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Published ahead of print on November 6, 2003, doi:10.1164/rccm.200307-979OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 220-226, (2004)
© 2004 American Thoracic Society

Opposing Effects of Short- and Long-term Stress on Airway Inflammation

Paul Forsythe, Cory Ebeling, John R. Gordon, A. Dean Befus and Harissios Vliagoftis

Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta; and Immunology Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Correspondence and requests for reprints should be addressed to Harissios Vliagoftis, M.D., Pulmonary Research Group, Room 550 HMRC, University of Alberta, Edmonton, AB, Canada, T6G 2S2. E-mail: hari{at}ualberta.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Between 20% and 35% of subjects with asthma experience asthma exacerbations during periods of stress. The biological mechanisms underlying these exacerbations are not clearly understood, and the role of psychologic factors in the pathophysiology of asthma remains controversial. We investigated the ability of psychologic stress to modulate airway inflammation and airway hyperresponsiveness (AHR) to methacholine in a murine model of asthma. Animals were exposed to a stressor daily for 3 (short-term stress) or 7 (long-term stress) days. After allergen challenge, AHR was assessed through plethysmography, and bronchoalveolar lavage cells were counted as a measure of inflammation. After short-term stress, inflammatory cell number was decreased compared with unstressed animals, whereas levels of interleukin (IL)-6, IL-9, and IL-13 were increased. Administration of a corticosteroid receptor antagonist, before stress, prevented the decrease in inflammatory cell numbers. In contrast, animals stressed for 7 consecutive days showed a significant increase in inflammatory cell numbers, which was independent of the glucocorticoid response, but no change in cytokine levels. AHR was not altered in stressed animals. Our results indicate that repeated exposure to stress over the long term engages different mechanisms than short-term stress and can exacerbate the chronic inflammatory responses of the airway.

Key Words: stress • inflammation • asthma • mouse

Prospective epidemiologic studies have indicated an association between life stress and health (1). However, the role of psychosocial factors, especially stress, in the pathophysiology of asthma remains controversial (2–5). Although there is no direct evidence of an effect of psychologic stress on pulmonary disorders, studies suggest that between 20% and 35% of subjects with asthma experience exacerbations of symptoms during periods of stress (4). Moreover, psychologic distress in children has been associated with asthma that is more difficult to manage (6), with more frequent and lengthier admissions to the hospital (7) and greater functional disability (8). Some patients with asthma experience increased bronchoconstriction in response to acutely distressful situations (9), and stress caused by academic examinations causes an increase in eosinophilic inflammation and interleukin (IL)-5 production in otherwise healthy subjects with mild asthma (10). Although the mechanisms linking anxiety and asthma are poorly defined, an important contributing factor may be stress-induced modulation of immune and inflammatory processes.

Stress can be defined as the psychophysiologic reaction of the body to a variety of emotional or physical stimuli that threaten homeostasis. A relationship between stress and inflammation has been proposed based on human studies showing that emotional stress exacerbates symptoms of inflammatory disorders such as rheumatoid arthritis and inflammatory bowel disease (11–14). It is well established that the inflammatory response is controlled by the central nervous system; however, communication between the brain and the inflammatory processes is complex, involving bidirectional signaling pathways interconnecting the central and peripheral nervous systems with endocrine and immune responses. Stress can modulate the immune response through activation of the hypothalamus–pituitary–adrenal (HPA) axis and the sympathetic nervous system leading to release of cortisol and catecholamines that can influence cell trafficking, proliferation, and function, including cytokine and inflammatory mediator production (15–17).

Conflicting results regarding modulation of the inflammatory response have been obtained using different experimental approaches to induce psychologic stress in rodents. These differences seem to be related not only to the inflammatory model used but also to certain characteristics of the stressful event that modulate its impact on the inflammatory process (15, 18–25). Duration of exposure to stress appears to be one important variable, and stress has markedly different effects on the immune system depending on whether it is acute or chronic. Dhabhar and McEwan demonstrated that acute psychologic stressors augment the immune response and result in adrenal hormone-mediated redistribution of immune cells into the bone marrow, lymph nodes, and skin (26). Acute stress also heightens antigen specific cell-mediated immunity, alters populations of T-cell subsets, and modulates mononuclear cell trafficking (16). In contrast, chronic stress seems to depress the migration of immune cells from the blood, an effect that correlates with the attenuation of responsiveness to corticosteroids (16, 26).

