Study objective: To examine whether bronchial hyperresponsiveness (BHR) in patients with chronic fatigue syndrome (CFS) is caused by immune system abnormalities.
Design: Prospective comparative study.
Setting: A university-based outpatient clinic (Vrije Universiteit; Brussels, Belgium).
Participants: One hundred thirty-seven CFS patients and 27 healthy volunteers.
Measurements: Pulmonary function testing, histamine bronchoprovocation test, immunophenotyping, and ribonuclease (RNase) latent determination.
Results: Seventy-three of 137 patients presented with BHR, of whom 64 had normal results of the histamine bronchoprovocation test. No significant differences were found in age or sex characteristics between the groups. There were no differences in the RNase L ratio, total lung capacity, or FE[V.sub.1]/FVC ratio between CFS patients with or without BHR. The group of patients in whom BHR was present (BHR+) differs most significantly from the control group with eight differences in the immunophenotype profile in the cell count analysis and seven differences in the percentage distribution profile. The group of patients in whom no BHR was detected (BHR-) only differed from the control subjects in CD25+ count and in the percentage of CD25+ cells. We observed a significant increase in cytotoxic T-cell count and in the percentage of BHR+ patients compared to BHR- patients, which is consistent with the significant reduction in percentage naive T cells.
Conclusions: These results refute any association between the cleaving of 80 kd RNase L and BHR. Immunophenotyping of our sample confirmed earlier reports on (chronic) immune activation in patients with CFS, compared to healthy control subjects. BHR+ CFS patients have more evidence of immune activation compared to BHR- patients. Inflammation and the consequent IgE-mediated activation of mast cells and eosinophils, as seen in asthma patients, is unlikely to be responsible for the presence of BHR in patients with CFS.
Key words: bronchial hyperresponsiveness; chronic; chronic fatigue syndrome; immunology; pathology; ribonuclease L
Abbreviations: BHR = bronchial hyperresponsiveness; BHR+ = bronchial hyperresponsiveness present; BHR- = no bronchial hyperresponsiveness detected; CDC = Centers for Disease Control and Prevention., CFS = chronic fatigue syndrome; CI = confidence interval; DTH = delayed-type hypersensitivity; FITC = fluorescein isothiocyanate; NK = natural killer; P[D.sub.20] = provocative dose of histamine causing a [greater than or equal to] 20% fall in FE[V.sub.1]; PBMC = peripheral blood mononuclear cell; RNase = ribonuclease; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis; 2-5A = 2',5' oligoadenylate; TLC = total lung capacity
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The chronic fatigue syndrome (CFS) is characterized by persisting fatigue causing physical and mental exhaustion. CFS is a multisystem disorder, in which the immune system is of cardinal importance. However, a single diagnostic test has not yet been established. Monitoring dysfunctions in different body systems assists in categorizing this heterogeneous population into clinically relevant subsets.
Airway hyperresponsiveness has been associated with fatigue, shortness of breath, and prolonged generalized fatigue following exercise, which are typical characteristics of CFS patients. In addition, De Meirleir et al (1) reported bronchial hyperresponsiveness (BHR) in 33 of 55 CFS patients (60%) who fulfilled the 1988 Centers for Disease Control and Prevention (CDC) case definition. (2) BHR was assessed using the histamine provocation test. We observed a significantly higher incidence of BHR in CFS patients compared to healthy control subjects (49% vs 19%, respectively) (unpublished data; manuscript under consideration). These two reports provide sufficient evidence for the increased prevalence of BHR in Belgian CFS patients. On the other hand, no attempt has been made to explain the presence of BHR in CFS patients. How does BHR fit into our current knowledge of the physiopathology of CFS? Does it conceal an unexplored mechanism?
Numerous investigators have reported chronic immune activation (3) and immune dysfunction (3-9) in CFS patients. Marked lymphocyte activation in CFS patients was first reported by Klimas et al (3) in 1990 and has been confirmed by many other investigators. On the other hand, a lack of consistency among investigators suggests immune abnormalities in subsets of CFS patients or in particular stages of the disease process. With regard to this, we searched for possible associations between immunity and BHR in CFS patients. We hypothesized that CFS patients with BHR would present more evidence of immune activation, compared to patients without BHR.
