in clear zones of disrupted sarcomeres (Figure 3D). See Table E2 in the online supplement for a summary of EM data.

Biochemical and Immunohistochemical Studies
IGF-I protein.

Diaphragm muscle IGF-I concentrations were significantly increased in EMP animals 4 days after LVRS (Figure 4)

compared with those of LVRS-D1 (p = 0.014) and EMP-sham (p = 0.026). No increment was noted 1 day after LVRS (Figure 4).

IGF-I immunoreactivity.

IGF-I immunoreactivity within individual fibers (Figure 5)

was analyzed to localize those fibers in which IGF-I expression was increased. The mean gray level intensity of IGF-I immunoreactivity within diaphragm fibers was significantly increased in LVRS-D4 hamsters compared with other groups (Figure 6; p < 0.05). In these animals, IGF-I immunoreactivity was disproportionately expressed in the different fiber types with immunoreactivity highest in type IIA fibers (55.3 ± 4.2%), followed by types I (28.6 ± 3.6%), and then by IIX fibers (16.1 ± 2.1%). This somewhat mirrored the distribution of fiber sarcolemmal injury after LVRS and the rank order for their fiber types (i.e., type IIA > I > IIX).

IGF-I mRNA.

Whereas the abundance of the main form of IGF-I (i.e., IGF-IEa) remained unchanged in the diaphragm muscle after LVRS, the abundance of the splice variant IGF-IEb was elevated approximately 3.2-fold compared with sham animals (Figure 7

; p < 0.01).

Contractile Studies

The specific force-frequency curve of the diaphragm in LVRS-D1 animals was significantly shifted downwards, with reductions in specific forces across the entire range of frequencies, compared with EMP-sham and EMP (no surgery) animals (Figure 8

; p < 0.05). For example, maximum specific force in the LVRS-D1 group was reduced by 42.5% and 29.3% compared with EMP and EMP-sham animals, whereas peak twitch force was reduced by 53.5% and 33.8%, respectively. No differences were observed between the groups with regard to Lo. Similarly, twitch characteristics (contraction time/time to peak twitch force; half-relaxation time) and diaphragm muscle fatigability were similar between the groups (see Table E3 in the online supplement).

In this study, the short-term effects of LVRS on the diaphragm of EMP hamsters included (1) focal sarcolemmal rupture and ultrastructural sarcomeric injury of diaphragm fibers; (2) profound force deficit; and (3) upregulation of IGF-I within diaphragm fibers. The diaphragm fibers most affected were those likely recruited during normal ventilatory behaviors (28), thus limiting the reserve capacity and function of the ventilatory pump after LVRS.

Mechanisms of Diaphragm Fiber Injury and Functional Sequelae

We postulate that LVRS would acutely impose a state of passive stretch (beyond Lo) on the foreshortened EMP diaphragm. In preliminary studies, we have demonstrated a 30% increase in resting diaphragm length immediately after LVRS in an EMP animal using implanted sonomicrometry crystals (Fournier and coworkers, unpublished observations). There is evidence that passive stretch of a muscle alone can result in muscle cell injury and impaired force generation (29, 30). Forceful superimposed concentric contractions would be expected to contribute to injury, as the strength of muscle contraction is an important determinant of skeletal muscle injury (31). The type of muscle contraction, another important determinant of injury, may also relate to force generation (32). In the early postoperative period after LVRS, high loads may be presented to the patient (early extubation postsurgery to avoid prolonged air leaks) necessitating increased force generation by the respiratory muscles to meet these demands. Whereas the above considerations could adequately explain diaphragm muscle fiber injury in our model, other possible factors will be briefly discussed. (1) Lengthening (eccentric) contractions of the diaphragm may occur secondary to abdominal (expiratory) muscle recruitment under conditions of load (33), tonic activity of inspiratory muscles (34), or with cough (35). Moreover, the magnitude of muscle injury is greater when lengthening contractions are initiated at a length longer than Lo (36). These circumstances may occur in the perioperative period after LVRS. Finally, the degree of muscle injury after lengthening contractions is greater in the elderly (37, 38), an age profile anticipated in an LVRS population. (2) With LVRS, the geometric conformational changes in the diaphragm could result in alteration in muscle fiber alignment, which together with an increase in chest wall recoil could augment fiber tensile force. Further, nonuniformity of stress and strain could produce regional lengthening of sarcomeres contributing to injury (39, 40).

