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Allergic Rhinitis, Asthma, Airway Biology, and Chronic Obstructive Pulmonary Disease in AJRCCM in 2004

Medical, Oncological, and Radiological Sciences, University of Modena, Modena, Italy; Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Respiratory Medicine and Thoracic Surgery, Glenfield Hospital, Leicester, United Kingdom; National Jewish Medical and Research Center, Department of Medicine and Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado; Division of Pulmonary and Critical Care Medicine, University of California–San Francisco, San Francisco, California; MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, United Kingdom; AP-HP, Reanimation Medicale, Hôpital Universitaire Henri Mondor, Creteil, France

Correspondence and requests for reprints should be addressed to Edward Abraham, M.D., University of Colorado Health Sciences Center, Division of Pulmonary Sciences and Critical Care Medicine, 4200 East 9th Avenue, Box C272, Room 5503, Denver, CO 80262-0001. E-mail: edward.abraham{at}uchsc.edu

	ALLERGIC RHINITIS AND NASAL DISORDERS

TOP ALLERGIC RHINITIS AND NASAL... ASTHMA AND AIRWAY BIOLOGY CHRONIC OBSTRUCTIVE PULMONARY... REFERENCES

 
Nasal Function
The eosinophil, found commonly in tissue in asthma, allergic rhinitis, and nasal polyposis, is generally believed to be cleared from tissue by a combination of apoptosis and subsequent phagocytosis. Uller and coworkers (1) used histochemical and ultrastructural techniques to examine human nasal polyp tissue and peripheral blood eosinophils for apoptosis, secondary necrosis, and cytolysis. They found that apoptotic eosinophils are exceedingly rare in human nasal polyp tissue. Although macrophages were seen commonly in tissue, histochemical techniques and transmission electron microscopy did not suggest engulfment of eosinophils. They found evidence that eosinophils migrate between epithelial cells into the airway lumen and that proinflammatory disintegration through cytolysis occurs as a primary event. The authors concluded that local turnover of tissue eosinophils in diseased airways occurs through pathways other than apoptosis, including paraepithelial migration and cytolysis.

Inflammation and Hyperreactivity
Watanabe and coworkers (2) raised the question of whether there might be other targets for therapeutic antibodies in a study of laboratory mice deficient in the IgG Fc receptor FcR11B. The gene-deficient mice developed more severe airway and nasal eosinophilia after allergen challenge than wild-type mice. This was associated with reduced interleukin 4 (IL-4) production by nasal mononuclear cells and decreased generation of allergen-induced IgE. These effects were not mice strain–specific and were independent of the route and type of sensitization. The authors suggested that modulation of expression and/or function of FcR11B might be a useful tool to inhibit allergic inflammation.

Treatment
Suissa and coworkers (3) assessed the clinical relevance of the systemic effect of inhaled and nasal corticosteroids in a nested case-control study with the particular merit of carefully controlling for oral corticosteroid use. The authors found no evidence that the recommended doses of inhaled and nasal corticosteroids increased fracture risk and that the risk of hip fracture was only increased in long-term users of more than 2,000 µg/day of beclomethasone or equivalent.

	ASTHMA AND AIRWAY BIOLOGY

TOP ALLERGIC RHINITIS AND NASAL... ASTHMA AND AIRWAY BIOLOGY CHRONIC OBSTRUCTIVE PULMONARY... REFERENCES

 
Genetics
Why some patients with asthma require high-dose corticosteroid therapy and others continue to have refractory symptoms is an important question. In vitro, corticosteroid insensitivity can be induced in peripheral blood mononuclear cells by IL-2 and IL-4, suggesting that the inflammatory milieu might be important. Torrego and coworkers (4) showed that this phenomenon was not related to an increased expression of the inhibitory corticosteroid receptor GR-ß in peripheral blood mononuclear cells from control subjects and subjects with stable and unstable asthma. Interestingly, peripheral blood mononuclear cells from subjects with unstable asthma had increased mRNA for the active glucocorticoid receptor GR-, but demonstrated a reduced antiproliferative effect of dexamethasone, suggesting impaired function of GR-. The authors speculated that this effect reflects IL-2– and IL-4–induced activation of the p38 mitogen-activated protein kinase, resulting in phosphorylation of GR and reduced glucocorticoid binding affinity and nuclear translocation of the GR.

Recent evidence suggests that alterations in the helper T-cell type 1 (Th1) cytokine pathway may be associated with susceptibility to allergic asthma. Birkisson and colleagues (5) examined a number of genes and protein expression in the Th1 pathway involved with IL-12 and IFN-, their subunits, and receptors, and found no evidence for a defect in these cytokine genes and responses as a primary event in asthma.

The IL-1 cluster on the human chromosome 2q12-2q14 has promising candidate genes for asthma and other inflammatory diseases. Gohlke and coworkers (6) conducted a study of single-nucleotide polymorphisms (SNPs) and found the diagnosis of asthma to be associated with SNPs in the IL-1 receptor antagonist and transforming growth factor ß₁ (TGF-ß₁) genes, respectively.

TGF-ß₁ is increased in the lungs of individuals with asthma and may modulate airway inflammation and remodeling. Silverman and colleagues (7) performed a case-control study of 527 patients with asthma and 170 without asthma. In the case of TGF-ß₁, the T allele of C-509T was not only associated with a diagnosis of asthma but also appeared to enhance gene transcription. Genes involved in the innate immune system were also explored.

Toll-like receptor 10 is a potential asthma candidate gene, because in early life, innate immune responses to inhaled allergens and pathogen-associated molecular patterns may influence asthma susceptibility. Lazarus and colleagues (8) reported that two SNPs in Toll-like receptor 10 were associated with asthma in two different populations (Nurses' Health Study and Childhood Asthma Management Program).

In a case-control analysis, Hizawa and colleagues (9) examined the genetic influence of promoter polymorphisms on the development of atopy and asthma in a Japanese population (n = 584). The authors reported that two different functional variations in the macrophage migration inhibitory factor (MIF) gene (–173G/C and –794[CATT]_5–8 repeat polymorphism) were associated with atopy but not asthma.

Genome scans for asthma have identified suggestive or significant linkages on 17 different chromosomes, including chromosome 12, region q13-23, housing the vitamin D receptor (VDR) gene. Poon and colleagues (10) reported that six variants of the vitamin receptor gene falling into two haplotype blocks were associated with asthma and four variants were associated with atopy in a Quebec cohort.

VDR polymorphisms have been associated with several immune-related diseases, and VDR and vitamin D itself modulate T-cell differentiation. VDR maps to chromosome 12q, near a region commonly linked to asthma.

Raby and colleagues (11) evaluated VDR as part of a 12q positional candidate survey and in response to observations of VDR polymorphism associating with asthma and atopy in a founder population of Quebec. Four of six variants were associated with asthma. Interestingly, and confusingly, one of these variants (and associated haplotype) was also seen in an increased frequency in a second cohort (517 females with asthma and 519 matched control subjects), but the association was reversed (i.e., appeared to confer protection rather than risk). Clearly, the relationships between genetic variability in the VDR and asthma are complex and not well understood.

Epidemiology
Burchard and colleagues (12) explored lung function, bronchodilator response, and asthma severity in subjects of Puerto Rican and Mexican ethnicity. They discovered that Puerto Rican individuals with asthma had reduced lung function, bronchodilator responsiveness (to albuterol), more severe asthma with greater morbidity, and longer asthma duration than Mexican individuals with asthma. These findings underscore the importance of racial/ethnic factors in asthma morbidity and response to therapy.

Glutathione is an antioxidant that mitigates oxidative stress in the lung. Acetaminophen results in a dose-dependent decrease in levels of glutathione in the lungs. Barr and colleagues (13), in a prospective cohort study of 121,700 women in the Nurses' Health Study, examined the relationship between use of acetaminophen and the development of adult-onset asthma. Participants were queried about the frequency of acetaminophen use in 1990. Those who reported physician-diagnosed asthma on questionnaires completed in 1990–1996 and in 1998 were defined as new cases. During 352,719 person-years of follow-up, there were 346 new cases of asthma. Increasing frequency of acetaminophen use was positively associated with newly diagnosed asthma (p = 0.006). The authors suggested that the historical trends in analgesic use may be responsible, in part, for the population-level increases in asthma prevalence in the United States.

Asthma and obesity are both common problems in the United States, and epidemiologic data suggest that obesity may be a risk factor for asthma. In a report of an NHLBI workshop, Weiss and Shore (14) summarized epidemiologic and pathophysiologic research linking obesity and asthma, and suggested future directions for research.

