Leukotrienes as Mediators of Airway Obstruction

JEFFREY M. DRAZEN

Departments of Medicine, Pulmonary Division, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts

	ABSTRACT

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The cysteinyl leukotrienes are potent mediators of airway narrowing derived from the lipoxygenation of arachidonic acid and the adduction of glutathione to this eicosanoid backbone. In lower animals and humans, the cysteinyl leukotrienes are among the most potent airway contractile substances ever identified. Furthermore, these moieties can be recovered from the urine during induced or spontaneous asthma attacks. Most important, inhibition of the synthesis of the leukotrienes or prevention of their action at the CysLT₁ receptor is associated with an improvement in the airway dysfunction that occurs in both induced and spontaneous asthma. These data indicate that the cysteinyl leukotrienes have a clinically significant role in the airway obstruction that characterizes asthma. Drazen JM. Leukotrienes as mediators of airway obstruction.

	INTRODUCTION

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The leukotrienes are a family of bioactive fatty acids that were originally identified in the 1970s (1) as the materials that constituted the biological activity previously known as slow-reacting substance of anaphylaxis, or SRS-A. Work done between the identification of SRS-A, in the late 1930s (6), and its structural elucidation as the leukotrienes indicated the potential importance of these molecules in airway disease (7, 8). However, without knowledge of the structure of SRS-A or access to inhibitors of the leukotrienes, it was impossible to assign, with any certainty, a role for the leukotrienes as mediators of airway obstruction in asthma. In the almost two decades since the discovery of the structure of the leukotrienes, a substantial body of data has accrued indicating an important role for these biomolecules in the airway obstruction that occurs in asthma. In this paper, we provide a summary of the leukotriene biochemistry and of data indicating that leukotrienes account for a clinically significant fraction of the airway narrowing in laboratory-induced as well as spontaneous asthma.

	LEUKOTRIENE BIOCHEMISTRY

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The leukotrienes were discovered as products of RBL-1 cells, a leukocyte cell line, and contain three conjugated double bonds, i.e., a triene, hence the name leukotriene. They derive from the ubiquitous membrane constituent arachidonic acid and are members of a larger group of biomolecules known as eicosanoids (9, 10) (Figure 1). Their synthesis is initiated through action of a family of enzymes known as phospholipases. When these enzymes are active, the arachidonic acid found esterified to cell phospholipids is cleaved from these phospholipids (11) and becomes available to serve as a substrate for 5-lipoxygenase (5-LO). For 5-LO to catalyze the formation of the leukotrienes, it must be translocated to the perinuclear membrane. This process is triggered by an increase in the level of intracellular calcium, which enhances the affinity of the 5-LO-activating protein (FLAP) for 5-LO (16- 19). FLAP is a highly hydrophobic membrane protein that serves as a binding site and cofactor for both 5-LO and arachidonic acid (20, 21). The creation of this trimolecular complex results in the appropriate conditions for 5-LO to adduct molecular oxygen to arachidonic acid to form leukotriene A₄ (LTA₄; 5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) (22).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Schematic diagram showing the biochemical steps in the production of the cysteinyl leukotrienes (LT). 5-LO = 5-lipoxygenase; FLAP = 5-LO-activating protein; AA = arachidonic acid; 5-HPETE = 5S-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid; Cys LT1 = cysteinyl LT receptor. (Reproduced with permission from Reference 139.)

In cells with a functional leukotriene C₄ (LTC₄) synthase (25), such as eosinophils, mast cells, and alveolar macrophages, glutathione is adducted at the C6 position of LTA₄ to yield the molecule known as LTC₄ (1). LTC₄ is transported from the cytosol to the extracellular microenvironment (26), where the glutamic acid moiety is cleaved by -glutamyltranspeptidase to form LTD₄ (2, 3); cleavage of the glycine moiety from LTD₄ by a variety of dipeptidases results in the formation of LTE₄ (4, 5). Because each of these molecules contains cysteine, they are known collectively as the cysteinyl leukotrienes; together these molecules constitute the material formerly known as SRS-A.

