New immunological approaches and cytokine targets in asthma and allergy REVIEW

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New immunological approaches and cytokine targets in asthma and allergy REVIEW
Copyright #ERS Journals Ltd 2000
European Respiratory Journal
ISSN 0903-1936
Eur Respir J 2000; 16: 1158±1174
Printed in UK ± all rights reserved
New immunological approaches and cytokine targets
in asthma and allergy
R.G. Stirling, K.F. Chung
New immunological approaches and cytokine targets in asthma and allergy. R.G. Stirling,
K.F. Chung. #ERS Journals Ltd 2000.
ABSTRACT: The aims of current asthma treatment are to suppress airway inflammation and control symptoms, and corticosteroids maintain a commanding position in
this role.
Steroids effectively suppress inflammation in the majority of patients but have little
impact on the natural history of this disease. In severe asthmatics, corticosteroids may
have relatively less beneficial effects. Recent advances in understanding the inflammatory and immunological mechanisms of asthma have indicated many potential
therapeutic avenues that may prevent or reverse abnormalities that underlie asthma.
As the roles of effector cells, and of signalling and adhesion molecules are better
understood, the opportunities to inhibit or prevent the inflammatory cascade have
In addition, there have been advances in the synthesis of proteins, monoclonal
antibodies and new small molecule chemical entities, which may provide valuable
flexibility in the therapeutic approach to asthma. The novel immunological approaches include the prevention of T-cell activation, attempts to influence the balance of
T-helper cell (Th) populations to inhibit or prevent Th2-derived cytokine expression,
and the inhibition or blockade of the downstream actions of these cytokines such as
effects on immunoglobulin-E and eosinophils. These approaches provide broad as well
as highly specific targeting, and also prospects for prevention and reversal of
immunological and inflammatory abnormalities associated with asthma.
Hopefully, the development of effective antiasthma agents with effects beyond those
provided by current therapies coupled with lesser side-effects will further address the
unmet needs of asthma.
Eur Respir J 2000; 16: 1158±1174.
Airway inflammation and remodelling
The chronic airway inflammation of asthma is characterized by infiltration of the airway wall and lumen by
diverse effector cells, including T-lymphocytes, eosinophils, monocytes/macrophages, mast cells, and occasionally, neutrophils [1±3]. The mobilization, activation and
trafficking of these effector cells to the airway are
controlled by a complex cytokine milieu derived from
activated CD4+ T-helper (Th) cells and also from other
resident airway cells including airway smooth muscle
and epithelial cells. T-helper cells of type 2 variety (Th2)
secrete a Th2 profile of cytokines, after cognate stimulation of the naive T-cell by antigen presenting cells, such
as the dendritic cell and the alveolar macrophage (fig. 1).
The Th2 cytokines include interleukins (IL)-4, -5, -9, -10
and -13. These cytokines promote various elements of
allergic inflammation (table 1), including propagation of
the Th2 phenotype, isotype-switching from immunoglobulin (Ig)-G1 to IgE synthesis, eosinophil mobilization,
maturation and activation and mast cell activation [4].
Additionally, the airway wall undergoes chronic structural changes labelled as remodelling, which include thickening of the airway smooth muscle due to hypertrophy
National Heart and Lung Institute,
Imperial College School of Medicine and
Royal Brompton and Harefield Hospital,
London, UK.
Correspondence: K.F. Chung
Dept of Thoracic Medicine
National Heart & Lung Institute
Dovehouse Street
Fax: 44 2073518126
Keywords: Adhesion molecules
gene therapy
Received: September 20 2000
Accepted after revision September 22 2000
and hyperplasia, myofibroblast activation with increase
in subepithelial basement membrane collagen deposition, angiogenesis and increase in submucosal blood
vessels and an increase in goblet cell numbers in the
airway epithelium [5±8]. These changes are also variably
influenced by Th2 cytokines, and certain growth factors
[9, 10].