Immune dysfunction in CFS patients is characterized by a deregulated 2',5' oligoadenylate (2-5A) synthetase/ribonuclease (RNase) L antiviral pathway. (4,5,10) In physiologic circumstances, type I interferon activates several cellular enzymes in an attempt to neutralize intracellular infections. Among these is the 80-kd RNase L, which blocks viral replication and triggers the cell to undergo apoptosis (programmed cell death). The activated RNase L is the end product of the 2-5A synthetase RNase L antiviral pathway. In subsets of CFS patients, however, a new dysfunctional low-molecular-weight (ie, 37 kd) RNase L has been identified in peripheral mononuclear blood cells (PBMCs). (4,10) By measuring and calculating the amount of low-molecular-weight protein relative to high-molecular-weight protein, we were able to quantify the deregulation of this intracellular pathway. Do CFS patients with marked BHR show associated deregulation of this antiviral pathway?
MATERIALS AND METHODS
Study Setting and Population
The study was conducted in Brussels, at a university-based outpatient clinic (Vrije Universiteit Brussel), and it was approved by the university hospital ethics committee. One hundred forty-seven consecutive patients seeking care for prolonged fatigue as a major complaint, and who fulfilled the 1994 CDC case definition, (11) were enrolled into the study. Subjects were excluded if they were < 18 or [greater than or equal to] 60 years of age. In order to fulfill the CDC criteria for CFS, clinically evaluated, unexplained, persistent or relapsing chronic fatigue that is of new or definite onset should result in a substantial reduction in previous levels of occupational, educational, social, or personal activities. (11) Furthermore, at least four of the following symptoms must have persisted or recurred during [greater than or equal to] 6 consecutive months and must not have predated the fatigue: impairment in short-term memory or concentration; tender cervical or axillary lymph nodes; muscle pain; multijoint pain; headache; unrefreshing sleep; and postexertional malaise for > 24 h. (11) Any active medical condition that may explain the presence of chronic fatigue prohibits the diagnosis of CFS. Therefore, all subjects underwent an extensive medical evaluation, consisting of a standard physical examination and medical history, an exercise capacity test, a symptom checklist (the Goldstein symptom checklist (12)), and routine laboratory tests. The laboratory tests included a CBC count, the determination of the erythrocyte sedimentation rate, a serum electrolyte panel, measures of renal, hepatic, and thyroid function, and rheumatologic and virologic screenings. When judged to be necessary, a structured psychiatric interview was performed. In a number of cases further neurologic, gynecologic, endocrinologic, cardiac, psychiatric, and/or GI evaluations were performed. When positive results were found in any of the evaluations that met the exclusion criteria according to Fukuda et al, (11) the patients were not included in this study. All patients completed a questionnaire, which included demographic information, date of onset, and current health status. Afterward, all subjects were examined by one physician (KDM), who interviewed the patients with respect to their signs and symptoms.
When all differential diagnoses were excluded, and the patients fulfilled the current CFS case definition, (11) patients were tested for the presence of BHR using a histamine provocation test. Pulmonary function testing and immunophenotyping were performed on the same day. In 80 randomly allocated, age-matched and sex-matched CFS patients (patients in whom BHR was present [BHR+], 40; patients in whom BHR was not detected [BHR-], 40), RNase L ratio determination was performed as well (Fig 1). Randomization procedures included stratification according to age, sex, and BHR status. Pulmonary function data were used as inclusion and exclusion criteria (ie, total lung capacity [TLC] of > 80% predicted; and FE[V.sub.1]/FVC ratio of > 70%). Sixty-four of 137 patients presented without BHR, and 73 had a positive histamine provocation test result. These two groups were matched for age as well as sex. All patients and control subjects were white. The demographic data for the sample are presented in Table 1.
[FIGURE 1 OMITTED]
Twenty-seven age-matched and sex-matched healthy volunteers were recruited among college students and hospital employees. Prior to blood collection, they were questioned about medication use and illnesses during the previous 3 months. Subjects participating in this study had to have been healthy for at least 3 months prior to blood withdrawal. In addition, participants were excluded if they were < 18 or [greater than or equal to] 65 years of age. The blood of healthy volunteers was used for immunophenotyping only, not for RNase L ratio determination. Since the aim of this study was not to estimate the prevalence of BHR in CFS patients, the control subjects did not undergo histamine bronchoprovocation or lung function testing.
