American Journal of Respiratory and Critical Care Medicine

In conditions characterized by airway inflammation, exhaled nitric oxide (eNO) levels are increased. Variable degrees of airway inflammation are present in stable lung transplant recipients (LTR), and may lead to airway remodeling and chronic graft dysfunction. The hypothesis tested is that in stable LTR, eNO concentrations would reflect the expression of inducible (iNOS) (but not constitutive [cNOS] nitric oxide synthase) in the bronchial epithelium as well as the degree of airway inflammation. We determined eNO concentrations in 20 stable LTR, free of infection, rejection, or obliterative bronchiolitis (OB). At routine bronchoscopy, we measured the differential cell count on bronchoalveolar lavage (BAL) and a quantitative assessment of iNOS and cNOS expression in endobronchial biopsies by immunohistochemistry. Mean ± SEM eNO concentrations in stable LTR were not significantly different from control subjects (13 ± 0.7 ppb versus 14.2 ± 0.49; p = 0.42). Percent BAL neutrophils was 11.5 ± 3.2 which was significantly higher than in a group of local control subjects (1.7 ± 0.6; p < 0.001). The bronchial epithelium and lamina propria contained abundant iNOS but cNOS was present only in the lamina propria. Using regression analysis, percent BAL neutrophils (r2 = 0.82; p < 0.0001) and iNOS expression in the bronchial epithelium (r2 = 0.75; p < 0.0001), but not in the lamina propria (r2 = 0.16; p = 0.08), were positively predictive of eNO. There was an inverse relationship between cNOS and eNO. We conclude that eNO concentrations although normal for the group, still reflect the degree of airway inflammation in stable LTR. Epithelial iNOS appears to be the major source of eNO and expression of cNOS may be downregulated with increasing iNOS expression. Gabbay E, Walters EH, Orsida B, Whitford H, Ward C, Kotsimbos TC, Snell GI, Williams TJ. In stable lung transplant recipients, exhaled nitric oxide levels positively correlate with airway neutrophilia and bronchial epithelial iNOS.

Nitric oxide (NO) is an important biological mediator (1). It acts as a neurotransmitter (2), may regulate smooth muscle tone (3), and may be a mediator of inflammation (4). It is synthesized from l-arginine by nitric oxide synthases (NOS) in two forms, constitutive (cNOS) and inducible (iNOS) NO synthase (5). NO produced in the lung can be measured in exhaled air (6). Exhaled NO (eNO) concentrations have been proposed as a noninvasive measure of inflammatory disease in the lung (7-11). Exhaled NO levels are increased in conditions associated with airway inflammation such as asthma (8, 9), bronchiectasis (10), and during upper respiratory tract infections (11). Nonetheless there is little evidence which directly correlates eNO concentrations with the severity of airway inflammation.

Variable degrees of airway inflammation are commonly present in stable lung transplant recipients (LTR) (12, 13). Previous studies have suggested that eNO concentrations in stable LTR are variable but not increased as a group when compared with control subjects (14, 15). While it has been suggested that eNO concentrations may be a non-invasive marker of airway inflammation (7), the precise relationship between airway inflammation and eNO concentrations is unclear and warrants further investigation. We set out to study stable LTR to examine those factors that may predict eNO concentrations and specifically to determine if a relationship exists between eNO and airway inflammation in the absence of overt disease. We chose stable LTR because the spectrum of severity of airway inflammation seen allows plausible regression analysis to be performed with the variable eNO concentrations that may be found.

We have previously shown that in stable LTR, bronchoalveolar lavage (BAL) neutrophilia is not equivalent to the degree of neutrophilia in endobronchial biopsies but may approximate it (12). We therefore used the measurement of BAL neutrophilia as an easily quantifiable and less invasive marker of airway neutrophilia.

When asthmatics are treated with inhaled corticosteroids, eNO concentrations return to normal (16) providing indirect evidence that the increased eNO concentrations reflect upregulation of iNOS which can be downregulated by corticosteroids (17). There is, however, little evidence directly linking eNO concentrations quantitatively with both iNOS and cNOS activity in bronchial epithelium and adjacent areas (18). We were interested in exploring the relationship between eNO and a quantitative assessment of iNOS and cNOS in stable LTR.