The exact link between the chronic inflammatory and
remodelling processes and the clinical presentation of
asthma is unclear in terms of the pathophysiological
mechanisms and of the contribution of the components of
these processes to bronchial hyperresponsiveness, airway
narrowing and the acute exacerbations that characterize the
disease. The overwhelming hypothesis is that these are
intricately related such that inhibition or prevention of the
inflammatory process should improve the control of
asthma, an idea supported by the potent actions of topical
corticosteroids in controlling the eosinophilic and lymphocytic inflammation of asthma, while being most
effective in restoring lung function and bronchial hyperresponsiveness [9, 10]. However, identification of the
key immunological and inflammatory processes that lead
to functional abnormalities and symptoms remain to be
Table 1. ± Summary of Th2 cytokine effects as predicted by cytokine stimulation and ablation studies (animal and human
data included)
Th response
Mucus hyperplasia
Mast cell activation
Surface markers
IL: interleukin; BHR: bronchial hyperresponsiveness; IgE: immunoglobulin-E; VCAM: vascular cell adhesion molecule; MHC: major
histocompatability complex.
Limitations of current therapy
Despite the introduction of new agents such as the
leukotriene inhibitors, corticosteroids remain the antiinflammatory drug of choice for the majority in the
treatment of chronic asthma [11, 12]. However, treatment
failure and drug-related side-effects pose serious limitations to their use [13]. Severe and difficult-to-treat asthmatics are characterized by ongoing symptoms and
frequent exacerbations despite the use of existing antiinflammatory and bronchodilator therapies. These patients are frequently prescribed corticosteroids to which
their disease is partially responsive and they endure the
adverse consequences of high dose inhaled and systemic
corticosteroid therapy [13, 14]. Amongst severe asthmatics, up to 25% demonstrate relative unresponsiveness
to the therapeutic but not to the adverse effects of
corticosteroids [15, 16]. The identification of novel therapeutic targets and subsequent development of specific
and safe therapeutic agents therefore, represents a major
challenge in asthma therapy. In addition to attempting to
reverse established changes in the airways resulting from
chronic inflammation and airway remodelling, the prevention of allergy and asthma remains an important consideration given the rising prevalence of these conditions
in many industrialized countries [17].
Immunological imbalance: T-helper cell balance
The vast majority of asthmatics have an atopic
background, in whom the inflammatory process of asthma
is assumed to be driven following sensitization and reexposure, or challenge by common aeroallergens. However, the inflammatory abnormalities in nonatopic asthma
also bear many similarities to those observed in the allergic
inflammatory process, including the presence of eosinophils, activated CD4+ T-cells, and the expression of Th2derived cytokines [18]. These findings suggest that most
asthmatics may benefit from an approach that targets
mechanisms of allergic sensitization and inflammation.
Allergens are taken up by specialized cells within the
mucosa such as dendritic cells (antigen-presenting cells),
and subsequently processed, following which peptide
fragments are presented to naive T-cells. The activation of
naive T-cells requires direct signalling by two distinct
pathways (fig. 1): firstly via the CD4+ T-cell receptor
through the antigen-presenting cell (APC)-bound antigen
to major histocompatability (MHC)-II complex and,
secondly, via the costimulatory pathway linked by the
B7 family and the T-cell bound CD28 [19]. T-cells
stimulated via the T-cell receptor (TCR) in the absence of
costimulatory signalling are incapable of IL-2 secretion
and subsequent activation and therefore, enter an anergic
state [20]. CD28 itself has two major ligands, B7.1, which
inhibits Th2 cell activation and development, and B7.2,
which induces T-cell activation, and Th2 development.
An important third ligand, cytotoxic T-lymphocyte
associated protein-4 (CTLA4), is expressed on activated
T-cells, binds CD28 with enhanced avidity and acts as a
negative regulator of T-cell function by inhibiting Th2
differentiation [21, 22]. In those who later develop an
allergic response, naive T-cells differentiate into the Th2
T-cell profiles in the newborn demonstrate a Th2 bias
suggesting that prenatal influences are involved in T-cell
priming [23]. Th1 cells form a natural counterbalance to
Th2 cells driving protective cell-mediated immunity
(CMI), and are induced on exposure to foreign agents
including protozoa and bacteria. Th1 responses are
characterized by the induction of CMI responses and
the synthesis of IgG2a, while Th2 responses are of the
humoral-type, inciting the production of IgE and IgG4.