Airway Inflammation
Animal models.
The importance of Th2 cytokines in acute mouse models of allergic asthma is supported by numerous studies. However, a similar level of importance of Th2 cytokines in human asthma has not yet been demonstrated. A study by Kumar and colleagues (15) suggests that Th1 cytokines, such as IFN-, may also be important in the full-blown allergic lung response. In this chronic disease model, anti–IL-5 was effective in improving elements of remodeling and eosinophil infiltration, whereas IL-13 impacted goblet cell hyperplasia with a minor effect on airway reactivity. However, interferon decreased total cellular inflammation and significantly reduced airway hyperresponsiveness. Studies looking at combinations of antibodies were not performed. These studies suggest that Th1 cytokines, in addition to Th2 cytokines, may be important in producing phenotypic changes in mice similar to those seen in asthma.

There is a need for alternative management strategies in patients whose asthma is inadequately controlled on inhaled corticosteroids. Toward and colleagues (16) examined two models of guinea pig models of airway disease: guinea pigs exposed to LPS or sensitized guinea pigs with atopy exposed to ovalbumen. Sildenafil (Viagra; Pfizer, Morris Plains, NJ) attenuated allergen- and LPS-induced airway hyperresponsiveness and inflammatory cell influx by a mechanism that appeared to be independent of endogenous nitric oxide. The authors call for clinical trials of phosphodiesterase-5 inhibitors in asthma and chronic obstructive pulmonary disease (COPD).

Infection with respiratory syncytial virus (RSV) has been associated with wheezing and childhood asthma, together with increased mucus production. In a murine model of prolonged airway responsiveness induced by RSV in animals that had allergic sensitization with ovalbumin, Hashimoto and colleagues (17) measured the expression of the mucin production genes Muc-5ac and gob-5 by histochemical analysis of lung tissue stained with antimucin antibodies and Western blot analysis to determine the mechanisms by which RSV infection induces airway hyperresponsiveness. Animals infected with RSV only had little expression of the two mucin production genes, whereas animals sensitized with ovalbumin and exposed during 5 days had increased expression, which was enhanced by concomitant RSV infection, but not inoculation with inactivated RSV. IL-10 levels were not associated with mucin production. However, increased lung levels of IL-17 after Day 5 were associated with increased expression of mucin genes in sensitized mice, but not in animals with RSV infection only. The results suggest that RSV infection in the presence of allergic inflammation may worsen airway responsiveness via increased expression of mucus production genes.

Bronchial and bronchoalveolar specimens.
As identified by other studies, the vascular basis of asthma is a research priority. Interest in this area is likely to be increased by the work of Kanazawa and colleagues (18) showing that vascular endothelial growth factor (VEGF) and the vascular permeability index were increased in patients with asthma, but not in patients with eosinophilic bronchitis. This difference is potentially of fundamental importance because eosinophilic bronchitis is associated with eosinophilic airway inflammation, but not the airway dysfunction that characterizes asthma.

Elliot and colleagues (19) studied isolated aggregations of lymphoid cells in the cartilaginous airways from postmortem tissues of nonsmokers and smokers, and nonfatal and fatal cases of asthma. These aggregations of lymphocytes were present in 70 to 100% of all cases, more often in distal airways, and confined to the outer airway wall in 80% of cases. However, aggregates with an area greater than 0.1 mm² were present more often in both groups of patients with asthma, whereas vascular structures were more common in cases of fatal asthma. Airways with aggregates had both increased airway dimensions and greater numbers of eosinophils and lymphomononuclear cells. The role played by these aggregates, and whether they are a cause or a result of persistent airway inflammation, remains to be determined.

Zhou and colleagues (20) evaluated changes in bronchial smooth muscle size and contractile gene expression following in vitro conditions of growth arrest versus proliferation. Antiproliferative factors, such as TGF-ß, are found in abundance in asthma and other diseases. The authors were able to show that, in transformed cells, growth arrest led to an increase in smooth muscle cell size and contractile/synthetic gene expression. Therefore, it would appear that the controversy is not yet settled.

The amount of airway smooth muscle has long been known to be increased in asthma. However, it remains controversial whether the increase is caused by hyperplasia or hypertrophy. Woodruff and coworkers (21) used endobronchial biopsy quantification of airway smooth muscle followed by laser microdisection, and concluded that the number of smooth muscle cells was increased in patients with asthma, but that there was no evidence for an increase in size or contractile mRNA related to the cells, as compared with the normal control subjects.

Fibroblasts have been reported in lavage fluid from patients with asthma. In this study by Larsen and coworkers (22), fibroblasts could be cultured from lavage fluid obtained from subjects with mild asthma, but not from control subjects. These fibroblasts exhibited a different phenotype from those obtained from endobronchial biopsies, having a different shape and greater mobility and producing greater amounts of fibrotic proteins. The actual tissue source of these lavage fibroblasts remains unclear.

Peng and colleagues (23) assessed the role for the signal transducer and activator of transcription 6 (STAT6) in mediating IL-13–stimulated eotaxin release in human airway smooth muscle cells using antisense oligodeoxynucleotides (ODNs). Effective downregulation of STAT6 protein occurred with antisense but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 ± 4% and 74 ± 7%, respectively, in cells transfected with antisense ODNs (p < 0.001) but not with a sense ODN or a scrambled ODN. In contrast, eotaxin release induced by IL-1ß was unaffected by STAT6 antisense ODN. Inhibitors of both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein kinase pathways abolished IL-13– and IL-4–dependent eotaxin release in STAT6 antisense ODN-transfected cells. By contrast, 25% of the response remained when each inhibitor was examined alone. These data indicate a role for both STAT6 and mitogen-activated protein kinase–dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Blood.
Morris and colleagues (24) studied the nitric oxide (NO) system in patients with asthma and control subjects and found a reduction in plasma concentrations of a number of amino acids, with the greatest decrease in arginine, the substrate for NO production. They also discovered an increase in serum arginase activity, suggesting an NO deficiency in patients with asthma that was postulated to induce airway hyperresponsiveness.

Exhaled NO.
International guidelines recommend that a diagnosis of asthma be based on a combination of clinical history, the presence of variable airflow obstruction, and response to a bronchodilator or to a trial of corticosteroids. Smith and coworkers (25) examined 47 consecutive patients with symptoms suggestive of asthma to assess the utility of exhaled NO (FE_NO) as a test for asthma. Sensitivities for peak flow, spirometry, and changes in these parameters after a trial of steroids were lower (0–47%) than for FE_NO (88%) and for sputum eosinophils (86%). The diagnostic accuracy when using FE_NO and sputum eosinophils was significantly greater. The authors concluded that single measurements of FE_NO or induced sputum in patients with undiagnosed chronic respiratory symptoms are strongly predictive of a diagnosis of asthma, and that FE_NO, because it is quick and easy to perform, may be readily incorporated into routine pulmonary function tests used to make the diagnosis of asthma.

With the increasing use of FE_NO as an indirect measure of airway inflammation, there is need for information about factors that can potentially alter FE_NO levels and therefore confound the measurement. In a randomized, double-blind, crossover study, Taylor and colleagues (26) administered either caffeinated or noncaffeinated coffee to 20 subjects with asthma (10 steroid-naive and 10 steroid-treated), and performed FE_NO measurements before and 30, 60, 120, and 180 minutes after ingestion. No significant changes in FE_NO occurred after caffeine, despite significant increases in serum caffeine levels. The authors concluded that caffeinated foods and beverages are unlikely to influence FE_NO measurements in subjects with asthma.

Current methods for measuring FE_NO generally do not distinguish between NO that originates from the airway versus alveolar compartments. Gelb and coworkers (27) measured FE_NO at three different expiratory flow rates (100, 150, and 200 ml/second) in 53 subjects with stable asthma taking inhaled corticosteroids, 13 subjects with stable asthma who were inhaled corticosteroid–naive, and 34 normal control subjects. Both bronchial NO and alveolar NO were increased in subjects with asthma compared with normal control subjects. There was no significant correlation between FEV₁ % predicted or lung elastic recoil and bronchial or alveolar NO. However, there was a significant correlation between NO bronchial flux and alveolar concentration. In 10 subjects with asthma who were taking inhaled corticosteroids, the addition of prednisone significantly decreased alveolar NO, but did not change total FE_NO or NO bronchial flux. The authors concluded that inflammation in asthma may behave differently in different parts of the lung, and suggested that even in patients with stable asthma taking inhaled corticosteroids there may be ongoing, prednisone-suppressible inflammation in the alveolar compartment.