Although there is mounting evidence from studies in animal models of a role for stress in the initiation and/or exacerbation of arthritis and inflammatory disorders of the gut (20, 27–29), there is a paucity of information on stress-induced modulation of allergic inflammation and function in the airway. In this study, we investigate the impact of repeated exposure to inescapable psychological stress on airway inflammation and hyperresponsiveness in a murine model of asthma and determine influence of duration of stress exposure on the immunomodulatory response. Portions of the data presented in this article have previously been published in abstract form (30, 31).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Adult male BALB/c mice (20–25 g) (Charles River Breeding Laboratories, St. Constant, PQ, Canada) were maintained in an automatic light/dark cycle (light periods of 12 hours) and fed water and chow ad libitum. Mice were acclimatized to the animal facility for 1 week before experimentation. Age-matched animals were used in all experiments. These experiments were in accordance with guidelines of the Canadian Council for Animal Care.

Restraint Stress
Each mouse was placed in an adequately ventilated 50-ml conical plastic tube (Corning, Corning, NY) for 1 hour at the same time each day (between 12:00 and 2.00 P.M.). Mice were not physically squeezed and felt no pain. This restraint allowed the mice to rotate from a supine to prone position, but not turn head to tail. This type of restraint is generally regarded as inducing psychologic stress on the animal (32, 33). Nonrestrained mice were left in their cages for the duration of the experiment.

Forced Swim Stress
Forced swim was used as it is regarded as a paradigm for depression and anxiety in animals (34, 35). Mice were placed in plastic tanks filled with water (25°C) to a height of 15 cm for 20 minutes. Animals were then towel dried and left in their home cage for 10 minutes before any further procedures. Control mice were left in their cages for the duration of the experiment.

Immunization and Airway Challenge with Antigen
Mice were sensitized to ovalbumin (OVA) with intraperitoneal injections containing 10-µg OVA and 150-mg Al(OH)3 in a total volume of 500-µl saline on Days 1 and 6. In the short-term stress protocol, mice were challenged by the airways with OVA (5% in saline) for 5 minutes by ultrasonic nebulization on Days 12 and 14 (Figure 1A) , whereas in the long term protocol, mice were challenged on Days 12 and 16 and 18 (Figure 1B). In both the short- and long-term protocols, a control group (n = 4) of mice was sensitized to OVA and challenged with saline alone.



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  		Figure 1. Diagrammatic representation of the protocols for immunization, challenge, and stress for both short-term (A) and long-term (B) experiments (see METHODS for details). BAL = bronchoalveolar lavage; OVA = ovalbumin.

 
Stress Protocols
Mice in the short-term stress group were exposed to restraint stress on 3 consecutive days starting immediately before the first OVA challenge (Figure 1A). Animals in the long-term stress group were exposed to restraint stress for 3 consecutive days starting immediately before their first OVA challenge and then to avoid habituation to a homotypic stressor mice were exposed to restraint stress immediately followed by swim stress for 4 consecutive days (Figure 1B). At 24 hours after the final antigen challenge, mice were assessed for airway hyperresponsiveness (AHR) and airway inflammation.

Corticosteroid Receptor Anatagonist Treatment
Where indicated, the corticosteroid receptor antagonist mifepristone (RU486) (Sigma Chemical Co., St. Louis, MO) in polyethylene glycol (Sigma) was administered intraperitoneally (6 mg/kg) 1 hour before every stress exposure. Vehicle alone was administered intraperitoneally to control mice.

Airway Inflammation
Airway inflammation was assessed by inflammatory cell counts in bronchoalveolar lavage (BAL) fluid. Cells were removed from BAL fluid by centrifugation at 200 x g for 15 minutes, and supernatants were stored at -80°C until evaluation of cytokine content. Cells were resuspended in phosphate-buffered saline (1 ml). BAL cells were stained with trypan blue, and viable cells were counted using a hemocytometer. Smears of BAL cells were prepared with a Cytospin (Thermo Shandon, Pittsburgh, PA) and stained with HEMA 3 reagent (Biochemical Sciences, Swedesboro, NJ) for differential cell counts, where a total of 200 cells were counted for each lavage.