Pulmonary Function Testing
Patients performed pulmonary function testing and provocation testing after peripheral blood was collected, following the protocol. Pulmonary function testing (SensorMedics; Bilthoven, the Netherlands) included spirometry (ie, peak expiratory flow, FE[V.sub.1], FVC, and forced expiratory flow after exhalation of 75% of FVC), single-breath carbon monoxide diffusing capacity test (ie, for CO transfer factor), body plethysmography (for TLC), and measurements of maximal inspiratory and expiratory pressure static against a closed valve as a measure of respiratory muscle strength. Pulmonary function tests and provocation tests were performed and analyzed following official guidelines, (13,14) Only FE[V.sub.1] and FE[V.sub.1]/FVC ratio results were used in this study (as inclusion and exclusion criteria).
Histamine Bronchoprovocation Test
We used histamine provocation tests, which were all performed in an identical manner by the same technician. Histamine is believed to be a nonspecific, immediately effective, chemical stimulus. The procedure starts with standard lung function tests (ie, spirometry, transfer factor, lung volumes, and lung resistance). After a 5-min break, patients performed a bronchial provocation test according to protocol (Table 2). Each step ends with a new lung function test, followed by a 2-min break. Stimulus administration is realized by means of aerosol dosimeters (MB3; Mefar SPA; Bovezzo, Italy) [flow, 10 mg/s; pressure of compression range, 1.6 to 1.8 kg/[cm.sup.2]]. BHR was considered to be present when the FE[V.sub.1] decreased by [greater than or equal to]20% compared to baseline values on inhalation of a cumulative dose of histamine of [less than or equal to]2 mg (ie, when the provocative dose of histamine causing a [greater than or equal to] 20% fall in FE[V.sub.1] [P[D.sub.20]] was [less than or equal to] 20 mg). A bronchodilator (albuterol [Ventolin; Glaxo Wellcome; Research Triangle Park, NC], two doses of 100 [micro]g) was administrated after the provocation test to patients with BHR. Histamine bronchoprovocation testing is a method with high reproducibility (15) and sensitivity. (16)
Immunophenotyping
Anticoagulated blood (Roche Biochemicals; Mannheim, Germany) was collected between 9:00 AM and 11:00 AM and was used for WBC enumeration, differential cell counts (Celldyn 4000; Abbott Laboratories; Abbott Park, IL), and flow cytometric studies. Lymphocyte populations were analyzed with dual color direct immunofluorescence (EPICS xl flow cytometer; Beckman Coulter; Miami, FL) with the aid of the computer software (System I; Beckman Coulter). Immunofluorescence is an immunologic technique that is used extensively to identify and count particular cells in suspension based on the different surface antigens. First, the cells are stained with different fluorescent reagents to detect surface molecules and are then put through a fluorescence-activated cell sorter that measures the fluorescence intensity. (17) The same procedure is repeated with different fluorescent reagents for each lymphocyte subset (Table 3).
One hundred microliters of whole blood was incubated, using the appropriate combination of monoclonal antibodies, for 25 min at 4[degrees]C. Then, RBCs were lysed using lysis buffer (Becton Dickinson; Franklin Lakes, NJ) for 7 min, spun down, and washed once with 2 mL phosphate-buffered saline solution. Resuspension was immediately followed by cell analysis. Commercially available (Becton Dickinson) phycoerythrin or fluorescein isothiocyanate (FITC) monoclonal antibodies were used and are listed in Table 3. Estimates of the absolute numbers of lymphocyte subsets were determined by multiplying peripheral lymphocyte counts by the percentage of each surface marker.
RNase/L Ratio Determination
The assay is performed by: (1) the preparation of a cytoplasmic extract of the patient's PBMCs, (2) the combination of this extract with a labeled probe that binds specifically to 2-5A binding proteins such as RNase L and the low-molecular-weight species, (3) sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and (4) densitometry to determine the relative quantities of 2-5A binding proteins. A numerical value is calculated by densitometry as the amount of low-molecular-weight protein that is present divided by the amount of native (ie, high-molecular-weight) RNase L present multiplied by a factor of 10. The next paragraph presents a more detailed description of the assay.