The cysteinyl leukotrienes are catabolized through three major mechanisms: (1) the formation of the N-acetyl derivative of LTE₄ (27); (2) the interaction of the leukotriene and hypochlorous acid to form the respective leukotriene sulfoxide and LTB₄ (28); and (3) -oxidation and -elimination with the progressive shortening of the  portion of the molecule (29). Each of these conversions is associated with a loss of bioactivity. Approximately 10% of the administered cysteinyl leukotriene appears in the urine as LTE₄ (30); such release can be quantified and used as an index of endogenous leukotriene availability (34).

A variety of physical, chemical, and immunologic stimuli have been shown to activate cells so that they can produce cysteinyl leukotrienes. Activation of mast cells through cross-linking of antigen-specific immunoglobin E (IgE) bound to FcRI (46, 47), hyperventilation of cold dry air (48), aspirin ingestion by aspirin-intolerant individuals (40, 49), hypoxia (52), hyperoxia (53), and exposure to platelet-activating factor (PAF) (54) are among the stimuli that cause lung cells to produce cysteinyl leukotrienes.

	LEUKOTRIENE RECEPTORS

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The cysteinyl leukotrienes and LTB₄ transduce their biological effects through stimulation at specific receptors. The receptor for the cysteinyl leukotrienes (CysLT₁) has been functionally characterized through comparisons of the contractile activity of the cysteinyl leukotrienes in a variety of smooth muscles in the presence of a number of specific receptor antagonists. However, as of October 1998, the CysLT₁ receptor has not been molecularly cloned; the receptor for LTB₄ has been functionally characterized and molecularly cloned (55).

LTB₄ (BLT) Receptors*

The BLT receptor, of which there appear to be no functional subtypes, is a 60-kilodalton (kD) cell-surface protein (56, 57) that predominantly transduces chemotaxis and cellular activation (21, 58, 59). Through its recent molecular cloning (55), it is now understood that the BLT receptor is G protein-coupled and has seven transmembrane-spanning segments. A number of chemically distinct, specific and selective antagonists have been identified with inhibitory concentration of 50% (IC₅₀) values of approximately 1-10,000 nM against a variety of LTB₄-mediated biological activities (60).

Cysteinyl Leukotriene Receptors

There are two subtypes of cysteinyl leukotriene receptor CysLT₁ and CysLT₂neither of which has been molecularly cloned. Only the CysLT₁ receptor has been extensively characterized in functional terms.

CysLT₁ receptor. The CysLT₁ receptor, previously known as the LTD₄ receptor or LTR_d (65), is a 45-kD membrane-associated protein found in a number of contractile tissues, including airway smooth muscle (66). Stimulation of the CysLT₁ receptor results in smooth muscle constriction, with signal transduction occurring in concert with phosphoinositide turnover (70). In the human lung in vitro, LTC₄ and LTD₄ exhibit an equal capacity to initiate smooth muscle constriction, while the biopotency of LTE₄ is lower by a factor of 10 (74). A number of chemically distinct, specific, selective antagonists have been identified with pA₂ values between 7 and 10 in functional assays using airway tissues (74, 79). The results of studies with such antagonists in humans are reviewed in LEUKOTRIENE PHARMACOLOGY.

CysLT₂ receptor. The CysLT₂ receptor, previously known as the LTC₄ receptor or LTR_c (65, 88), is found in the vascular smooth muscle of the human lung (89) as demonstrated by the functional antagonist BAY u9773 (92). It has not been studied in humans in vivo.

	LEUKOTRIENE PHARMACOLOGY

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Contractile Responses Initiated by Cysteinyl Leukotrienes

The cysteinyl leukotrienes are potent airway smooth muscle contractile agonists in both animals and humans. Indeed, the ability of these molecules to produce long-lasting airway obstruction at very low concentrations is the basis for the interest they arouse among pulmonary biologists. Because extensive reviews of the pharmacology of the cysteinyl leukotrienes have been published (93), only certain aspects are reviewed here.