Cytokine Assays
ELISA procedures were used to detect IL-6, IL-9, IL-10, IL-13, and IFN-{gamma}, as described previously (36).

Airway Responsiveness
At 24 hours after the final OVA challenge, bronchial reactivity to aerosolized methacholine was measured using the Buxco whole-body plethysmograph (Buxco Electronics, Troy, NY) as described (37). Unrestrained, conscious mice were placed in the chambers, and after 10 minutes of stabilization, increasing concentrations of methacholine (2 to 32 mg/ml) were aerosolized for 3 minutes each, and mean enhanced respiratory pause values were obtained over 5-minute periods.

Statistical Analysis
In comparing cell number or cytokine levels, analysis of variance was used to determine the level of differences among all groups followed by the Bonferroni posttest for control versus stress comparisons. Differences in AHR were determined by F test analysis that compared the entire curves of each treatment group. From this F test, a p value was generated. In all cases, a p value of less than 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Populations in BAL Fluid
Total cell numbers in BAL fluids were significantly increased 24 hours after the final OVA challenge in sensitized mice compared with saline-challenged mice (1.9 ± 0.3 x 106 and 0.13 ± 0.02 x 106, respectively, n = 12, p = 0.0002) (Figure 2A) , thus confirming that challenge with OVA was efficient. Although the cell population in BAL fluid from saline-challenged mice was almost exclusively alveolar macrophages, OVA challenge caused a dramatic increase in the proportion of eosinophils (Figure 2B). OVA-challenged mice exposed to restraint stress in the short-term protocol had a decreased number of cells recovered in BAL fluid compared with unstressed animals (0.78 ± 0.12 x 106 and 1.98 ± 0.35 x 106, respectively, n = 12, p = 0.004) (Figure 2A). This corresponded to a significant decrease in both eosinophil (0.52 ± 0.12 x 106 vs. 1.56 ± 0.32 x 106, n = 12, p = 0.006) and macrophage numbers (0.22 ± 0.05 x 106 vs. 0.38 ± 0.13 x 106, n = 12, p = 0.047) (Figure 2C); however, the change in proportion of eosinophils and macrophages in the total cell population was not statistically significant when compared with unstressed animals (Figure 2B).



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  		Figure 2. Effect of repeated exposure to stress for 3 consecutive days on total (A) and differential (B) cell counts (macrophages, eosinophils, neutrophils, and lymphocytes) in BAL fluid from OVA-sensitized male mice 24 hours after challenge with nebulized OVA or saline. The absolute numbers of macrophages and eosinophils in OVA challenged mice are also shown (C). Solid columns represent unstressed mice, whereas open columns represent stressed mice. Each column represents the mean ± SEM (n = 12). *p < 0.05; **p < 0.01 compared with unstressed control.

 
Exposure to stress in the long-term protocol produced markedly distinct changes in BAL cell populations compared with the short term. After 7 days of repeated exposure to stress, total BAL cell numbers were significantly increased compared with unstressed animals (8.1 ± 0.6 x 106 and 3.8 ± 0.5 x 106, respectively, n = 12, p = 0.008) (Figure 3A) . This increase in total cells corresponded to an increase in all cell types, as the proportions of various cell populations did not change significantly (Figure 3B).



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  		Figure 3. Effect of repeated exposure to stress for 7 consecutive days on total (A) and differential (B) (macrophages, eosinophils, neutrophils, and lymphocytes) cell counts in BAL fluid from OVA-sensitized male mice 24 hours after challenge with nebulized OVA. Solid columns represent unstressed mice, whereas open columns represent stressed mice. Each column represents the mean ± SEM (n = 12). *p < 0.05 compared with unstressed control animals.

 
To address the question of whether the addition of swim stress was responsible for the difference in stress-induced modulation of BAL cell numbers between the short- and long-term protocols, we amended the short-term protocol to include exposure to both restraint and swim stress. The changes in numbers of cells recovered from BAL fluid in mice exposed to both restraint and swim stress for 3 consecutive days were similar to those exposed to restraint alone (from 1.63 ± 0.45 x 106 in unstressed to 0.47 ± 0.10 x 106 in restraint and swim-stressed animals, n = 4, p = 0.013), suggesting that the distinct responses to stress in the short- and long-term protocols were due to the duration of exposure, rather than quality, of stress.