In the present study, significant force deficit was observed one day after LVRS. It is likely that diaphragm force generating capacity would be even further curtailed by Day 4 after LVRS based on enhanced ultrastructural abnormalities noted and well-established literature in which injury and force deficit peak 3–4 days after the insult (41). In the present study, no significant sarcolemmal rupture was noted in EMP-sham animals. However, mild ultrastructural changes were observed in 13.7% of myofibers in EMP-sham hamsters. This may account for the nonsignificant fall in diaphragm specific force noted in these animals subjected to anesthesia, bilateral thoracotomy, mechanical ventilation during surgery, long-acting opiate analgesia post-surgery, and the recovery period from anesthesia (reduced food intake and activity). It is not known if EMP (no surgery) hamsters would also show diaphragm ultrastructural changes similar to EMP-sham animals. A recent study reported ultrastructural changes in the diaphragm of the EMP hamster (42). However, age may be an important factor enhancing susceptibility to muscle injury (37, 38, 43). In the present study, animals were 10–10.5 months of age, compared with 15–16 months in the Machiels study (42). Our animal model does not simulate all aspects of chronic obstructive pulmonary disease, in which a prominent inflammatory component has recently been highlighted (44). Thus, some of the mechanisms for diaphragm injury reported in chronic obstructive pulmonary disease (45, 46) may differ from the animal model.

One would predict, however, that recovery of impaired contractile function would occur over time. Indeed, 9 weeks after LVRS in EMP hamsters, Marchand and colleagues (47) reported no differences in maximum specific force between EMP animals subjected to LVRS or sham surgery. Furthermore, Shrager and colleagues (48) also reported similar maximum forces 5 months after LVRS or sham surgery in EMP rats.

Injury, Regeneration and Diaphragm Fiber Remodeling: Biochemical Considerations

The IGF axis has been shown to play an integral role in the regenerative processes occurring after muscle injury (11). Whereas IGF-I may be important in mediating myoblast proliferation and satellite cell activation with injury and repair (11, 49), increased expression of IGF-II is believed to promote myoblast differentiation (11). Paoni and coworkers (50) evaluated the time course of IGF gene expression in hind-limb muscles after injury and reported an early rise in IGF-I mRNA peaking at Day 7 and returning to baseline values after Day 10. By contrast, IGF-II expression increased only at Day 7, with levels falling to baseline by approximately Day 15 (50). In the current study, the early increase in IGF-I protein expression in the presence of diaphragm fiber injury after LVRS mirrors changes in the IGF axis described above regarding muscle cell repair. Of interest, the time-course increase in IGF-I immunoreactivity within muscle fibers after eccentric contractions and injury (51) was similar to our findings. In the present study, localization of augmented fiber IGF-I immunoreactivity tended to mirror the fiber-specific distribution of injury. Furthermore, Reynders and colleagues (52) reported increased expression of myogenin mRNA in the EMP diaphragm after LVRS. IGF-I has been reported to induce myogenin (53, 54), and both IGF-I and myogenin are upregulated in skeletal muscle with regeneration (55).