Within 4 h of phlebotomy, PBMCs were separated from heparinized blood (30 mL) by density gradient centrifugation (Ficoll-Hypaque; Beckman Coulter). In addition, PBMCs were stored at -70[degrees]C until cytoplasmic extraction preparation. The latter was performed in the presence of protease inhibitors aprotinin, leupeptin, pefabloc-SC, and anticoagulated blood (Roche Biochemicals; Mannheim, Germany). Protease inhibitors were required for preventing proteolytic cleavage. Standard laboratory procedures were used to separate serum from coagulated blood and to store it at -70[degrees]C until analysis. A modified Bradford assay method (Bio-Rad Laboratories; Hercules, CA) was used for the quantification of total proteins in the patients' cell extracts and serum. The probe specifically attaches to 2'-5'A binding proteins like 80-kd RNase L and 37-kd RNase L. Two hundred micrograms of PBMC extract was incubated with a metaperiodate (final concentration, 10 mmol/L; pH 4.75) oxidized 2-5A trimer radiolabeled at the 3' end with (32)P-pCp as the receptor ligand, at 2 to 4[degrees]C for 15 min. In addition, it was covalently attached to the binding proteins by the addition of cyanoborohydride (20 mmol/L in 100 mmol/L phosphate buffer; pH 8.0). This reduction reaction was allowed to progress for 20 min at 2[degrees]C to 4[degrees]C. SDS-PAGE buffer and a tracking dye were added to the samples and were incubated at 95[degrees]C for 5 min followed by separation using standard SDS-PAGE with a 4% stacking gel and a 10% separating gel. The gel was dried, and autoradiography was used to detect the radioactivity of the marked probe (Molecular Imager Fx; Bio-Rad Laboratories). Densitometric analysis of the autoradiographs was followed by quantification of any present 2'-5' A binding proteins using specialized software (Quantity One Software; Bio-Rad Laboratories). The RNase/L ratio was counted using the following equation: RNase/L ratio = [low-molecular-weight RNase L]/[high-molecular-weight RNase L] x 10.
Statistical Analysis
All the data were administered using a database program (Excel 2000; Microsoft; Redmond, WA). The data were coded and transferred to the University of Newcastle (Callaghan, Australia) where the statistical analysis was performed. Data distributions were evaluated for violations of normality. Subject characteristics were assessed using [chi square] test probability and Student t test. Symptom variation was assessed by odds ratio analysis. Univariant group differences were assessed on untransformed data using the Student t test. Immunophenotyping profiles were assessed by forward stepwise discriminant function analysis. The patient classification capacity of the discriminant function module was used to assess the patient compliance within each model. This allowed an evaluation of the predictive capacity of the different immunophenotypes in determining a potential diagnosis of BHR. These data were processed using several different types of software (Access 2000 and Excel 2000; Microsoft; and Statistica, version 5.1; Statsoft; Tulsa, OK).
RESULTS
One hundred thirty-seven CFS patients and 27 healthy control subjects met the inclusion criteria. Sixty-four of the 137 patients presented without BHR, and 73 patients had a positive histamine provocation test result. No significant differences were found in the mean ([+ or -] SD) age (control group, 36.3 [+ or -] 11.4 years; BHR-, 38.0 [+ or -] 8.2 years; BHR+, 37.6 [+ or -] 9.4 years) or the percentage of men (control group, 18.5%; 95% confidence interval [CI], 3 to 34%; BHR-, 25.0%; 95% CI, 11 to 39%; BHR+, 12.5%; 95% CI, 2 to 23%) among the three groups. The prevalence of symptoms differed between the two groups. Patients with BHR presented significantly more often with fatigue that was made worse by physical exercise, recurrent flu-like illness, thyroid inflammation, and painful lymph nodes (Table 4). On the other hand, BHR--patients with CFS appeared to have GI symptoms, paralysis, spatial disorientation, and rashes more often than BHR+ patients with CFS (Table 4). However, no differences were observed concerning the respiratory signs and symptoms (data not shown).
There were no differences in the mean RNase L ratio (BHR-, 19.7 [+ or -] 5.9; BHR+, 6.5 [+ or -] 1.4), TLC (BHR-, 100.1 [+ or -] 1.7% predicted; BHR+, 103.9 [+ or -] 1.6% predicted), or FE[V.sub.1]/FVC ratio (BHR-, 82.4 [+ or -] 1.0%; BHR+, 80.2 [+ or -] 0.9%) between BHR- and BHR+ CFS patients; however, the RNase L ratio approached statistical significance (p = 0.060). There was no difference in the number of patients who had an RNase L ratio of > 2 when comparing BHR- and BHR+ patients (odds ratio, 1.1; 95% CI, 0.4 to 2.9; p = 0.81). Thus, no relationship was found between the RNase L ratio and BHR.