Cytokine Levels in BAL Fluid
After exposure to stress in the short-term protocol, levels of IL-6 in BAL fluid were increased significantly compared with unstressed animals (85.6 ± 12.3 and 41.5 ± 9.8 pg/ml, respectively, n = 10, p = 0.0035), as were levels of the proinflammatory cytokines IL-9 (337.6 ± 57.6 vs. 203.9 ± 47.9 pg/ml, n = 12, p = 0.03) and IL-13 (260.3 ± 24.6 vs. 191.9 ± 36.5 pg/ml, n = 12, p = 0.04) (Figure 4) . Levels of IL-10 and IFN-{gamma} were not significantly altered by exposure to stress (124.3 ± 34.8 vs. 156.8 ± 24.9 pg/ml, n = 10, and 195.5 ± 45.5 vs. 210.8 ± 23.0 pg/ml, n = 12, respectively). Unstressed mice in the long-term protocol did not have significantly different cytokine levels compared with those in the short-term protocol. However, exposure to stress for 7 consecutive days did not result in significant changes in the levels of IL-6, IL-9, IL-10, IL-13, or IFN-{gamma} compared with unstressed control subjects (Table 1) .



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  		Figure 4. Effect of repeated exposure to stress for 3 consecutive days on levels of the cytokines interleukin (IL)-6, IL-9, and IL-13 in BAL fluid from OVA-sensitized male mice 24 hours after challenge with nebulized OVA. Solid columns represent unstressed mice, whereas open columns represent stressed mice. Each column represents the mean ± SEM (n = 12). *p < 0.05; **p < 0.01 compared with unstressed control animals.

 

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  		TABLE 1. The effect of repeated exposure to stress for 7 consecutive days on cytokine levels in bronchoalveolar lavage fluid from ovalbumin-sensitized male mice 24 hours after challenge with nebulized ovalbumin

 
Effect of Corticosteroid Receptor Antagonist Pretreatment
Treatment with RU486 did not influence the total numbers or differential populations of cells recovered from BAL fluid in unstressed mice. However, the decrease in total BAL numbers observed after 3 consecutive days of stress was ablated in those animals treated with RU486 before stress exposure (Figure 5A) . The increase in total BAL leukocytes observed after exposure to 7 days of stress was unaltered by prior treatment with the corticosteroid receptor antagonist (Figure 5B). Treatment with RU486 before stress exposure did not change BAL cytokine levels in either the short- or long-term protocols nor did it augment the increase in levels of IL-6, IL-9, or IL-13 seen after stress in the short-term protocol (data not shown).



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  		Figure 5. Effect of intraperitoneal administration of the glucocorticoid receptor antagonist RU486 (6 mg/kg) on total cell counts in BAL fluid in OVA-sensitized and challenged male mice exposed to stress for 3 (A) and 7 (B) consecutive days. RU486 was administered 1 hour before each stress exposure. Each column represents the mean ± SEM (n = 8). *p < 0.05 compared with no stress/no RU486 control animals.

 
Airway Response to Methacholine
As shown in Figures 6A and 6B , challenge of sensitized mice with OVA resulted in an enhancement of AHR to methacholine when compared with saline-challenged mice in both short- and long-term protocols. However, repeated exposure to stress for either 3 or 7 days did not alter airway responsiveness to methacholine, as assessed by changes in enhanced pause.



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  		Figure 6. Effect of repeated exposure to stress for 3 (A) and 7 (B) consecutive days on airway responsiveness to methacholine, as assessed by changes in enhanced pause (Penh) in OVA-sensitized male mice 24 hours after challenge with nebulized OVA or saline. Each data point represents the mean ± SEM (n = 12). **p < 0.01. Control = filled circles; stress = filled squares; saline = open circles.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have studied the effect of stress on two major characteristics of asthma: airway inflammation and AHR. We show that exposure to inescapable restraint stress for 3 consecutive days inhibits allergen-mediated cellular influx in BAL fluid of OVA-sensitized mice. This stress-induced reduction in BAL cells can be prevented by prior treatment of the animals with the glucocorticoid receptor antagonist RU486, indicating that the response is mediated through corticosterone released after HPA axis activation.