Neural and psychologic mechanisms.
Studies suggest that between 20 and 35% of asthma exacerbations occur during periods of stress. Forsythe and colleagues (28) examined the effect of short-term (3 days) or long-term (7 days) stress on airway inflammation and airway hyperresponsiveness to methacholine in a murine model. When allergen challenge was performed after short-term stress, the number of inflammatory cells in bronchoalveolar lavage fluid was decreased compared with unstressed animals, but levels of IL-6, IL-9, and IL-13 were increased. The decrease in inflammatory cell numbers was prevented by pretreatment with a corticosteroid receptor antagonist. In animals subjected to 7 days of stress, there was an increase in inflammatory cell numbers, which was independent of the glucocorticoid response, but no change in cytokine levels. Airway hyperresponsiveness did not change in either group of stressed animals. The authors concluded that long-term stress may engage different mechanisms than short-term stress, and can exacerbate the chronic inflammatory responses of the airways.

Review articles.
Clinicians and researchers with an interest in asthma should not miss the occasional essay titled "A Century of Asthma" by McFadden (29).

Busse and colleagues (30) reported on the future direction in asthma research in this NHLBI workshop article.

Airway Hyperreactivity
Animal models: antigen challenge.
One intriguing question is the extent to which virus-specific IgE might contribute to airway dysfunction. Dakhama and colleagues (31) provide some evidence that it might by showing that lung infection with RSV in mice was associated with increased mRNA for IgE and for IgE high- and low-affinity receptors. Moreover, RSV induced mast cell degranulation in vitro. Passive sensitization with virus-specific IgE was associated with an exaggerated airway response to RSV infection. The extent to which RSV-specific IgE contributes to human disease needs further study.

Critical inflammatory and immune processes involved in airway inflammation and hyperresponsiveness require activation of receptor tyrosine kinases. Berlin and colleagues (32) administered imatinib mesylate (Gleevac; Novartis, Basel, Switzerland), a nonspecific tyrosine kinase inhibitor that blocks BCR-ABL, c-KIT, and PDGFRA kinase activity, to allergic mice before allergen challenge. Imatinib attenuated allergen-induced airway hyperreactivity, accumulation of eosinophils, and levels of IL-4, IL-13, CCL2, CCL5, and CCL6. However, serum IgE levels, airway goblet cell number, or mucus gene (MUC5AC) expression were not reduced. The authors suggested that activation pathways, such as those involving receptor tyrosine kinase, may be important targets for future studies of the inflammatory response in asthma, and for future therapy.

In examining the events associated with allergen-induced airway reactions, Abraham and colleagues (33) explored the role of the integrin 1ß1 (very late antigen-1, CD49a/CD29) in early and late airway reactions to antigen in allergic sheep. An aerosolized antibody to the 1 chain administered 30 minutes before allergen inhalation challenge blocked the allergen-induced late airway response (but not the early airway response) and the associated airway hyperresponsiveness, as well as the recruitment of lymphocytes, neutrophils, and eosinophils induced by segmental allergen challenge. These data suggest the importance of this receptor in allergen-induced airway inflammatory reactions.

Leigh and coworkers (34) reported that, whereas IL-13 appeared to be important in airway hyperresponsiveness in mice who underwent acute antigen sensitization and challenge, it appeared to be unimportant in the increase in airway wall remodeling (increase in goblet cells, subepithelial collagen deposition, and -smooth muscle actin staining) in mice that had chronic (4-week) antigen exposure. This report stresses, yet again, important differences in acute versus chronic animal models of antigen exposure and "asthma."

Chemical and antigen challenge.
To examine the contribution of bone marrow–derived factors to differences in the allergen-induced eosinophilic airway inflammation observed in subjects with asthma with an isolated immediate airway response (early responders) versus those with both an early and late response (dual responders), Dorman and colleagues (35) examined blood, bone marrow aspirates, and sputum in 26 subjects with mild, stable asthma. In dual responders, allergen inhalation was associated with a rapid onset of IL-3–dependent eosinophilopoiesis in the bone marrow, detectable as early as 5 hours, and sustained IL-5–dependent eosinophilopoiesis at 12 and 24 hours. IL-5 protein levels increased in serum and bone marrow at 12 and 24 hours in dual responders only. There was significant correlation between IL-5–responsive eosinophil/basophil colony-forming units and IL-5 protein levels in the bone marrow, suggesting that both airway and blood eosinophilia in dual responders are sustained by an IL-5–responsive, eosinophil-differentiating process in the bone marrow. At 48 hours after allergen, bone marrow IL-5 and eosinophilopoiesis decreased, in association with increased IFN-. Because these changes occurred only in dual responders who developed peripheral blood and airway eosinophilia and prolonged airway hyperresponsiveness, the authors concluded that bone marrow eosinophilopoiesis and airway eosinophilia play a role in allergen-induced airway hyperresponsiveness.

The fate of eosinophil progenitor cells in the bone marrow after allergen challenge is not well characterized. Dorman and colleagues (36) used flow cytometry to measure CD34⁺ and CD34⁺IL-5R⁺ cells in induced sputum from subjects with asthma and normal control subjects before and 7 and 24 hours after allergen inhalation. At baseline, the proportion of airway CD34⁺ cells was greater in subjects with asthma than in normal control subjects. After allergen challenge, CD34⁺ cells in sputum increased at 7 hours in all subjects with asthma, and remained elevated only in dual responders. The proportion of CD34⁺ cells expressing IL-5R increased in dual responders only; this change occurred in conjunction with increases in IL-5 protein. The authors suggested that CD34⁺ progenitor cells migrate from the bone marrow to the airways, where, in the presence of IL-5, they differentiate into eosinophils.

Erpenbeck and colleagues (37) tested the hypothesis that administration of a natural porcine surfactant given before segmental allergen challenge of subjects with asthma would reduce the subsequent airway inflammatory reaction. This hypothesis stems, at least in part, from the observation that surfactant deficiency occurs in at least some patients with asthma. Surprisingly, they observed that surfactant treatment augmented eosinophilic inflammation, plus eotaxin and IL-5 levels, 24 hours after allergen challenge. This increase in Th2-related inflammatory factors was associated with decreased levels of IFN- and IFN- staining T cells. The mechanism or mechanisms by which this occurs and whether other surfactants would also have this proinflammatory effect remain to be determined.

Liu and collaborators (38) reported the generation of both Th1 and Th2 chemokines in human patients with asthma challenged segmentally with antigen. There was a correlation between numbers of airway (bronchoalveolar lavage) eosinophils and the Th2 cytokines IL-5 and IL-13, whereas the concentration of airway lymphocytes correlated with both Th2 and Th1 chemokines (monokine-induced by IFN-, IFN-–inducible protein-10). Furthermore, concentrations of both types of chemokines were higher in those subjects with asthma who displayed a late, as well as early, airway reaction after whole lung allergen inhalation, in comparison with subjects with asthma who only displayed an early airway response after allergen. These results suggest that activation of both Th1 and Th2 pathways are greater in these dual-responding patients with asthma.

Other Pathophysiologic Mechanisms in Asthma
Tachykinins and neural activity.
The contribution of nonadrenergic, noncholinergic nerves to asthma pathogenesis remains poorly understood. Yoshihara and colleagues (39) performed an in-depth pharmacologic study of the impact of cannabinoid receptors to inhibit capsaicin-induced contraction of tracheal rings in guinea pigs. In these studies, cannabinoid receptor 2 activation appeared to have the greatest inhibitory effects, primarily through diminishing release of tachykinins from sensory nerves, as cannabinoid receptor activation had no impact on neurokinin A–induced smooth muscle contraction. Further studies in other animal and human models with these cannabinoid receptor agonists should prove of interest.

A commonly used cancer drug, paclitaxel, known to induce severe hypersensitivity reactions, which include severe bronchospasm and respiratory distress, appears to induce these effects through sensory nerves, including both neurokinin (NK)-1 and NK2 pathways. In a study of paclitaxel in rats by Itoh and coworkers (40), the authors could not induce the reaction in mast cell–deficient rats. However, both NK1 and NK2 receptor antagonists were effective in blocking the reactions. These findings might also suggest that other "presumed" mast cell–mediated reactions may be driven by sensory nerves.

Groneberg and coworkers (41) determined the expression of transient receptor potential vanniloid 1 (TRPV-1) in the airways of patients with chronic persistent cough of diverse causes and with an enhanced capsaicin cough response. Airway mucosal biopsies were obtained by fiberoptic bronchoscopy in 29 patients with chronic cough and 16 healthy volunteers without a cough. Immunostaining for nerve profiles with antiprotein gene product-9.5 antibody showed no increase in nerve profiles in the airway epithelium of patients with chronic cough; however, with an anti–TRPV-1 antibody, there was a fivefold increase of TRPV-1–staining nerve profiles (p < 0.001). There was a significant correlation between capsaicin tussive response and the number of TRPV-1–positive nerves within the patients with cough. The findings indicate that TRPV-1 receptors may contribute to an enhanced cough reflex and the cough response in chronic persistent cough of diverse causes.