Table 5 shows the results of comparisons of analysis of variance, multivariate analysis of variance, and Tukey honestly significant differences tests among the three groups. The BHR+ group had eight differences in the immunophenotype profile in the cell count analysis (CD25+, CD3+HLADR+, CD19+CD5+, CD4+ CD45RA-, CD3+CD16+CD56+, and CD19+ were increased in the BHR+ patients, while CD2+ and CD4+CD45RA+ were significantly depleted compared to healthy control subjects) and seven differences in the percentage distribution profile (exactly the same as for the cell count analysis, except for CD19+ cells). The BHR- patients only differed from the control subjects in one variable, the CD25+ count and percentage. Comparison of the BHR+ and BHR- groups showed only two differences (ie, the increase in the CD8+CD11b- count and percentage, and the reduction in the percentage of CD4+CD45RA+ cells). Thus, the BHR+ CFS patients had evidence of greater immune activation than did the BHR- CFS patients and the control subjects.
The data also were assessed by discriminant function analysis to determine the profile differences and the number of patients who complied with the immunophenotypic profile determined for each group. Figure 2 shows the canonical scatter plot and the group separation statistics for each group. The discriminant function models were strong for both the percentage and cell count analyses (Table 5), with the percentages of CD25+ count (p < 0.02) and CD2+ count (p < 0.03) being the percentage discriminant variables and the CD25+ count (p <0.008) being the cell count discriminant variable.
[FIGURE 2 OMITTED]
Discriminant function analysis of the differences between the BHR+ and BHR- patients also was assessed. The percentage model was strong (Wilks [lambda], 0.73; F(6,73), 4.45; p < 0.0007) with the percentage of CD4+CD45+RA+ cells (p < 0.009) being the primary discrimination variable, followed by the percentage of CD8+CD11b+ cells (p < 0.03) and the percentage of CD3+HLADR+ cells (p < 0.05). The cell count model was strong (Wilks [lambda], 0.74; F(6,73), 4.37; p < 0.0008) with the CD8+CD11b- cells (p < 0.02) being the primary discrimination variable followed by the CD4+CD45+RA- cells (p < 0.004) and the CD3+HLADR+ cells (p < 0.13).
The 73 patients with BHR were assessed to establish any association between the immune parameters and the histamine provocation test response. The P[D.sub.20] showed a positive correlation with the suppressor cells/natural killer (NK) cell subset (CD8+CD11b+: percentage, r = 0.32; p < 0.006; count, r = 0.30; p <0.01) and NK cells (CD3-CD16+CD56+: percentage, r = 0.33; p < 0.004; count, r = 0.27; p < 0.03). Conversely, the P[D.sub.20] correlated negatively with the memory CD4- cells (CD4+CD45RA-: count, r = -0.28; p <0.02; percentage, r = 0.24; p <0.05), the activated cells (CD25+: count, r = -0.25; p <0.03), and cytotoxic t cells (CD8+CD11b-: count, r = 0.24; p <0.05).
DISCUSSION
BHR has been defined as an exaggerated bronchoconstrictive response of smooth muscles with airway narrowing in response to a small quantity of a nonallergic stimulus that does not provoke such a reaction in healthy subjects. (18) The stimulus can be physical, chemical, or pharmacologic. (15) Airway hyperresponsiveness is characteristic for asthmatic patients but is not specific, as it is also frequently observed in patients with COPD, (19) bronchiectasis, (20) cystic fibrosis, (21) tuberculosis, (22) and sarcoidosis. (23) The concept of using this physiologic response as a diagnostic tool originates from the early 1940s. (24) Bronchial responsiveness to histamine is a method with high reproducibility (15) and sensitivity. (16)
The aim of this study was to reveal the mechanism behind the presence of BHR in CFS patients, rather than estimating its prevalence. Inflammation and consequent IgE-mediated activation of mast cells and eosinophils as seen in asthma patients is unlikely to be responsible for BHR in CFS patients. Indeed, our sample revealed no results in favor of IgE-mediated hypersensitivity. No significant differences in CD19+ or CD19+CD5+ cells were observed between BHR- and BHR+ patients. Immunophenotyping revealed a significant increase in the cytotoxic T-cell count and in the percentage of BHR+ patients compared to BHR- patients, which is consistent with the significant reduction in the percentage of naive T cells. The increase in cytotoxic T cells was confirmed by the correlation analysis (ie, P[D.sub.20] negatively correlated with the CD8+CD11b- count in the BHR+ group). Cytotoxic T cells are crucial in the immune defense action against infected cells. (25) These results may therefore indicate an immune reaction against an intracellular pathogen in BHR+ CFS patients. Candidates are for instance cytomegalovirus, (26-28) adenovirus, (29) or Mycoplasma, (30-32) each of which has been linked to CFS. The antigens of the pathogen may well induce a pathologically delayed-type hypersensitivity (DTH) response, a truly hypersensitive condition characterized by a prolonged DTH response in reaction to a persistent intracellular antigen. (33) On the other hand, the T cells activated in the DTH response are mainly CD4+. Only in a few cases can CD8+ cells be held responsible. DTH responses have been studied in CFS by means of DTH skin tests. However, conflicting results have been reported, (34-37) with more evidence in favor of a reduced DTH skin response. Seeing as no serologic or culture evidence of current or past infections was provided in this study, the infection-induced BHR theory remains hypothetical.