In marked contrast to the decrease in BAL cell number observed after 3 days of stress, 7 days of repeated stress exposure led to a more than twofold increase in total cells recovered in BAL fluid. This enhancement of inflammatory cell influx is similar to that seen after prenatal or chronic psychologic stress in rats (23, 24). Prior treatment with RU486 did not prevent the stress-induced increase in cell influx observed after 7 days of repeated stress exposure, indicating that the effect is mediated via a mechanism independent of the action of glucocorticoids. The phenomenon of bipolar effects (enhancement or reduction) of stress on the immune or inflammatory response depending on the duration of exposure has been reported previously. Although acute stress exacerbates delayed-type hypersensitivity reactions in rats and mice, chronic stress attenuates this response (19, 26), and repeated but not acute stress suppresses inflammatory plasma extravasation (22). However, in general and in keeping with our observations, exacerbation of inflammatory diseases has been correlated to chronic or repeated exposure to a stressor, rather than short-term or acute stress (20, 25, 27–29, 38). Furthermore, our results suggest that in the airway, as in other compartments of the body, as duration of exposure to stress increases, different physiologic mechanisms are engaged and exert qualitatively distinct effects on the inflammatory response. That the immunomodulatory response to stress involves several independent pathways is indicated in a rat model of colitis where the antagonist of the HPA axis response, {alpha}-helical corticotropin-releasing factor-(9–41) cannot block enhancement of inflammation induced by repeated restraint stress (20). Furthermore, in humans, stress-induced suppression of T-cell mitogenesis and increase in circulating CD8+ and natural killer cell numbers (39, 40) appear to be mediated by the autonomic nervous system (41).

Chronic or repeated long-term exposure to stress leads to adaptation of the HPA axis response, including downregulation of glucocorticoid receptor expression, consequent loss of negative feedback on hypothalamic and pituitary secretion of corticotrophin-releasing factor and adrenocorticotropin hormone, respectively (42), and blunting of the corticosteroid response (26, 42, 43). An impaired HPA axis response has been associated with an enhanced inflammatory response after stress in a rat model of colitis (38).

A decrease in glucocorticoid response consequent to chronic stress may not only lead to a loss of the associated immunosuppressive effects but may also unmask proinflammatory actions of other stress-induced mediators such as corticotropin-releasing factor and catecholamines (44–48). The proinflammatory effects of corticotropin-releasing factor are mediated, at least in part, through mast cell activation (47, 48), and in contrast to glucocorticoids, catecholamine hormones have been shown to increase blood leukocyte numbers in rats and humans. The catecholamine-induced increase in blood granulocyte numbers may be mediated by {alpha} and ß adrenergic receptors and counteracted by corticosterone acting at the type II adrenal steroid receptor (16, 47). This has led to the suggestion that the absolute number of specific blood leukocytes may be significantly affected by the ambient concentrations of epinephrine, norepinephrine, and corticosterone. A similar interplay of catecholamines and glucocorticoids in the lung may influence the airways' immune response and differences in concentrations, and ratios of these and other stress mediators may explain the distinct inflammatory response to stress of varied duration observed in our study. Indeed, Dobbs and colleagues demonstrated that both corticosterone and catecholamine-mediated mechanisms were involved in the stress-induced suppression of antiviral cellular immunity (49). Further studies suggested that coordinated interactions between these two physiologic response mechanisms are required to optimize development of a restraint stress-induced immune response to experimental influenza infection (50).