We postulate that stretch and injury provide stimuli for diaphragm muscle length readaptation after LVRS in EMP hamsters. Further passive stretch could reduce the workload of the adapting diaphragm, as active fiber shortening would be aided by recoil from passive elastic properties of the diaphragm under stretch. Shrager and coworkers (48, 56) reported that LVRS restored normal force-length relationships in EMP rats after 5 months, by the addition of sarcomeres in series, as has been well demonstrated in limb muscles (57, 58). Similarly, passive stretch also increased IGF-I mRNA levels and protein content (59, 60). Combined stretch and electrical stimulation in limb muscles further increased IGF-I concentration (59). It is of interest that after 4 days, the newly added sarcomeres at the end of the muscle fibers were mainly of types I and IIA (58). In addition, Goldspink and coworkers (59, 61, 62) have reported on the increased expression of IGF-I splice variants in limb muscles of the rabbit subjected to stretch. Specifically, they describe two main isoforms of IGF-I expressed in skeletal muscle, one which is similar to the main liver IGF-IEa and another isoform that has an additional E domain insert that is designed for local autocrine/paracrine action within muscle (IGF-IEb; also termed mechanogrowth factor). Of interest, the IGF-IEb isoform was markedly upregulated in the rabbit extensor digitorum longus muscle after stretch for 6 days, whereas very little of this splice variant could be detected in the absence of stretch (61). Hill and coworkers. (49) reported an early rise in IGF-IEb mRNA after local muscle injury induced by stretch and electrical stimulation. This preceded evidence of satellite cell activation and the later expression of IGF-IEa mRNA (49). The increase in the IGF-IEb isoform in the diaphragm of EMP hamsters subjected to LVRS in the present study strongly supports the concept of acute muscle stretch after LVRS. Although not measured at Day 1 after LVRS, it is likely that an early rise in IGF-IEb mRNA would have preceded the significant rise in IGF-I protein observed 4 days after LVRS. Thus, local IGF-I action may be an important factor responsible for the enhanced protein synthesis and muscle growth known to accompany stretch, contributing to diaphragm fiber remodeling (i.e., length readaptation) in the long term. This mirrors the increase in diaphragm length noted clinically in patients 3 and 6 months after LVRS (5).

Clinical Implications

Several important clinical implications and strong inferences arise from our data in the animal model. Diaphragm fiber injury after LVRS occurred to the greatest extent in those fibers usually recruited during normal ventilatory behaviors or after the imposition of some load (28). Thus, injury of mainly types IIA and I fibers would be expected to not only curtail force generation but also endurance capacity, as these fibers are the most fatigue resistant (63). Injury of type IIX fibers would be expected to limit short-term, high-force generating potential. The reduction in diaphragm muscle specific forces observed in our study after LVRS would be coupled by further functional geometric decline in force production in vivo due to diaphragm stretch. Thus, the total force generating capacity of the diaphragm in vivo would be even further curtailed. Thus, in the short term, one would expect a compromised ventilatory pump with decreased functional-force reserve and endurance capacity and enhanced susceptibility to diaphragm muscle fatigue and task failure particularly under conditions of increased load (64).

These data highlight how vulnerable the ventilatory pumps of LVRS patients are likely to be early in the postoperative period. This is especially so in patients with very limited ventilatory reserve at baseline, together with the common practice of avoiding mechanical or noninvasive means of ventilatory support postoperatively as a means of preventing or further aggravating air leaks. Indeed, hypercapnia in the early postoperative period after LVRS is extremely common despite normocapnia at baseline. Whereas there are several factors contributing to hypercapnia (pain and splinting, narcotic respiratory depression, impaired load compensation, altered breathing pattern, etc.), significantly compromised diaphragm function almost certainly contributes.

In conclusion, LVRS in the EMP hamster model results in short-term diaphragm fiber injury and impaired diaphragm contractility. We postulate that acute diaphragm stretch post LVRS predisposes not only to fiber injury, but is, in addition, an important mechanotransductive stimulus for fiber regeneration and remodeling as the diaphragm adapts to reduced lung volume.

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Correspondence and requests for reprints should be addressed to Michael I. Lewis, M.D., Cedars-Sinai Medical Center, Division of Pulmonary/Critical Care Medicine, 8700 Beverly Boulevard, Room 6732, Los Angeles, CA 90048. E-mail:

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