Leukotriene Contractile Airway Activity in Humans

In vitro. Contractile studies of airway smooth muscles isolated from the bronchi of specimens removed at surgery for pulmonary carcinoma have shown that LTC₄ and LTD₄ possess nearly equal activity as contractile agonists (75, 77, 96, 97) with median effective concentration (EC₅₀) values of approximately 10 nM; this is about one-thousandth of the EC₅₀ values for histamine in the same tissues. LTE₄ is about one-tenth as active as a contractile agonist as LTC₄ and LTD₄ in these tissues (75).

Nonasthmatic subjects in vivo. Inhalation of aerosols generated from solutions of cysteinyl leukotrienes results in airway obstruction in nonasthmatic humans. The airway obstruction is reflected as decreased specific airway conductance (SGaw) or as decreased flow rates (measured from partial or full expiratory flow-volume curves) (98). LTC₄ and LTD₄ are nearly equipotent in their capacity to elicit airway obstruction in intact humans. When nebulizer leukotriene concentrations on the order of 10 µM are used to create an aerosol, inhalation of the aerosol reduces the maximal expiratory flow rate (measured from a partial flow-volume curve, ₃₀-P) by 30% in nonasthmatic subjects (Table 1). Approximately 30-fold greater concentrations of LTE₄ in the nebulizer are required to achieve an equivalent physiological effect on airway patency in nonasthmatic subjects. With respect to other inhaled contractile agonists, LTC₄ and LTD₄ are approximately 3,000 times and LTE₄ approximately 30 times more potent than histamine as bronchoconstrictor agonists (99). When complete dose-response curves for leukotrienes are constructed, it can be shown that the plateau response to LTD₄ is greater than that to methacholine (107).

View this table: [in this window] [in a new window]   TABLE 1 POTENCY OF INHALED LEUKOTRIENES AS BRONCHOMOTOR AGONISTS IN ASTHMATIC AND NONASTHMATIC SUBJECTS,* AS INDICATED BY THE GEOMETRIC MEAN NEBULIZER CONCENTRATION REQUIRED TO DECREASE THE 30-P BY 30-40%

Asthmatic subjects in vivo. Patients with asthma also exhibit bronchoconstrictor responses when they inhale aerosols generated from solutions of cysteinyl leukotrienes (100, 101, 106, 108, 109); the concentrations of LTC₄, LTD₄, and LTE₄ required in an aerosol generator to decrease the ₃₀-P by approximately 30% are given in Table 1. The bronchoconstrictor responses of subjects with asthma to these leukotrienes are all manifest within 3-5 min after aerosol inhalation; the duration of the effect is related to the severity of the bronchospasm but is on the order of 20-30 min when the decrement in the ₃₀-P is 30%. Although patients with asthma are more sensitive to the obstructive effects of inhaled leukotrienes (i.e., have a response at a lower inhaled dose) than they are to the effects of reference agonists such as histamine or methacholine, they exhibit a lesser degree of hyperresponsiveness to the two classes of agonists than do normal subjects (Table 1). One possible explanation for this differential sensitivity is that the airways of patients with asthma are repeatedly stimulated with the cysteinyl leukotrienes and therefore become tachyphylactic to their actions.

	RECOVERY OF LEUKOTRIENES FROM BIOLOGICAL FLUIDS

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It has been technically difficult to devise artifact-free, sensitive, and specific assays for LTB₄ or cysteinyl leukotrienes in blood or plasma (110). In contrast, it has been possible to devise such assays for the presence of LTE₄ in the urine. Since a fixed fraction of exogenously administered LTC₄ or LTE₄ appears in the urine as authentic LTE₄ (30, 36, 111), a number of investigative groups have successfully used the urinary excretion of LTE₄ as an index of the production of the cysteinyl leukotrienes; these studies are detailed below. In contrast, no methods to detect the production of LTB₄ in vivo have been applied in disease states.

Nonasthmatic Subjects

Nonasthmatic subjects have measurable amounts of LTE₄ in their urine (34, 43, 44, 114); the source(s) and the biological significance of this LTE₄ excretion have not been established. It is known that the excretion rate of LTE₄ does not vary systematically over the course of a day.