Infection and immunology.
Whether dendritic cells in asthma are polarized to enhance Th2 inflammation is not yet clear. However, a study by Long and colleagues (42) identified differences between monocyte-derived dendritic cells from those in allergic subjects with asthma, as compared with normal nonallergic subjects. Because prostaglandin E2 has been associated with enhancement of Th2 inflammation, this basal increase in monocyte-derived dendritic cells may have importance in perpetuating Th2 inflammation.

The chemokine eotaxin (CCL11) is elevated in biological fluids in patients with asthma, and correlates with eosinophilia, but its role as a chemoattractant in asthma is not well defined. Dent and coworkers (43) collected sputum from healthy subjects and from subjects with mild, stable-moderate, unstable-moderate, and severe asthma, and measured eotaxin levels and eosinophil chemotactic activity. Eotaxin was significantly increased in sputum from subjects with moderate and severe asthma, but not in those with mild asthma. Chemotactic activity was significantly increased in all subjects with asthma compared with healthy control subjects. However, a high-affinity neutralizing antibody to eotaxin inhibited chemotactic activity only in sputum from subjects with moderate and severe asthma. The authors concluded that the contribution of eotaxin to eosinophil chemotaxis in the airway increases with increasing asthma severity.

Bruder and colleagues (44) established a model to study the role of CD4⁺ T cells, as well as their regulation and immune mechanisms, in producing pulmonary inflammation. In this model, a self-antigen expressed in the lung of mice bearing a major histocompatibility complex-class-II–restricted T-cell receptor specific for the antigen was studied. These transgenic mice developed progressive interstitial pneumonitis characterized by massive lymphocytic and plasmacytic infiltration of alveolar septa, a picture resembling some interstitial lung diseases. Pulmonary inflammation reached a plateau in older mice with prominent formation of lymphoid follicles, reduced interstitial inflammation, and the induction of regulatory T cells at the site of inflammation. This model has the potential to increase our understanding of the evolution of a pulmonary inflammatory response induced by activated CD4⁺ cells with the subsequent induction of regulatory T cells.

Ali and colleagues (45) studied the effects of T-cell peptide epitopes derived from the major cat antigen Fel d-1 when given to cat-allergic subjects with asthma by inhalation. In a previous set of experiments, they showed that intradermal injection of peptides resulted in an isolated late airway response in cat-allergic subjects with asthma, with tolerance to subsequent doses of peptide, demonstrating that late airway responses could be obtained via a mechanism involving T cells alone, without the necessity of a prior early IgE-mediated/mast cell response. In this article, the authors demonstrated that inhalation of T-cell peptides also produced an isolated late airway response, but that this response was accompanied by airway inflammation (sputum eosinophilia), without induction of tolerance to a subsequent airway challenge with peptides. Therefore, these investigators have defined a unique set of human models to study T-cell–dependent late reactions, both with and without tolerance.

Fajardo and coworkers (46) characterized the pattern of proteins increased in mice lungs and bronchoalveolar lavage fluid in animals sensitized and challenged with ovalbumin by proteomic analysis. Among the proteins elevated were glycolytic enzymes, glucose-regulated protein 78kD, prolyl-4-hydroxylase, peroxiredoxin 1, arginase, cathepsin S, and Ym2, a protein with unknown function. This article not only helps to define the pattern of proteins that respond to allergic inflammatory reactions in the lung but also points out an underappreciated relationship between allergic inflammation and the induction of hypoxia-related gene products.

Histone acetylation status is believed to be important in the regulation of inflammatory gene expression and its inhibition by glucocorticoids. Cosío and colleagues (47) showed that alveolar macrophages from patients with asthma, but not peripheral blood mononuclear cells, have increased histone acetylase and decreased histone deacetylase activity when compared with control subjects. The authors suggested that one of the mechanisms of the increased inflammatory response seen in asthma is reduced histone deacetylase and increased histone acetylase activity at the site of the disease. Blood granulocytes (mainly neutrophils) had low histone deacetylase activity, offering a potential explanation for the limited ability of glucocorticoids to regulate neutrophil-mediated inflammatory responses.

To study the kinetics of memory of T lymphocytes in response to allergen, Aronica and coworkers (48) generated T-cell receptor–transgenic CD4⁺ vector cells in vitro and transfected these into naive recipient mice, which were then allowed to resume a quiescent state. Inhalation of protein antigen activated these antigen-specific Th2 donor cells, resulting in pulmonary inflammatory and airway hyperreactivity. The susceptibility to pulmonary inflammation was correlated with the size of the input of Th2 cells, but inflammation in this model was not affected by Th1 cells. The reactivation of these antigen-experienced cells by inhaled antigen did not change the cytokine balance of recipient-derived T cells recruited to the lung. These data suggest that quiescent memory Th2 cells can create susceptibility to allergic pulmonary inflammation in a manner refractory to inhibition by Th1 cells or endogenous inhibitory mechanisms.

To assess the mechanisms involved in allergic-induced hyperreactivity and airway remodeling, Leigh and coworkers (49) studied wild-type and IL-4–, IL-5–, and IL-13–deficient (^–/–) mice sensitized and studied 4 weeks after chronic allergen exposure. The study showed that wild-type mice developed sustained airway hyperreactivity and airway remodeling after chronic antigenic allergen exposure. However, both IL-4^–/– and IL-13^–/– mice were protected from development of sustained airway hyperreactivity and aspects of airway remodeling. This was not the case for IL-5^–/– mice, which developed sustained airway hyperreactivity and airway remodeling similar to that seen in wild-type mice. These results indicate that IL-4 and IL-13, but not IL-5, are critical for the development of sustained hyperreactivity and airway remodeling after allergen exposure.

Remodeling.
Because mouse models of asthma, even chronic ones, still do not completely reflect the human disease, primate models of asthma have received considerable attention. In mouse models of asthma, immunostimulatory CpG oligonucleotides have been shown to diminish Th2-associated allergic responses. In this study by Fanucchi and colleagues (50), many of the phenotypic airway changes associated with asthma in humans (eosinophil and mast cell infiltration, goblet cell hyperplasia, and airway hyperresponsiveness) were attenuated by the inhalation of these oligonucleotides in dust mite–sensitized rhesus monkeys. Interestingly, the monkeys were treated after sensitization and initial exposure, with inhibition of allergic responses even at this late stage.

Not all inflammatory responses to allergen are closely associated with Th2 pathways. Terada and colleagues (51) measured increases in the acute inflammatory response element, thrombin, in the airways of subjects with asthma after segmental allergen challenge. This was accompanied by increases in fibronectin and TGF-ß. Furthermore, highly diluted lavage fluid was able to enhance fibroblast proliferation, and that effect was blocked by a specific inhibitor of thrombin. These studies suggest that some of the remodeling events occurring in allergic asthma may occur through thrombin-related pathways.

Oxidants.
Cross-sectional and case studies have suggested that diet may play an important role in the origin and progression of asthma. Rubin and coworkers (52) measured serum levels of antioxidants in 7,505 youth in the Third National Health and Nutrition Examination Survey. They found that an SD increase in ß-carotene, vitamin C, and selenium was associated with a 10 to 20% reduction in asthma prevalence. Serum vitamin E had little or no association with asthma. The apparent benefit of selenium was even greater in youth with passive exposure to cigarette smoke. The authors suggested that serum antioxidants may be important in the prevention of asthma onset and/or the progression of asthma.

Nasal inflammation.
Because individuals with asthma usually have inflammation in the nose, Pinto and colleagues (53) hypothesized that treatment with an intranasal steroid would reduce nasal inflammation and further decrease nasal conditioning capacity. In a randomized, double-blind, placebo-controlled, two-way crossover study on 20 subjects with asthma, the authors compared the effect of treatment with intranasal budesonide for 2 weeks on nasal conditioning. Treatment with budesonide caused no significant effect on nasal conditioning as compared with placebo. In the subgroup of nonsmoking subjects, budesonide caused a significant reduction in nasal conditioning. The results suggest that nasal inflammation in nonsmoking individuals with asthma increases the conditioning capacity, and reducing it with an intranasal steroid worsens the ability of the nose to condition air. In addition, smoking causes an increase in nasal conditioning capacity by non–steroid-dependent factors.