We therefore set out to test the following two hypotheses: first, that in stable LTR, eNO concentrations would increase in direct proportion to the degree of BAL neutrophilia that is commonly present (13), and second, that eNO concentrations would reflect both the intensity of iNOS immunostaining and its proximity to the airways, i.e., iNOS in the bronchial epithelium but not iNOS in the lamina propria.

Study Population and Inclusion Criteria

Twenty LTR (mean age 49 ± 3 yr; 12 male) were studied. All were clinically stable at a median of 288 (range 90 to 550) d post-transplant. At the time of the study, all patients were at or near their best postoperative lung function (mean percent of best FEV1 post-transplant 98.6 ± 0.3%). There were 13 bilateral sequential LTR (bronchiectasis [seven patients], cystic fibrosis [four], and emphysema [two]) and seven patients had received single lung transplants (emphysema [six] and pulmonary fibrosis [one]). In the single LTR, the bronchoscopic study involved only the allograft.

Patients were studied at the time of routine surveillance bronchoscopies. Six additional patients were not recruited owing to the presence of clinically significant infection or rejection which required treatment. The 20 patients comprising the study group were afebrile, and had no recent change in physical signs, peripheral blood leukocyte count, or chest radiograph. In addition, their airways were clear of purulent secretions on bronchoscopic examination. In all cases, gram stains for bacteria were negative and cytomegalovirus (CMV) cellular inclusions were not detected in either BAL or transbronchial biopsy samples. Although microorganisms were occasionally isolated after BAL fluid culture, these were considered to be commensals and not treated.

The study was approved by The Alfred Hospital ethics committee and written informed consent was obtained from each patient.

Bronchoscopic Procedures

At bronchoscopy, BAL, transbronchial biopsies (TBB), and endobronchial biopsies (EBB) were obtained. For BAL, a total of 180 ml of warmed (37° C) phosphate-buffered saline solution was instilled in the right middle lobe or lingula in three 60-ml aliquots, with immediate aspiration at a negative pressure of approximately −80 mm Hg. On average 6 EBB specimens were taken from lower lobe subcarinae using alligator forceps (FB 15C; Olympus, Tokyo, Japan) as previously described (12). TBB were taken in the same way from several segments as part of the normal follow-up surveillance protocol of LTR and were assessed for the presence of acute rejection or opportunistic infections by standardized criteria (19).

A volume of 10 ml of pooled BAL fluid was retained for microbiological testing. The BAL total and differential cell count was determined by a standard method (12). Based on local normal values obtained from nonsmokers (12), lavage cell differential was considered normal if values were as follows: lymphocytes ⩽ 20%, neutrophils ⩽ 4%, and eosinophils ⩽ 2%.

Immunohistochemistry

Immediately after bronchoscopy, EBB were snap-frozen in a liquid nitrogen–isopentane slurry. One EBB taken from each subject was chosen on the basis of morphological criteria assessed after staining a section with Quick Dip (Histo Labs, Melbourne, Australia). Immunohistological staining for iNOS and cNOS was performed on duplicate 7-μm sections, approximately 35 μm apart, using monoclonal mouse anti-human iNOS and cNOS antibody, respectively (Transduction Laboratories, Lexington, KY) and amplified using a biotinylated anti-mouse and avidin–biotin horseradish peroxidase complex method (12). Isotype control immunoglobulins (IgG2a and IgG1; Dako, Glostrup, Denmark) and nasal polyps were used as negative and positive controls respectively. Staining was done in one run by a single experienced person.

Five nonoverlapping high-power fields were counted from each of the two sections of each biopsy. The slides were coded and counted by one blinded observer using a computerized image analyzer (Image Pro Plus 3.0 for Windows; Media Cybernetics, Silver Spring, MD). In the lamina propria, positive NOS staining was scored if it was exclusively intracellular or localized to vessel wall. Results were expressed as positive cells/mm2 or positive vessels/mm2. The between-section coefficient of variability was < 12% for all measurements. Slides from a subset of patients were recoded and counted on a second occasion. The mean intraobserver coefficient of variability was < 10% for all measurements.