Asthmatic Subjects

Induced asthma. In patients with allergen-induced asthma, allergen challenge has been associated with the enhanced recovery of urinary LTE₄ during the early-phase but not the late-phase response (34, 44, 49, 115, 116); the magnitude of the decrease of the FEV₁ after allergen challenge, and the amount of LTE₄ recovered in the urine are directly related (35). In contrast, most (42, 115) but not all (38, 117) investigators have been unable to recover elevated amounts of LTE₄ in the urine after the induction of asthma with an exercise stimulus.

In the absence of known aspirin exposure, patients with aspirin-induced asthma excrete three to four times as much LTE₄ in their urine as non-aspirin-sensitive patients with asthma. After aspirin challenge (40, 41, 49, 50), urinary LTE₄ levels increase dramatically in aspirin-sensitive but not non- aspirin-sensitive patients with asthma. The LTE₄ levels in the urine of aspirin-sensitive patients with asthma after aspirin exposure can be quite highoften 10 times those found in non- aspirin-sensitive patients with asthma.

Chronic stable asthma. Asano and coworkers (118) measured urinary LTE₄ excretion in eight patients with mild chronic stable asthma. The average FEV₁ of these patients was 72% of that predicted, and their only asthma treatment was the intermittent use of inhaled -agonists. Sixteen consecutive 6-h urine samples were obtained from each of these patients during a 4-d observation period on a metabolic ward. The mean urinary level of LTE₄ in this group was 110.0 ± 59.2 picograms (pg) LTE₄/mg creatinine, significantly higher than that for nonasthmatic subjects, 83.8 ± 38.2 pg LTE₄/mg creatinine (p < 0.05). The level of LTE₄ in the urine exceeded the mean level found in nonasthmatic subjects by two standard deviations in at least one of the 16 samples obtained from seven of the eight asthmatic patients.

Spontaneous asthma. In the absence of laboratory-administered asthma-eliciting challenges, the evidence for a role for leukotrienes in the pathogenesis of asthma, as indicated by the recovery of LTE₄ from the urine, is less than compelling. Taylor and coworkers (34) recovered increased amounts of LTE₄ from the urine of only a fraction of patients with acute severe asthma who presented for emergency treatment; however, in many subjects with asthma of equal severity, the amount recovered was not elevated. Drazen and coworkers (114) demonstrated that, among individuals presenting to an emergency service for the treatment of asthma, all those with acutely reversible airway narrowing had elevated urinary levels of LTE₄. This observation suggests that acute spontaneous bronchospasm is associated with leukotriene excess.

	EFFECTS OF AGENTS THAT INTERRUPT THE 5-LO PATHWAY IN ASTHMA

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Among the lines of evidence suggesting that the cysteinyl leukotrienes are important in the pathogenesis of asthma are those arising from clinical trials of agents inhibiting either the action of LTD₄ at the CysLT₁ receptor or the action/translocation of 5-LO. In the former group are montelukast (Singulair), pranlukast (Onon or Ultair), and zafirlukast (Accolate) (119- 127); in the latter are zileuton (A64077, Zyflol), an inhibitor of 5-LO, and MK886, an inhibitor of FLAP (50, 128). These agents have been used in studies of three conditions: induced asthma, asthmatic bronchoconstriction, and chronic stable asthma.

Laboratory-induced Asthma

Asthma has been induced in the laboratory by three distinct strategies: cold air or exercise, antigen inhibition, or aspirin administration.

Exercise- or cold air-induced asthma. Exercise- or cold air- induced asthma is created in the laboratory by exposing subjects to exercise or cold air stimuli of increasing intensity until bronchoconstriction is induced. In order to examine the importance of leukotrienes in such induced asthma, challenges are conducted while patients are receiving treatment with either an active agent or a placebo (121, 127, 128, 131). Each of the anti-leukotriene agents noted above has been shown to inhibit the induced asthmatic response. Even though the LTD₄ receptor antagonists used in these trials differ by a factor of approximately 10 in their capacity to inhibit the action of LTD₄, they exhibit similar effectiveness in inhibiting exercise- or cold air-induced asthma. This observation suggests that only a component (about 50%) of exercise- and cold air-induced airway obstruction is leukotriene-mediated.