Treatment
Glucocorticoids.
Our understanding of the role and complexity of T cells in asthma exacerbations, the pathogenesis of asthma, and other airway and interstitial diseases, and factors inducing pathologic responses versus immune tolerance, continues to evolve. Castro and colleagues (54) studied "asthma exacerbations," defined by falls in PEF (25%) and FEV₁ (15%) induced by withdrawal of inhaled corticosteroids. Thirteen of 25 asthmatics studied experienced an exacerbation defined by these criteria. Although both groups of subjects with asthma (exacerbating and nonexacerbating) experienced an increase in eosinophils present in bronchial biopsies, an increase in airway T cells (both CD4⁺ and CD8⁺) was observed only in subjects with asthma with an exacerbation defined by these physiologic criteria, accompanied by an increase in CCL5 in airway epithelium without activation of nuclear transcription factor NF-B. This pattern of immunologic responses was suggested to resemble that occurring after viral infection, rather than a classic Th2-driven eosinophilic response, and indicates a role for airway T cells in mediating asthma exacerbations.

One potential explanation for apparently refractory symptoms and airway dysfunction, particularly in clinical practice, is poor treatment adherence. Krishnan and colleagues (55) evaluated this using a range of measures, including an electronic medication monitor, in 60 adults who had been discharged after an episode of acute severe asthma. Poor adherence (< 50% doses taken) was present in one half of the population studied when an assessment was made at 1 week. When present, it was associated with significantly worse symptom control. Perhaps the most important message of this study was that poor adherence was best detected using the electronic medication monitor; self-reported adherence, canister weight, and pill count all had a low sensitivity for detecting poor adherence.

A novel approach to management of patients with severe asthma and persistent eosinophilic airway inflammation is the use of the long-acting corticosteroid triamcinalone. ten Brink and colleagues (56) showed that 2 weeks after an intramuscular injection of 120 mg of triamcinalone, all patients with refractory symptoms and eosinophilic airway inflammation, despite high-dose inhaled and sometimes oral corticosteroid, had an improvement in symptoms, improved FEV₁, and a reduction in the sputum eosinophil count. The study participants were apparently compliant with inhaled and oral treatment before entry into the study, suggesting that intramuscular triamcinolone might have additional properties. However, this management approach might be particularly useful in high-risk patients with poor treatment adherence and persistent eosinophilic airway inflammation.

The use of high-dose inhaled corticosteroids brings into focus the potential adverse effects of therapy and the importance of careful study of systemic toxicity. A good example of the latter is the thorough assessment of adrenal function and bone metabolism after escalating doses of fluticasone and mometasone described by Fardon and colleagues (57). This study showed approximately equivalent effects of fluticasone and mometasone on a microgram basis on overnight urinary cortisol–creatinine ratio and other markers. These findings are not consistent with the view that mometasone has negligible systemic bioavailability.

Leukotriene inhibitors.
Mendes and coworkers (58) investigated the effects of inhaled fluticasone and oral montelukast on airway blood flow in a double-blind, crossover study. They assessed airway blood flow using a previously validated soluble inert gas uptake method before and after treatment for 2 weeks with montelukast, fluticasone, their combination, and placebo in 12 subjects with mild intermittent asthma. The investigators found that both agents reduced bronchial blood flow modestly and equally; the effects were not additive and were not associated with improvement in FEV₁, suggesting that vascular modulation can be assessed independently of lung function in patients with mild asthma. The authors suggested that the impact of montelukast and fluticasone on airway blood flow were caused by an antiinflammatory effect of the agents.

To examine the mechanisms by which cysteinyl leukotrienes contribute to airway eosinophilia, Parameswaran and colleagues (59) examined sputum and blood eosinophils, bone marrow eosinophil progenitors, and levels of chemotactic and eosinophilopoietic cytokines in 15 subjects with mild asthma who were treated with the cysteinyl leukotriene-1 receptor antagonist, pranlukast, for 2 weeks. Pranlukast decreased mean sputum eosinophils from 5.3 to 0.7%, and blocked the allergen-induced increase in airway eosinophils by decreasing both eosinophil progenitor cells (IL-5–responsive eosinophil cfu and CD34⁺CCR3 cells) in the bone marrow and the levels of eosinophilopoietic and chemotactic cytokines (IL-5, regulated on activation normal T-cell expressed and secreted, or RANTES, eotaxin) in the airway. On the basis of their data, the authors suggested that cysteinyl leukotrienes play a role in the IL-5 signal transduction pathway.

Immunotherapy.
Djukanovic and coworkers (60) investigated the effects of anti-IgE (omalizumab) on airway inflammation in atopic asthma in a placebo-controlled, double-blind, parallel group study. Sixteen weeks of treatment with greater than 0.016 mg/kg omalizumab by subcutaneous injection every 4 weeks was associated with a significant reduction in induced sputum and bronchial mucosal eosinophils, biopsy CD3⁺, CD4⁺, and CD8⁺ cells, and cells staining positively for IL-4. Interestingly, there was no effect of omalizumab treatment on FEV₁ or airway hyperresponsiveness, emphasizing once again that these cardinal features of asthma can be disassociated.

Management plans and education.
Perhaps one of the most fundamental questions in asthma is the extent to which control can be achieved. In a large trial of over 3,400 patients, Bateman and coworkers (61) showed that total control, which was defined as no day or night symptoms, no exacerbations, and a PEF of more than 80% predicted for more than 7 of 8 weeks, was achieved in 41% of patients with uncontrolled asthma after 12 months' treatment with salmeterol/fluticasone; minimal symptoms, or well controlled asthma, were achieved in 71%. Exacerbation rates were reduced to a very low level, particularly with salmeterol/fluticasone. Asthma control was achieved at a lower dose of fluticasone and more quickly with salmeterol/fluticasone than with fluticasone alone, although a substantial number of patients required escalation of therapy to high-dose corticosteroid therapy and an important minority appeared to have persistent symptoms and/or airway dysfunction.

Specific Clinical Scenarios
Acute severe and fatal asthma.
Although the concept of airway–parenchymal uncoupling leading to loss of elastic recoil in asthma has been discussed for many years, a pathologic correlate for this process has not been described. Mauad and colleagues (62) present compelling data to suggest that there is both loss of the number of alveolar attachments and a decrease in the total elastin content in the same region. Although no correlates were made with inflammatory processes or physiologic changes, this study strongly supports structural changes in the distal airways, which may contribute to both loss of elastic recoil and, perhaps, fatal asthma.

Cough.
Airway inflammation has been previously demonstrated in patients with chronic cough; it has been suggested that the mechanism underlying chronic cough may be through the release of tussive mediators that activate afferent sensory nerve endings. Birring and coworkers (63) measured concentrations of both proinflammatory and tussive mediators (histamine, cysteinyl leukotrienes, prostaglandin D₂ and E₂, and IL-8) in the supernatant from induced sputum in 20 patients with cough-variant asthma or eosinophilic bronchitis, 20 patients with nonasthmatic cough, 22 patients with idiopathic chronic cough, and 18 control subjects. Sputum histamine concentrations were higher in patients with idiopathic cough (8.0 ng/ml) and cough-variant asthma/eosinophilic bronchitis (10.2 ng/ml) than in normal subjects (2.6 ng/ml, p < 0.01). All categories of chronic cough had significant elevations of prostaglandin D₂ and E₂ in sputum. These data indicate that the inflammatory and tussive mediators are released in patients with chronic cough and also that similarities in the mechanism of chronic cough might occur in a diverse range of conditions.

Psychopathology.
Asthma morbidity and mortality are often associated with delay in seeking treatment during severe asthma exacerbations, and poor perception of the severity of an attack has been proposed as a mechanism underlying treatment delay. Eckert and coworkers (64) examined the ability to perceive externally applied resistive loads, as well as the symptoms experienced after methacholine-induced bronchoconstriction, in 16 individuals with stable asthma who were exposed to 34 minutes of isocapnic hypoxia, hypercapnia, or isocapnic normoxia. The perceived magnitude of externally applied resistive loads was reduced throughout hypoxia compared with normoxia, and there was a trend for a progressive decline during hypercapnia. Scores for difficult breathing, chest tightness, and breathlessness were 25 to 30% lower when methacholine was administered after hypoxia compared with normoxia or hypercapnia. The authors concluded that a short period of sustained hypoxia and possibly hypercapnia may impair sensations of respiratory load, and that the effects of hypoxia persist for at least 10 minutes after returning to normoxia.

Diet.
A review of the literature relating diet to asthma prevalence and progression and speculation on future direction is provided by McKeever and Britton (65) in their Pulmonary Perspective.

Occupational Asthma
The American Thoracic Society (66) published a document titled "Guidelines for Assessing and Managing Asthma Risk at Work, School, and Recreation."