The sensitization of the CNS is characterized by an aggravated response to a low threshold of stimulation (ie, hyperexcitability), as seen in BHR. The proinflammatory cytokine interleukin-1[beta] is known to play a major role in inducing cyclooxygenase-2 and prostaglandin E2 expression in the CNS. (38,39) The up-regulation of cyclooxygenase-2 and prostaglandin E2 sensitizes peripheral nerve terminals. The elevated CNS reactivity inhibits functioning of regulatory pathways for the autonomic, the endocrine, and the immune systems. (40) Central sensitization has already been suggested to occur in CFS (38) and fibromyalgia syndrome. (41-43)
Suhadolnik and colleagues (5) were the first to report evidence for the up-regulation of the 2-5A synthetase/RNase L antiviral pathway in a sample of 15 CFS patients. Two articles (4,10) have indicated the complex nature of this deregulated mechanism in subsets of CFS patients. These studies have suggested an absence of the high-molecular-weight 2-5A binding proteins (80 kd), which leaves us with a new, low-molecular-weight protein (37 kd). By measuring and calculating the amount of low-molecular-weight protein relative to high-molecular-weight protein, we are able to quantify the deregulation of the antiviral pathway. We hypothesized that the observed BHR in subsets of CFS patients could be attributed to a deregulation of the 2.5A synthetase/RNase L pathway. These results do not support a strong association between the cleavage of 80-kd RNase L and BHR, however, the results should be interpreted with caution as the RNase/L ratio does not reveal all components of the 2-5A synthetase/RNase L antiviral pathway. The RNase/L ratio only addresses the relationship between 37-kd and 80-kd RNase L. The bioactive 2.5 A synthetase concentration, the RNase L activity, the RNase L inhibitor concentration, and the absolute amount of low-molecular-weight and high-molecular-weight RNase L are other key components of this antiviral pathway. Future research should address the possible interactions between these components of the 2.5A synthetase/RNase L antiviral pathway and BHR in CFS patients.
One can argue that the methods that were used were not adequate to monitor the pathophysiology of BHR. Indeed, peripheral blood changes may not correspond to the alterations in the capillaries surrounding the lung alveoli. Additionally, smoking has been found to be a risk factor for BHR, (44) although this has been refuted by others. (45) In our study, no attempt was made to stratify comparison groups according to smoking history, because in an unpublished study neither the incidence nor the degree of BHR were different in CFS smokers compared to CFS nonsmokers.
Taken together, these results suggest that BHR in CFS patients is triggered by an immune reaction that is characterized by an increase in the number of cytotoxic T cells and a reduction in the number of naive T cells. Inflammation and the consequent IgE-mediated activation of mast cells and eosinophils are unlikely to be responsible for BHR in CFS patients. Future research should further explore these mechanisms.
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* From the Department of Human Physiology (Mr. Nijs, and Drs. De Becker and De Meirleir), Faculty of Physical Education and Physical Therapy, the Division of Hematology and Immunology (Dr. Demanet), and the Respiratory Division (Dr. Vincken and Mr. Schuermans), Academic Hospital, Vrije Universiteit Brussel, Brussels, Belgium; and the Collaborative Pain Research Unit (Dr. McGregor), Department of Biological Sciences, Faculty of Science, University of Newcastle, Callaghan, New South Wales, Australia.
Manuscript received December 14, 2001; revision accepted September 11, 2002.
Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).
Correspondence to: Jo Nijs, MSc, Vakgroep MFYS/Sportgeneeskunde, AZ-VUB KRO gebouw-1, Laarbeeklaan 101, 1090 Brussels, Belgium; e-mail: Jo.Nijs@vub.ac.be
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