The scoring of NOS staining within the bronchial epithelium proved more difficult, and was based on a previously validated semiquantitative method (20). It was performed by two blinded observers. The staining within the epithelium was assessed for both intensity and extent in each of five high-power fields from the two sections of each biopsy using a visual analogue scale of 0 to 5 in 0.5 increments. The mean of both observers' score for intensity and extent in each area was added so that a maximum score of 100 was possible [(5 + 5) × 10] for each biopsy. The mean difference ± 2 SDs between the two observers' scores for intensity and extent was 0.3 ± 0.7 and 0.5 ± 0.9 respectively. The mean intraobserver coefficient of variation was 6% for intensity and 12% for extent of staining.

eNO Measurements

eNO was measured immediately prior to bronchoscopy using a rapidly responding, highly sensitive chemiluminescence analyzer (model 270 B; Siever, Boulder, CO) with a resolution of 0.3 parts per billion (ppb) of NO and response time (0–95% rise time) of 0.7 s. The sampling rate was 250 ml/min for all measurements. The analyzer was calibrated using medical grade NO at concentrations of 0 ppb (Nitrogen High Purity Gas; Air Liquide Australia, Melbourne, Australia) and 900 ppb (CIG Special Gases; Chatswood, Australia) diluted by a mass flow calibrator (Advanced Pollution Instrumentation, San Diego, CA).

NO measurements were performed as previously described (21). Patients inhaled NO-free air (Medical Air; Air Liquide Australia, Melbourne, Australia). As NO concentrations are highly flow-dependent (21), a constant flow rate of 5.95 L/min was maintained against a fixed resistance ensuring closure of the internal nasal route thus eliminating nasal NO contamination of the exhaled gas (22).

The single breath test is characterized by a short NO peak followed by a longer NO plateau. The plateau concentration of NO when performed by the method described has been shown to be reproducible and to reflect lower airway NO release (23). The mean plateau concentration of three technically acceptable measurements was recorded. eNO levels performed in the same standardized method were also recorded on 50 healthy nonsmoking control subjects without history of asthma or allergic rhinitis.

Lung Function

Spirometry was performed immediately prior to bronchoscopy. A computerized rolling-seal spirometer (SensorMedics, Yorba Linda, CA) was used to measure flow–volume loops.

Statistical Analysis

Data are expressed as means (± SEM) unless otherwise indicated. Comparisons between groups were made using the unpaired Student's t test for parametric data and the Mann-Whitney test for nonparametric data. A correction for multiple testing (Tukey) was applied. To determine parameters that predicted the eNO concentration (dependent variable), univariate and multivariate regression analyses were performed.

A standard bronchoscopy with BAL, EBB, and TBB was performed in all 20 patients at time of clinical, radiologic, and physiologic stability. None of the study group had evidence of significant [A2 mild or higher grade (19)] rejection, and organisms were cultured from the BAL from four patients. In the absence of any other clinical, radiologic, or laboratory supportive evidence, they were all considered to be commensals. None of the four received antimicrobial therapy and no adverse sequelae resulted.

Mean ± SEM eNO concentrations for the lung transplant group was 13.0 ± 0.7 ppb (range 6.4 to 18.3) compared with 14.2 ± 0.49 ppb (range 11.2 to 16.1) in normal control subjects (p = 0.42). In the lung transplant group neither the underlying disease nor transplant procedure (single versus bilateral lung) affected the eNO level.

BAL cell differentials are shown in Figure 1. Mean (SEM) percent neutrophils, lymphocytes, and eosinophils were 11.5 (3.2) (range 0.1 to 55%), 14.1 (2.1), and 0.5 (0.2) respectively; all other cells being alveolar macrophages. The percent BAL neutrophils in our group was significantly higher than percent BAL neutrophils found when using the same lavage procedure in a cohort of 20 local control subjects (1.7 ± 0.6; p < 0.001). A relative BAL neutrophilia was present in 10 patients, relative lymphocytosis in two, and mixed lymphocyte and neutrophil pattern in two.

The immunohistochemical findings on the EBB of individual patients are shown in Table 1, and illustrative examples of staining for iNOS and cNOS are shown in Figures 2A and 2B respectively. iNOS was present in both the bronchial epithelium and within macrophages and neutrophils within the lamina propria of all LTR. It was not found in the endothelium. Bronchial epithelium did not contain cNOS in 17 subjects and in the other three it was very faint. On the other hand, cNOS staining was present to a significant degree in both the lamina propria cellular population and endothelium.