The importance of identification of occupational sensitizers when evaluating a patient with asthma is emphasized by the work of Malo and Ghezzo (67). This important longitudinal observational study of 80 patients with confirmed occupational asthma investigated recovery of airway hyperresponsiveness after cessation of exposure using complex statistical modeling. The analysis showed that progressive improvement in airway responsiveness to histamine occurred after cessation of exposure. The most rapid improvement was seen in the first 2.5 years (0.27 natural logarithms of PC₂₀/year), but improvement or 0.09 natural logarithms of PC₂₀/year was evident for several years after this. In this study, in contrast to others, the duration of exposure, the type of occupational sensitizer, and the duration of symptoms before diagnosis did not relate to the rate of recovery in airway hyperresponsiveness.

Identification of occupational asthma is problematic. Girard and coworkers (68) showed disappointing sensitivity (34.8%) and specificity (62.5%) of work and off-work PEF records in identifying occupational asthma in patients with a positive specific inhalation challenge, even when results were carefully scrutinized by five experts. The authors speculated that the poor validity of PEF monitoring in comparison to earlier studies might reflect the more widespread use of inhaled corticosteroids and long-acting ß₂ agonists by patients undergoing evaluation for occupational asthma. One potential solution is to incorporate induced sputum analysis because the addition of a significant work-related change in sputum eosinophil count improved the sensitivity of PEF monitoring. It also facilitated the potentially important observation that patients exposed to an irritant developed sputum neutrophilia. The clinical relevance of this observation needs to be investigated.

Airway Obstruction
In a State of the Art review article, Ernst and coworkers (69) examined the current approaches to the workup and treatment of patients suffering from central airway obstruction.

	CHRONIC OBSTRUCTIVE PULMONARY DISEASE

TOP ALLERGIC RHINITIS AND NASAL... ASTHMA AND AIRWAY BIOLOGY CHRONIC OBSTRUCTIVE PULMONARY... REFERENCES

 
Genetics
COPD is a complex human disease influenced by multiple genes, environmental factors, and gene-by-environment interactions. Although cigarette smoking is known to be the most important factor influencing the development of the disease, little is known about the genes involved in the pathogenesis of COPD. DeMeo and coworkers (70) performed an autosomal whole genomewide linkage scan in 72 pedigrees ascertained through a proband with severe, early-onset COPD, focusing the analysis on forced midexpiratory flow, in particular on FEF_25–75 and FEF_25–75/FVC spirometric phenotypes. A linkage was found between chromosome 2 and 12, and FEF_25–75 and FEF_25–75/FVC. Interestingly, when the analysis was stratified by cigarette smoking, the logarithm of the odds score on chromosomes 2 and 12 increased and new unreported regions on chromosomes 16, 20, and 22 were identified. These results suggest that genes in these regions may be involved in gene–smoking interactions potentially relevant to COPD susceptibility.

Skeletal muscle dysfunction is an important complication of COPD associated with reduced quality of life, increased utilization of health care resources, and increased mortality. Angiotensin II is involved in the regulation of production of cytokines, growth factors, and hormones, such as insulin, which might be involved in the development of skeletal muscle dysfunction and cachexia, and a polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to be associated with skeletal muscle dysfunction in normal subjects. Hopkinson and colleagues (71) found that the D allele of the ACE gene was associated with greater quadriceps strength in patients with COPD, and that this effect was independent of the severity of the disease. The authors also found that skeletal muscle weakness was indeed related to ACE genotype in patients with COPD, providing an interesting insight into the mechanisms of skeletal muscle dysfunction in COPD.

COPD is a heterogeneous disorder with variable morphologic, physiologic, and clinical phenotypes. To identify potential genetic factors of susceptibility to COPD, Guerassimov and colleagues (72) examined five different strains of mice after 6 months of exposure to cigarette smoke. Elastance, extent of emphysema, and the inflammatory cell and cytokine profile were measured in each animal. The results of the study show a remarkably different susceptibility of the five strains to the development of smoking-induced emphysema, with the AKR/J strain being highly susceptible, the C57BL/6/j and SJ/l strains mildly susceptible, and the NZWLac/j strain resistant. The degree of susceptibility was determined not only by the increase in airspace enlargement but also by the decrease of elastance and the pattern of inflammatory cells. T cells and a Th1 cytokine profile were present only in the lungs of highly susceptible animals, suggesting that the inflammatory cell and cytokine profile may be an important determinant of the phenotype in response to cigarette smoke exposure. These results suggest that identification of resistant and susceptible strains for the development of emphysema could be useful for genomic studies of emphysema susceptibility in mice and eventually in humans.

To investigate the role of placenta growth factor (PlGF) in the pathogenesis of pulmonary emphysema, Tsao and coworkers (73) generated PlGF transgenic mice resulting in constitutive overexpression of PlGF. The authors demonstrated that overexpression of PlGF in mice causes structural and functional alterations similar to human emphysema, with enlarged air spaces and enhanced pulmonary compliance, not associated with pulmonary inflammation. Overexpression of PlGF resulted in increased apoptosis of type II pneumocytes in the alveolar septa, a finding that was confirmed by in vitro analysis, showing that exogenous PlGF inhibited proliferation and promoted cell death of type II pneumocytes. Furthermore, PlGF transgenic mice showed decreased production of VEGF (a driver of vascular formation), suggesting that epithelial cell death affects endothelial cell fate. These results suggest that PlGF may play an important role in pulmonary emphysema through its action on type II pneumocytes and on endothelial cells via VEGF. The authors concluded that this mouse model has revealed a novel noninflammatory pathway in pulmonary emphysema.

₁-Antitrypsin Deficiency
Mal and colleagues (74) reported a case of recurrence of pulmonary emphysema in a patient with ₁-antitrypsin deficiency. They described the case of a 49-year-old white man who underwent single lung transplantation for emphysema related to ₁-antitrypsin deficiency and to superimposed smoking. After several rejection episodes in the first year, the patient remained stable without evidence of graft dysfunction for more than 10 years, but he resumed light smoking at 8 years after transplant. At 11 years after transplant, recurrence of emphysema on the grafted side was diagnosed on computed tomography (CT) of the thorax, and 1 year later, the patient developed a moderate decline in lung function. A significant amount of unopposed elastase activity, possibly induced by smoking, has probably played a central role in the recurrence of emphysema.

To determine whether variations in the distribution of emphysema may be associated with discordance between airflow obstruction and impairment of gas exchange that usually occur in patients with COPD, Parr and colleagues (75) used quantitative CT to assess emphysema severity and distribution in 119 subjects with ₁-antitrypsin deficiency (PiZ phenotype) and grouped them according to distribution pattern. In the 102 subjects with emphysema, they found that 65 had a predominantly basal pattern ("basal") and 37 (36%) had greater involvement of the upper regions ("apical"). As compared with subjects with apical distribution, subjects with basal distribution of emphysema had lower FEV₁ (mean difference, 9.9% predicted; 95% confidence interval, 3.8–16.0; p = 0.002) and milder impairment of gas exchange (Pa_O2 mean difference, 0.5 kPa, 0.03–0.1; p = 0.016), as well as alveolar–arterial oxygen gradient (mean difference, 0.7 kPa; 0.2–1.2; p = 0.007). The authors concluded that the use of single physiologic parameters as a surrogate marker of emphysema severity may be misleading.

Risk Factors
Postmortem studies of patients who died in the Warsaw Ghetto during World War II suggested that death from starvation was associated with pulmonary emphysema. To determine whether anorexia nervosa is associated with an increased risk of pulmonary emphysema, Coxson and coworkers (76) examined body mass index (BMI), pulmonary function, and CT of the chest in 21 subjects with anorexia nervosa and 16 control subjects. They found greater CT measurements of emphysema in subjects with anorexia, a correlation between the BMI and the CT measures of emphysema in all subjects examined, and a correlation between diffusing capacity and CT measurements in subjects with anorexia. These results demonstrate emphysema-like changes in the lungs of chronically malnourished subjects.

Cellular, Molecular, and Anatomic Abnormalities
To clarify the pathogenetic mechanisms of cachexia in COPD, Itoh and coworkers (77) examined the plasma levels of ghrelin, a novel growth hormone–releasing peptide, which has been shown to improve energy balance by reducing fat use and stimulating food intake. Plasma ghrelin levels were reported to be significantly higher in underweight patients with COPD (n = 26) than in normal-weight patients with COPD (n = 24) and healthy control subjects (n = 13). Moreover, plasma ghrelin levels correlated negatively with BMI and positively with catabolic factors (tumor necrosis factor [TNF-], norepinephrine) and indexes of hyperinflation (residual volume [RV%], RV/total lung capacity [RV/TLC%]). Considering the positive energy effects induced by ghrelin, the authors hypothesized that the increased ghrelin levels observed may represent a compensatory mechanism under catabolic–anabolic imbalance in cachectic patients with COPD.