Allergen-induced asthma. In the laboratory, allergens introduced into an aerosol can be used to induce asthma in subjects known to be allergic to that agent. In allergic subjects, inhalation of the aerosol elicits an early-phase bronchoconstriction, which occurs within minutes of exposure. In addition, about 10-40% of patients have a secondary bronchoconstrictor response (known as the late-phase antigen response) 4-8 h after allergen exposure. Each of the clinically tested LTD₄ receptor antagonists has inhibited the early phase of antigen-induced bronchoconstriction in the antigen-induced asthma model. The more potent LTD₄ receptor antagonists also inhibit the late-phase antigen response (120, 122, 125, 132). There is a direct relation between the potency of the antagonist and its capacity to inhibit the antigen response.

Clinical trials in the allergen challenge model with zileuton, an inhibitor of 5-LO, or with MK886, an inhibitor of the association of 5-LO with FLAP, have had varied results. In one trial, zileuton had a small and statistically insignificant effect on the early-phase antigen response and no effect on the late-phase response (116), while in another trial MK886 (129) significantly inhibited the early-phase response.

Aspirin-induced asthma. Products of 5-LO action are of particular importance in the physiological events of aspirin-induced asthma. In this syndrome, aspirin-sensitive patients develop bronchospasm as well as naso-ocular and gastrointestinal symptoms after ingesting aspirin, while they develop only bronchospasm after inhaling aerosols created from lysine aspirin.

In a clinical trial in which the inhaled leukotriene receptor antagonist SKF104353 was used for pretreatment, many patients toleratedwithout developing significant bronchospasmall doses of aspirin that had previously caused a bronchospastic reaction (124). However, in this trial, neither systemic (i.e., nonpulmonary) symptoms nor urinary LTE₄ excretion was altered. In another trial in which patients inhaled lysine aspirin, oral treatment with ICI204219, a leukotriene receptor antagonist, inhibited the bronchospastic response usually observed in such subjects. Finally, in another study the 5-LO inhibitor zileuton was given orally for 1 wk prior to the systemic administration of aspirin (130). In this trial, both the bronchospastic and the systemic reactions to aspirin were ablated; moreover, the elevated urinary excretion of LTE₄ associated with systemic aspirin challenge was significantly reduced. These data demonstrate that products of the 5-LO pathway are the primary effector molecules in aspirin-induced asthma.

Dahlen and coworkers (51) obtained additional evidence for this hypothesis by demonstrating that the systemic administration of a CysLT₁ receptor antagonist is associated with improvement in lung function in individuals with aspirin-sensitive asthma (ASA) induced in the absence of specific ASA provocation. These findings are consistent with a causal link between LTE₄ availability and deranged lung function in aspirin-sensitive patients with asthma.

On the basis of these data, one could conclude that the mediators of asthmatic airway narrowing vary depending on the inciting stimulus used. The exercise/cold-air response is only partially mediated by leukotrienes; the antigen response is largely, but not wholly, leukotriene-mediated, while the aspirin response is entirely leukotriene-mediated.

Inhibition of Asthmatic Bronchoconstriction

If patients with moderate persistent asthma are not given bronchodilator medications, many develop spontaneous reversible bronchoconstriction over the ensuing 6-12 h. This so-called asthmatic bronchoconstriction has been used as a model for examination of the role of leukotrienes in spontaneous airway narrowing in asthma. For example, in a group of patients with moderately severe asthma, most of whom were using inhaled steroids, the administration of zafirlukast, an antagonist of the action of LTD₄ at the CysLT₁ receptor, resulted in a 5-10% improvement in the FEV₁ (133). In this study, inhalation of a -agonist aerosol after zafirlukast administration resulted in an increase in the FEV₁ by 20-30%. Thus, the -agonist had a bronchodilator effect two to three times as great as that associated with the LTD₄ receptor antagonist. However, it is important to note that the effects of the -adrenergic agonist were additive with the effects of the LTD₄ receptor antagonist, which indicates that distinct contractile mechanisms are involved in each response. Gaddy and coworkers (134) and Impens and coworkers (135) used a similar trial design but administered structurally distinct CysLT₁ receptor antagonists intravenously; the results of these studies essentially duplicated the findings of Hui and Barnes (133). The 5-LO inhibitor zileuton given to subjects with asthmatic bronchoconstriction (130) produced a 10-15% increase in the FEV₁.