Table 1. eNO CONCENTRATIONS AND ENDOBRONCHIAL BIOPSY IMMUNOHISTOCHEMICAL FINDINGS IN INDIVIDUAL PATIENTS

No.LTxeNO (ppb)Epithelial iNOS Score* Lamina Propria iNOS (cells/mm2 )Lamina Propria cNOS (cells/mm2 )Endothelial cNOS (vessels/mm2 )
 1BSLT13.117.541.225.338.0
 2BSLT11.712.540.736.042.4
 3(R)SLT 9.2 1.317.644.563.5
 4BSLT18.327.566.820.220.2
 5BSLT13.211.298.455.355.3
 6BSLT15.319.234.411.823.6
 7(R)SLT 7.9 4.129.251.851.8
 8BSLT11.3 3.124.030.466.9
 9BSLT11.3 9.433.256.067.2
10BSLT14.527.542.134.039.5
11(R)SLT16.622.561.027.421.9
12(L)SLT17.125.036.4 7.815.6
13(R)SLT15.318.826.256.028.0
14BSLT 6.4 6.824.033.027.6
15BSLT12.913.7532.422.017.6
16(R)SLT14.020.552.120.220.2
17BSLT 9.7 3.122.343.030.7
18(L)SLT14.017.824.934.417.2
19BSLT17.620.036.411.629.0
20BSLT 9.912.546.629.847.7

Definition of abbreviations: BSLT = bilateral sequential lung transplant; LTx = lung transplant procedure; (R) or (L) SLT = right or left single lung transplant.

* Intensity and extent of iNOS staining in bronchial epithelium.

To determine which factors predicted eNO concentration, regression analysis was performed with eNO as the dependent variable. The results are summarized in Table 2. iNOS staining in the epithelium was strongly associated with eNO levels as was percent BAL neutrophilia. There was a linear relationship between epithelial iNOS score and eNO (Figure 3A; r2 = 0.74, p < 0.0001). Although a significant linear relationship between percent BAL neutrophil and eNO could be determined (r2 = 0.57, p < 0.001), the curve of best fit suggested that the relationship was logarithmic (Figure 3B; r2 = 0.81, p < 0.0001).

Table 2. RESULTS OF REGRESSION ANALYSIS WITH eNO AS THE DEPENDENT VARIABLE

UnivariateMultivariate
rp Valuer2 p Value
iNOS epi score0.860.00001iNOS epi score0.420.0001
iNOS l.p.0.400.08%BAL neutrophil0.190.0068
cNOS l.p.−0.530.017iNOS l.p.0.020.48
cNOS endothelial−0.540.014cNOS l.p.0.0020.79
%BAL neutrophil0.760.0001cNOS endothelial0.010.54
%BAL lymphocyte−0.070.76%BAL macrophage0.030.43
%BAL macrophage−0.660.016Days post LTx0.050.15
Age−0.010.96Actual FEV1 0.010.64
Cyclosporine level0.150.54
Days post Ltx−0.360.12
Actual FEV1 −0.170.52
% predicted FEV1 0.010.96

Definition of abbreviations: cNOS = constitutive nitric oxide synthase; epi Score = intensity and extent of iNOS in bronchial epithelium; iNOS = inducible nitric oxide synthase; l.p. = lamina propria; LTx = lung transplant procedure.

cNOS staining in lamina propria and endothelium and percent BAL macrophages were negatively associated in univariate analysis with eNO concentrations (Table 2). When examined by univariate analysis there was a trend toward a positive association between iNOS staining in the lamina propria and eNO concentrations (r2 = 0.16; p = 0.08). Neither actual FEV1 nor percent predicted FEV1 were associated with eNO concentrations.

All potentially significant variables on univariate analysis were examined for interrelationships by multivariate stepwise analysis. Only epithelial iNOS and percent BAL neutrophilia remained predictive of eNO concentrations.

eNO concentrations are increased in conditions associated with airway inflammation. Variable degrees of airway inflammation are known to be present in stable LTR. Previous studies have suggested that eNO concentrations are increased in some stable LTR but not in this group as a whole. We examined the eNO level in 20 stable LTR to systematically determine if a relationship exists between eNO, indices of airway inflammation, and NOS expression in the absence of overt disease. We confirmed that in stable LTR, eNO concentrations are not increased when compared with control subjects. However, we found that the variability of eNO concentrations between stable LTR was directly associated with the expression of bronchial epithelial iNOS and with the degree of BAL neutrophilia.