In an in-depth study, Bolton and colleagues (78) performed a comparative evaluation of markers of inflammation and tissue destruction, BMI, fat-free mass, and fat mass with bone mineral density in patients with and without COPD. The fat-free mass, fat mass, and bone mineral density were evaluated using dexa scanning, as might be done to determine osteoporosis in postmenopausal women. The authors were able to identify individuals with COPD who had low fat-free mass or fat mass (often with a normal BMI), who had bone mineral density in the osteoporotic or osteopenic range. These subjects were significantly more likely to be present in the COPD group than in the control group. Although there were some differences in inflammatory and tissue destruction markers, the best predictors for osteopenia/osteoporosis were low fat-free or fat mass index. This study strengthens the argument that individuals with COPD, especially those with more severe loss of lung function, are at risk for development of osteoporosis/osteopenia. With more widespread use of inhaled steroids in this population, closer observation of patients with COPD for these comorbid processes is indicated.

Lung Inflammation
The pathogenesis of cigarette smoke–induced emphysema is an area of intense investigation. To investigate the role of TNF- in the development of emphysema and inflammatory cell recruitment, Churg and coworkers (79) exposed wild-type and TNF- receptor knock-out mice (TNFRKO) to cigarette smoke for 6 months. TNFRKO mice exhibited approximately 70% protection against the development of emphysema and had 65% fewer neutrophils than wild-type mice. The levels of metalloproteases (MMP-2, MMP-9, MMP-12, MMP-13, and MT1-MMP) were markedly increased by cigarette smoking in wild-type mice, but some increased MMP activity was present even in TNFRKO mice. The authors concluded that, at least in the mouse, emphysema has two components: the majority of matrix breakdown and emphysema is mediated via TNF- and neutrophils, with neutrophil elastase as the presumed major effector, and MMPs (at least MMP-12 and possibly other MMPs) serving to release active TNF-. The residual matrix breakdown has a different pathogenesis that may be related to other types of proteases and/or direct MMP destruction of matrix.

Lapperre and coworkers (80) sought to objectively examine the heterogeneity of COPD by categorizing the various functional and inflammatory features of COPD into separate, complementary domains without a priori assumptions. The factor analysis performed on physiologic and inflammatory data from 114 patients with moderate to severe COPD, not treated with inhaled steroids, resulted in a four-factor structure explaining 63.6% of the total variance. The four factors included the following: FEV₁, FEV₁/inspired vital capacity, and hyperinflation (Factor 1); ß2 response, total serum IgE, airway hyperresponsivenes, and Kco (Factor 2); exhaled NO (Factor 3); and percentage of sputum eosinophils and neutrophils (Factor 4). These four factors indicate that airflow limitation, a feature commonly associated with asthma and airway inflammation, is a separate, largely independent dimension that characterizes patients with COPD.

CT scans can be used to "phenotype" patients with COPD. Patel and colleagues (81) suggested that those patients with COPD with evidence for bronchiectasis, usually in the lower lobes, were at risk for greater levels of bacterial colonization, higher sputum IL-8 levels, and a more prolonged course once an exacerbation occurred. However, there was no relationship between frequency of exacerbations and CT phenotype. Larger scale studies are needed to determine whether more aggressive clearance therapy in these patients could improve their level of colonization and risk for more severe exacerbations.

Pathophysiologic and Radiologic Studies
Parr and colleagues (82) detailed the differences over time on CT scans in a cohort of patients deficient in ₁-antitrypsin. The authors astutely recognized that, in a group of individuals with a disease that should be getting progressively worse over time, the CT scans were showing improvement. When the studies over time were corrected with the use of an anatomic phantom, which benchmarked bone, water, and air, the expected progression of emphysema, with the accompanying decrease in lung density became apparent. Although this study has direct applicability to follow-up of patients with emphysema, it is likely that long-term CT follow-up of many lung diseases would benefit by benchmarking the CTs to a known correction factor.

Respiratory Muscles
Richardson and coworkers (83) assessed skeletal muscle function in patients with COPD (n = 6) during both cycle and single-leg knee-extensor exercise in comparison with that of age- and activity-matched healthy control subjects. Arterial and femoral venous blood sampling, thermodilution blood flow measurements, and needle biopsies allowed the assessment of muscle oxygen supply, utilization, and structure. Maximal work rates and single-leg O₂max during bicycle exercise were significantly reduced in patients with COPD as compared with control subjects (0.37 ± 0.1 vs. 0.63 ± 0.1 L/minute). This difference in O₂max disappeared during knee-extensor exercise, whereas maximal work capacity was reduced (flywheel resistance: 612 ± 81 vs. 923 ± 198 g in subjects with COPD and control subjects, respectively). Patients with COPD had a greater proportion of less efficient type II muscle fibers as compared with control subjects, whereas muscle fiber cross-sectional areas, capillarity, and mitochondrial volume density were not different between the groups. This study reveals significant changes in skeletal muscle structure and function in patients with COPD and suggests that those patients have a tendency for inefficient work economy in skeletal muscle, becoming more evident during knee-extensor exercise, when muscle function is not overshadowed by decreased lung function.

Peripheral Muscles
To investigate the role of systemic oxidative stress in the reduced quadriceps endurance of patients with COPD, Koechlin and coworkers (84) conducted a randomized, double-blind, crossover study measuring dynamic quadriceps endurance tests at 40% of maximal strength after oral treatment with either the antioxidant N-acetylcysteine or placebo. Venous blood was sampled before and immediately after exercise, as well as 6 hours later. Endurance time improved by 25% after N-acetylcysteine treatment compared with placebo (p < 0.05). Oxidant release by stimulated phagocytes decreased after N-acetylcysteine treatment (p < 0.05). No change in the antioxidant system was observed. Lipid peroxidation, an index of oxidative stress, was significantly increased 6 hours after exercise in the placebo-treated patients' condition (p < 0.05), but not after N-acetylcysteine treatment. Advanced oxidized protein products, another index of oxidative stress, were also increased 6 hours after exercise by 139 ± 27% in the placebo group, but only by 54 ± 19% after N-acetylcysteine treatment (p < 0.05). These results suggest that oxidative stress may be involved in the reduced quadriceps endurance of patients with COPD.

Drug Therapy
Glucocorticoids.
To evaluate the potential for inhaled corticosteroids to adversely impact bone density, Lee and Weiss (85) analyzed a predominantly male Veterans' Affairs–based population of over 40,000 patients with COPD. Cases with nonvertebral fractures were matched with control subjects. Supporting results from previous studies, veterans using more than 700 µg of inhaled steroids/day (beclomethasone equivalent) were at significantly higher risk of fracture than those on no steroids or on lower doses. This study suggests that specific guidelines for monitoring should be developed.

The use of either high-dose inhaled corticosteroids and/or oral corticosteroids could have beneficial impact beyond the lungs, with an impact on systemic inflammation as well as local lung inflammation. Systemic inflammation has recently been identified as a risk for coronary artery disease. In the study by Sin and colleagues (86), patients with mild to moderate COPD treated with high-dose inhaled or oral steroids lowered their C-reactive protein levels in blood by over 70%. The levels of C-reactive protein correlated with levels of IL-6, but IL-6 was not significantly impacted by corticosteroids. The mechanism for the systemic effects are not clear but are likely related to absorption of the inhaled steroids, leading to systemic effects similar to those observed on bone. Whether the use of inhaled corticosteroids in COPD can impact the high rate of coronary disease found in this population awaits large-scale trials.

To study the potential relevance of the interactions between COPD, use of inhaled corticosteroids, and osteoporosis, Scanlon and colleagues (87) randomized over 400 subjects with mild to moderate (not severe) COPD to therapy with 600 µg of triamcinolone or placebo twice daily. Bone mineral density was analyzed at the femur and lumbar spine sequentially over 3 years. Over 3 years, there was a significant loss of bone mineral density at the femoral neck, but not the lumbar spine as compared with the placebo arm. There were also significantly more patients treated with inhaled corticosteroids who had a greater than 6% decline in bone mineral density as compared with the placebo group. Relationships were seen with female sex and the level of compliance with therapy.