Taken together, these data indicate that a clinically significant component of asthmatic bronchoconstriction is directly due to the action of leukotrienes at their receptors and that the stimuli resulting in leukotriene synthesis are continuously activated in patients with this form of airway obstruction. This latter point is evidenced by the fact that zileuton was as effective as leukotriene receptor antagonists in reversing asthmatic bronchoconstriction. Thus, there appears to be ongoing activation of the 5-LO pathway in patients with chronic persistent asthma.

Studies in Chronic Stable Asthma

The archival literature contains a number of reports of trials of at least 6-wk duration in which the effects of agents active on the 5-LO pathway have been evaluated in a blinded, randomized, placebo-controlled manner in patients with chronic stable asthma. In the first of these reports, LY171883, an antagonist at the CysLT₁ receptor that shifts the LTD₄ dose- response curve about fivefold in nonasthmatic subjects, was given to patients with mild asthma in a 6-wk, parallel group, placebo-controlled trial (119). Patients receiving the LTD₄ receptor antagonist had a significant increase in FEV₁ (approximately 250 ml) during the trial. Moreover, while they were receiving treatment with LY171883, their asthma symptoms decreased.

Spector and colleagues published the results of a 6-wk trial in which zafirlukast, a CysLT₁ receptor antagonist, was administered to patients with mild-to-moderate chronic persistent asthma (136). At the initiation of randomized, placebo-controlled treatment, the mean FEV₁ of the patients in the trial was about 65% of predicted. Over the randomized active treatment period, there was a 10-15% improvement in the FEV₁ group receiving zafirlukast, 40 mg bid, which was significantly greater than the improvement noted in the placebo treatment group (Figure 2). The magnitude of the therapeutic effect was directly proportional to the plasma levels of zafirlukast. There were also statistically significant improvements in asthma symptoms and a decrease in rescue -agonist use.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Percentage change ± SEM in FEV1 from Week 0 (randomization) through Week 6 (end point) for subjects who received zafirlukast (10 mg, n = 66; 20 mg, n = 67; 40 mg, n = 67) or placebo (n = 66). *p  0.01 between 40 mg and placebo. (Reproduced with permission from Reference 136.)

Thirteen- and 26-wk treatment trials in which the effects of zileuton on asthma control in patients with chronic stable asthma, whose average FEV₁ was about 60% of predicted on enrollment, have been reported (137, 138). In these trials, treatment with zileuton resulted in a 15-20% improvement in the FEV₁ that was sustained for the duration of the trial. Active treatment was associated with an improvement in asthma-specific quality of life, decreased rescue -agonist use, and, most important, a more than twofold reduction in the number of asthma exacerbations requiring oral corticosteroid rescue (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Percentage of patients who required corticosteroid treatment for asthma exacerbations. Groups are stratified by FEV1 as a percentage of predicted on entry into the study. A total of 111 patients had an FEV1 greater than 70%, 187 had an FEV1 of 50% to 70%, and 103 had an FEV1 less than 50%. *p < 0.05 versus placebo. (Reproduced with permission from Reference 138.)

	SUMMARY

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These data demonstrate that the leukotrienes mediate changes in the airway consistent with the pathobiology of asthma. Even more important, clinical trials with agents active on the 5-LO pathway show that interruption of this pathway can alleviate asthmatic airway obstruction and decrease both the need for rescue -agonist use and the number of steroid-requiring asthma exacerbations (139).

Thus, our accrued database clearly indicates that, among the mediators known to be released during asthmatic activation of the airway, the cysteinyl leukotrienes participate in a meaningful way in the airway obstruction of asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Jeffrey M. Drazen, M.D., Chief, Pulmonary Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. E-mail: jmdrazen{at}rics.bwh.harvard.edu.

	References

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