We confirmed previous reports (14, 15) that eNO concentrations in stable LTR as a group are not higher than those of control subjects. This presumably reflects that when compared with asthma or post-transplant obliterative bronchiolitis (OB), the degree of airway inflammation is mild (12) but also that eNO concentrations may be reduced by the use of chronic immunosuppression (16). The eNO concentrations that we found in normal control subjects were similar to those of other investigators (21) when using a similar expiratory flow rate. The range and variability of eNO concentrations were greater in the lung transplant group compared with the control group, which may reflect the spectrum of BAL neutrophilia that we found in the stable LTR. We chose stable LTR precisely because of this spectrum of BAL neutrophilia that varied from neutrophil concentrations that were equivalent to those found in local control subjects to neutrophil levels that were moderately increased. This spectrum, in a group with a wide range of eNO concentrations maximizes the potential to determine what, if any, relationship exists.

Increased proportion of inflammatory cells (neutrophils and/or lymphocytes) in BAL was present in 14 of 20 (70%) LTR. This predominantly reflected an elevation in percent BAL neutrophils. Mean percent BAL neutrophils in our group was significantly higher than in a cohort of 20 local control subjects. Significant airway infection was excluded by recruiting subjects in the prospectively screened absence of overt infection, as assessed by clinical and microbiological criteria. The finding of elevated BAL neutrophils in this study confirms previous work in our laboratory (12) and others (13), suggesting that even in stable LTR, neutrophilic airway inflammation is common and a characteristic of the condition per se.

We are, to our knowledge, the first group to have shown that eNO concentrations are directly associated with the severity of BAL neutrophilia. eNO levels were increased in direct association with percent BAL neutrophils. Further, this relationship persisted when examined by multivariate regression analysis. The relationship appeared to be logarithmic. We analyzed differential cell counts because of the additional inherent variability in performing total cell counts. Nonetheless, a significant relationship was also found between eNO and log absolute BAL neutrophil count (r2 = 0.56, p < 0.001).

The development of post–lung transplant OB is the commonest cause of late graft failure and is characterized by initially inflammatory and subsequently proliferative phases (24). In patients with OB, when compared with stable LTR, there is more intense airway inflammation (12, 13) and eNO concentrations are higher than in either control subjects or stable LTR (14, 15). It has been postulated that increasing airway inflammation in stable LTR predisposes to the development of airway remodeling and OB (12,13). A longitudinal study of this cohort is being performed to examine the role of serial eNO measurements in predicting the development of worsening airway inflammation and subsequently the early detection of OB.

We found that EBB contained abundant iNOS but not cNOS in the bronchial epithelium. Constitutive NOS was present only in the lamina propria cellular population and localized to the endothelium. We examined EBB because previous work has suggested that in LTR, there is pan-airway inflammation (12, 13, 25) and because TBB do not consistently provide adequate airway samples (19).

We quantified NOS expression in different areas of the airway and present the novel finding that when examined univariately, eNO concentrations were directly associated with epithelial iNOS and the relationship with iNOS expression in the lamina propria failed to reach statistical significance. Further, on multivariate analysis the relationship between eNO and iNOS in the bronchial epithelium persisted but there was no relationship between eNO and iNOS in the lamina propria. Saleh and colleagues (26) found that in asthmatics, eNO concentrations correlated significantly with a semiquantitative assessment of iNOS expression in bronchial epithelium and to a lesser extent lamina propria. However, they did not perform multivariate regression analysis so that the relative role of each area was unclear.

Our findings confirm that in stable LTR, in association with BAL neutrophilia, upregulated bronchial epithelial iNOS rather than iNOS in the lamina propria is best correlated to the measured eNO concentrations. Because of its unpaired electron, NO is highly reactive (1). It seems probable that NO produced by upregulated iNOS in the lamina propria is less able to diffuse into the airway lumen, and thereby contribute to measured eNO concentrations, than NO produced in the airway epithelium. This is consistent with the findings of Fisher and colleagues (14) who found that in a group of LTR, the presence of perivascular inflammation (i.e., acute graft rejection) alone was not sufficient to cause an increase in eNO if the pulmonary epithelium was not involved.