Despite a decrease in inflammatory cell influx after 3 days of restraint stress, it is interesting to note that the levels of several inflammatory cytokines in lavage fluid increased. IL-6 levels were increased approximately twofold compared with unstressed mice. IL-6 is a proinflammatory cytokine that has regulatory effects on the HPA axis (51–53). IL-6 release during inflammation is dependent on the major HPA axis mediator corticotropin-releasing factor (54), and increased circulating levels have been reported during physical, psychologic, and inflammatory stress in many species (55). IL-9 and IL-13 are also increased in stressed compared with unstressed mice after OVA challenge. Both of these proinflammatory cytokines are thought to cause airway epithelial damage, AHR, goblet cell hyperplasia, and mucus hypersecretion (56–59). Given the well established ability of glucocorticoids to downregulate proinflammatory cytokine production and the corticosterone-dependent decrease in inflammatory cell influx into the BAL fluid observed in our model, the increase in these cytokines appears paradoxical. However, mild stress and glucocorticoids at low levels induce production of the cytokine macrophage inhibitory factor (60). Macrophage inhibitory factor overrides glucocorticoid-induced suppression of gene expression of proinflammatory cytokines, including IL-6 and IL-8 (60, 61). However, our observation that the increase in levels of the cytokines IL-6, IL-9, and IL-13 after restraint stress is not prevented by pretreatment with RU486 indicates the action of corticosteroids is not required to mediate this response. Notably, alveolar macrophages isolated from stressed rats show a marked increase in IL-1 and tumor necrosis factor secretion that is unrelated to corticosterone and adrenocorticotropin hormone levels (62), and the decrease in IL-1{alpha} seen in airways of influenza-infected rats after restraint stress is unaffected by treatment with RU486 (63). This indicates that mechanisms distinct from HPA axis, perhaps involving the sympathetic nervous system, can regulate cytokine responses to stress. These findings also suggest that despite a decrease in inflammatory cell influx after short-term exposure to stress, resident cells may be at a higher level of activation and stimulated to release more proinflammatory mediators.

One of the defining characteristics of asthma is AHR that increases after exposure to allergen. Although the mechanisms underlying this heightened responsiveness are still largely unknown, it is thought to result from a complex inflammatory cascade involving several cell types, including T lymphocytes and eosinophils. In many animal models, development of altered airway function has been linked to eosinophil accumulation in the lungs (64, 65). Despite the stress-induced changes in eosinophil numbers and increase in IL-9 and IL-13 in BAL fluid, we did not see augmentation of AHR after either 3 or 7 days of exposure to stress. It should be noted that in this study we used whole-body plethysmography to assess AHR and did not directly measure lung resistance. It has been demonstrated that airway responsiveness after antigen challenge shows a different time course depending on whether enhanced pause or resistance is measured (66). One potential explanation for this discrepancy is the contribution of nasal resistance to the enhanced pause measurement. For this reason, it is possible that nasal resistance could be masking changes in lung resistance caused by stress.

However, a causal relationship between eosinophilic inflammation and AHR is far from established, and there is evidence that AHR can be induced by at least two entirely independent pathways, one involving an eosinophil-dependent process and another that requires the activation of mast cells (64, 65, 67, 68). Similarly, IL-10 appears critical to the expression of AHR in the mouse model of eosinophilic airway inflammation (69). Although this requirement for IL-10 is downstream of the eosinophil inflammatory cascade, the fact that IL-10 levels did not change in OVA-sensitized and -challenged mice after either 3 or 7 consecutive days of stress exposure may be a significant factor in our failure to observe changes in AHR.

Overall, our data support the hypothesis that repeated exposure to stress over the long term engages different mechanisms than short-term stress. In the short-term, the antiinflammatory effects of corticosterone are apparent leading to a decrease in inflammatory cells in lavage fluid. However, in the long-term cell influx is exacerbated possibly because the antiinflammatory response to endogenous corticosterone is lost. Independently of the glucocorticoid response, short-term stress increases the levels of some proinflammatory cytokines in the BAL. Despite the modulation of the inflammatory response, neither short-term nor long-term stress resulted in changes in AHR. Here it is notable that in studies of the effects of academic examination stress on otherwise healthy mild subjects with asthma, modulation of the cellular immune response and increased eosinophilic airway inflammation and production of IL-5 are not associated with significant changes in lung function (10, 70). It is possible that the major effect of repeated stress is to exacerbate the chronic inflammatory response. In humans, this may translate to increased long-term damage of the airway and gradual deterioration in function through remodeling.


   FOOTNOTES
 
Supported by the Alberta Heritage Foundation for Medical Research, Canadian Institutes of Health Research.

Conflict of Interest Statement: P.F. has no declared conflict of interest; C.E. has no declared conflict of interest; J.R.G. has no declared conflict of interest; A.D.B. has no declared conflict of interest; H.V. has no declared conflict of interest.

Received in original form July 17, 2003; accepted in final form November 3, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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