Inhaled diuretics.
To investigate the effect of inhaled furosemide on the sensation of dyspnea during exercise, Ong and coworkers (88) performed a double-blind, randomized, crossover study in 19 patients with moderate to severe COPD. Inhalation of furosemide alleviated the sensation of dyspnea induced by constant-load exercise testing (mean dyspneic visual analogue scale score: 33.7 ± 25.2) as compared with placebo (visual analogue scale score: 42.4 ± 24; p = 0.014). Moreover, furosemide induced a small but significant improvement in mean FEV₁ after exercise testing (FEV₁ 45.37 ± 16.66% predicted) as compared with placebo (FEV₁ 43.53 ± 14.53% predicted, p < 0.05). The authors concluded that the overall reduction in dyspneic sensation produced by furosemide during endurance testing might be caused, at least in part, by its bronchodilator effect, but also suggested that other mechanisms may be involved, such as effects on the airway epithelium, sensory nerve endings, or an inhibition of mediator release from inflammatory cells

New drugs.
Men with COPD have a high prevalence of low testosterone levels and dysfunction of the muscles of ambulation, which may contribute to muscle weakness and exercise intolerance. To determine the effect of testosterone supplementation on body composition and muscle function, Casaburi and coworkers (89) performed a randomized, placebo-controlled, 10-week trial in 47 men with COPD (mean FEV₁ 40% predicted) and low testosterone levels (mean 320 ng/dl). Subjects were randomized to four groups: placebo/no training, testosterone/no training, placebo/resistance training, or testosterone/resistance training. Testosterone injections (100 mg of testosterone enanthate weekly) yielded a mean increase of 271 ng/dl in the nadir serum testosterone concentration. The lean body mass increase averaged 2.3 kg with testosterone alone and 3.3 kg with combined testosterone and resistance training (p < 0.001). Increase in one-repetition maximum leg-press strength averaged 17.2% with testosterone alone, 17.4% with resistance training alone, and 26.8% with testosterone plus resistance training (p < 0.001). These results show that testosterone increases lean body mass and strength in patients with COPD, and that the effect is amplified by concomitant resistance training. The results suggest that testosterone supplementation may be an appropriate therapy, in conjunction with rehabilitative programs, for patients with COPD who have muscle weakness.

Other Therapies
Lung volume reduction.
To investigate whether passive diaphragm stretch after lung volume reduction surgery (LVRS) results in fiber injury, Lewis and colleagues (90) studied the short-term impact of LVRS on diaphragm muscle in hamsters with emphysema induced by intratracheal instillation of pancreatic porcine elastase and control animals. Analysis of diaphragm fiber injury, specific force, and insulin-like growth factor I (IGF-I) expression were assessed 4 days after LVRS. Sarcolemmal rupture was evident in 10.9% of fibers in the LVRS group at Day 1, and reduced to 1.6% in the LVRS group at Day 4. Ultrastructural analysis revealed focal abnormalities involving over one third of fibers in LVRS group animals. IGF-I immunoreactivity was increased in the LVRS group at Day 4, localizing to types IIA and I fibers. These structural changes were associated with marked force deficit in the LVRS group. Thus, short-term effects of LVRS on the diaphragm of emphysematous hamsters included focal sarcolemmal rupture and ultrastructural sarcomeric injury of diaphragm fibers, profound force deficit, and upregulation of IGF-I within diaphragm fibers. The diaphragm fibers most affected were those likely recruited during normal ventilatory behaviors, thus limiting the reserve capacity and function of the ventilatory pump after LVRS. These results suggest a markedly compromised ventilatory pump acutely after LVRS.

Outcome
Wilkinson and coworkers (91) followed over 1,000 exacerbations occurring in 128 patients with COPD over a 6-year period. Patients kept diary cards of symptoms and peak flows. Those individuals that sought medical care earlier in the course of their exacerbation had shorter exacerbations. More frequent treatment of the exacerbations led to improved quality of life, whereas delaying treatment increased the risk of hospitalization. Whether patient diaries and peak flow readings will be indicated in "poor perceivers/noncomplainers" in COPD requires further study.

Microbiology
Nontypeable Haemophilus influenzae colonizes the respiratory tract of patients with COPD, often presenting with a pattern in which periods of negative sputum cultures are preceded and followed by isolation of apparently identical strains. To investigate whether the strains preceding and following episodes of negative cultures were indeed the same, Murphy and colleagues (92) performed molecular typing on isolates of H. influenzae collected monthly in a prospective study. During a 7-year study involving 104 patients, they detected 122 episodes of negative cultures lasting 1 month or more, which were preceded and followed by isolation of an apparently identical strain of H. influenzae. Seventeen such episodes of negative cultures, lasting 6 months or more, were studied in detail to test the hypothesis that these periods of negative cultures represented continuous colonization by the same strain of H. influenzae. Molecular typing by three independent methods established that the strains preceding and following the episodes of negative cultures were indeed identical. Strain-specific H. influenzae DNA was detected in some of the sputum samples that had yielded negative cultures. These results show that some patients with COPD are persistently colonized with H. influenzae, and that sputum cultures underestimate the frequency of colonization of the respiratory tract by H. influenzae. The study also suggests that chronic bacterial colonization may significantly contribute to airway inflammation and to the course and pathogenesis of COPD.

To test the hypothesis that the immune response to the homologous (infecting) strain of H. influenzae may have limited ability to kill other (heterologous) H. influenzae strains, and thus that it may protect against recurrent exacerbations caused by homologous strains, Sethi and coworkers (93) collected sputum and serum samples in 81 patients with COPD monthly and during exacerbations. They found that, after exacerbation, an immune response to homologous H. influenzae occurs in 61% of cases with newly acquired strains as compared with 21% of cases with preexisting strains (odds ratio 4.4; confidence interval 95%, 1.8–10.8; p = 0.001). New bactericidal antibodies developed after COPD exacerbations were highly strain-specific, showing bactericidal activity for only 12% of heterologous strains. These results suggest that, after an exacerbation of COPD associated with H. influenzae strain, serum antibodies to the infecting strain develop in the majority of cases with newly acquired but homologous strains, and that the immune response to the homologous strain may not protect against infectious exacerbations by heterologous strains of H. influenzae. These observations further support the role of H. influenzae in a large proportion of COPD exacerbations and, by showing the limited immune response to heterologous strains of H. influenzae, may explain the mechanism of recurrent exacerbations with H. influenzae in COPD. In fact, although patients produce strain-specific antibodies to homologous strains of H. influenzae after exacerbations, the immune response leaves the host susceptible to reinfections by other heterologous strains of H. influenzae.

To determine whether Pneumocystis colonization is associated with COPD and its severity, Morris and colleagues (94) conducted a cross-sectional analysis using nested polymerase chain reaction, and examined Pneumocystis colonization rates in surgical lung biopsies from 68 smokers with a wide range of pulmonary function. Pneumocystis colonization was detected in 37% of patients with very severe COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stage IV) as compared with 5% of smokers with normal lung function or less severe COPD (Stages 0 to III; p = 0.004) and with 9% of control subjects (p = 0.007). Subjects with Pneumocystis colonization showed more severe airway obstruction than Subjects without colonized Pneumocystis (median FEV₁, 21 vs. 62% predicted, p = 0.006). Patients with very severe COPD (GOLD IV) were the strongest predictor of Pneumocystis colonization (odds ratio 7.3, 95% confidence interval, 2.4–22.4; p < 0.001), independent of smoking history. The authors concluded that there is a strong association between Pneumocystis colonization and severity of airflow obstruction in smokers, suggesting a possible pathogenic link with COPD progression.

	FOOTNOTES

 
Conflict of Interest Statement: L.F. has participated in Advisory Boards for Altana, Aimirall, Pfizer, Roche, GlaxoSmithKline (GSK), Merck, Sharp, and Dohme (MSD), and Chiesi Farmaceutical and received grants from Altana-Byk Guiden, Aimirall, AstraZeneca (AZ), Bayer, Blofutura, Boehringer Ingelheim, Chiesi Farmaceutical, GSK, MSD, Miat, Pfizer, and Schering Plough in 2001–2004 and owned stock in GSK in 2000–2003; S.P.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.P. has received $5,000 from GSK and $2,000 from AZ for participation as a speaker and received $160,000 as a grant from GSK; S.E.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; W.M. has been reimbursed for travel by GSK, Zambon, AZ, Boehringer Ingelheim, Pfizer, and Micromet and has received honoraria from GSK, AZ, Zambon, and Pfizer for participating as a speaker at scientific meetings. He serves on Advisory Boards for GSK, Pfizer, Aimirall, Amgen, Bayer, and Micromet and serves as a consultant for Pfizer and SMB Pharmaceuticals. He has received research grants to support work carried out in his laboratory from SMB, Pfizer, Ceremedix, GSK, Chugi, and Novartis; F.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.A. has been a consultant for Eli Lilly in 2002 and 2003 and also received clinical research contracts from Eli Lilly.

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