In our group, cNOS was inversely related to the eNO concentration. When examined by multivariate analysis, this relationship disappeared suggesting that cNOS expression was reduced in association with increased iNOS expression. It is possible that downregulation of cNOS may reflect a negative feedback response resulting from the iNOS-derived increased NO production. This is in keeping with the findings of Ravichandran and colleagues who found that increased NO downregulates cNOS activity (27). However, although NO may downregulate cNOS activity it is unlikely to affect the enzyme protein level. It is more likely that, as suggested by others (28– 32), the downregulation of cNOS expression that we found reflects the underlying inflammation per se. Additionally, it is possible that chronic immunosuppression with cyclosporine and azathioprine may downregulate cNOS expression.

Although we did not study different population groups, it would appear that the localization and intensity of NOS staining may be dependent upon the population studied. In conditions associated with marked airway inflammation such as asthma, iNOS was similarly found to be expressed in the airway epithelium (25). Watkins and coworkers found that iNOS but not cNOS was expressed in association with inflammation in the airway epithelium of six patients undergoing surgical resection for lung cancer (28). In most studies of normal subjects free of inflammation (29-32), cNOS rather than iNOS was predominant in the airway wall.

Previously we have examined EBB from 12 nonsmoking, nonatopic control subjects. We found that iNOS staining in the bronchial epithelium of stable LTR exceeds that of control subjects (mean ± SEM, 14.7 ± 1.84 versus 3.4 ± 0.6 (20); p < 0.01). Furthermore, cNOS staining was present in the bronchial epithelium of all 12 control subjects compared with only three of 20 stable LTR (p < 0.03) and cNOS staining in the lamina propria of control subjects exceeded that of stable LTR (51.8 ± 7.9 cells/mm2 versus 32.5 ± 3.3 cells/mm2; p < 0.02). Therefore our findings are consistent with the view that compared with airways of control subjects, the presence of airway inflammation is associated with upregulation of iNOS and perhaps downregulation of cNOS.

Our findings suggest that bronchial epithelial iNOS production is the major source of eNO in conditions in which airway inflammation is present. However, by using multivariate analysis we found that changes in bronchial epithelial iNOS expression accounted for only 42% of the variability of eNO. Further, iNOS staining was present in inflammatory cells in the lamina propria. This suggests that inflammatory cells are, at least in part, a source for eNO and perhaps more importantly that human inflammatory cells, as suggested by others (33), are capable of generating functional NO.

A potential criticism of our findings is that we examined single as well as bilateral lung recipients. Changes in regional flow rates (34) may independently affect eNO concentrations which may be of particular relevance to single LTR in whom the transplanted and native lung are likely to have different flow characteristics. However, we found that the relationship between eNO, BAL neutrophilia and iNOS was present if either single or bilateral LTR were examined independently, suggesting that despite this potential concern our results remain valid.

Although mean eNO concentrations in stable LTR were not different from that of control subjects, the variability and range of eNO concentrations was. Stable LTR as a group displayed concentrations that were both higher and lower than that of control subjects. As eNO concentrations were highly correlated in stable LTR with both percent BAL neutrophilia and bronchial epithelial iNOS, the highest eNO concentrations were found in those with the highest percent neutrophils in the lavage. The low eNO concentrations were found in those in whom BAL neutrophilia was absent (Figure 3B). We would postulate that these concentrations were significantly lower than those found in control subjects because of the associated downregulation of cNOS which, in the absence of airway inflammation, is likely to be the major source of eNO (7, 16, 17).

eNO concentrations are being used as a noninvasive measurement of airway inflammation and there is indirect evidence that this is valid (7-11, 18). We have shown that in stable LTR, eNO concentrations are highly dependent upon the severity of BAL neutrophilia and the intensity and extent of expression of iNOS in the bronchial epithelium but not in the subepithelial area. We found that, in this group, in association with increasing iNOS expression and BAL neutrophilia, cNOS is downregulated. Our results therefore confer biological plausibility to the contention that eNO is a valid noninvasive measure of airway inflammation and suggest that serial measurements of eNO concentrations may have a role in the early detection of conditions, such as OB, in which graft airway inflammation is prominent.

Supported by Alfred Hospital Research Trust, Glaxo Wellcome (UK), Department of Respiratory Medicine Scholarship, Alfred Hospital Whole Time Medical Specialists Trust, and National Health and Medical Research Council (Australia).

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Correspondence and requests for reprints should be addressed to Dr. Trevor Williams, Head, Lung Transplant Service (Medical), Department of Respiratory Medicine, Alfred Hospital, Commercial Road, Prahran, Victoria 3181, Australia. E-mail:

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American Journal of Respiratory and Critical Care Medicine
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