Signal 2
Signal 1
Fig. 1. ± T-helper cells differentiate from the naive state following
interaction with the antigen-presenting cell. The Th2 phenotype is
characterized by secretion of the cytokines interleukins -4, -5, -9 and
-13, while the Th1 cells secrete interferon-c, and interleukin (IL)-2. IL-4
directly stimulates Th2 differentiation while IFN-c promotes the Th1
phenotype. APC: antigen presenting cell; MHC: major histocompatibility complex; TCR: T-cell receptor; CTLA4: cytotoxic T-lymphocyte
associated protein-4; Ag: antigen.
Th1 responses inhibit Th2 responses through the production of cytokines such as IL-12 and interferon gamma
(IFN-c) (fig. 2). Abnormal polarization of these responses
may result in tissue pathology. The excessive expression
of Th1 cytokines is noted in autoimmune conditions
including experimental diabetes mellitus [24, 25] and
multiple sclerosis [26, 27], while excessive Th2 expression is observed in the atopic diathesis. There is evidence
for a preferential skewing to expansion of the CD4+ Th2
lymphocyte subset in allergic processes [3, 28] and this is
a likely crucial forerunner to the development of allergic
disease. During the course of maturation of the normal
infant however, increased Th1 expression occurs, and the
Th2 imbalance is overcome [29]. Delay or failure of this
Th1 response may result in Th2 persistence and atopy or
atopic disease, and accordingly in infants destined to
become atopic, an impaired production of IFN-c by
circulating lymphocytes is observed [30].
The "hygiene hypothesis" postulates a diminished induction of Th1 responses as a potential explanation for the
rising prevalence of atopy and asthma [23, 31, 32]. Crosssectional surveys have identified inverse relationships
between prior microbial exposure and development of
atopy [23, 33, 34]. Further, respiratory allergy appears
less frequently in those heavily exposed to orofaecal and
foodborne microbes [32]. Thus, improved hygiene, early
infection and antibiotic use, and a westernized or semisterile diet may facilitate atopy by influencing exposure to
commensals and pathogens that stimulate immune cell
populations such as gut-associated lymphoid tissue [35,
36]. Thus, early environmental exposure may be a
determinant of the development of atopy in the adult [37].
The identification of ways to prevent, control or even
reverse the process of Th2 immunodeviation has become
a focus for the development of new strategies to control
asthma and allergies.
T-cell immunomodulators
Cyclosporin A and the functionally-related, tacrolimus
(FK506), are powerful immunosuppressant agents used
widely to prevent immune rejection in organ transplantation. These compounds inhibit T-cell growth by creating a
block in the G0 phase of development through inhibition of
T-cell growth factors including IL-2 [38]. Both agents
strongly inhibit mitogen-stimulated IL-5 release by
inhibition of transcription [39, 40]. Cyclosporin A and
Table 2. ± Novel strategies for the inhibition and prevention of allergic asthma
Prevention of T-cell activation
Prevention of reversal of
Th2 expression
Inhibition of Th2 cytokines/
Three main thrusts of research into new therapies have
developed: 1) preventing T-cell activation; 2) prevention or
reversal of Th2 polarization, and; 3) ablation of the effects
of Th2 cytokines and downstream mediators (table 2).
Many of the potential ways of achieving these objectives
have been investigated in animal (mainly murine) models,
and therefore, their applicability to human asthma and
allergic diseases remains unclear given the inherent
variability within varied species.
Promotion of Th1 cytokines/
Prevention of T-cell activation
Inhibition of downstream
Antiinflammatory cytokines
Inhibition of eosinophil
migration and activation
Fig. 2. ± Activation of T-helper cells by the antigen presenting cell
(APC) requires dual signalling via the major histocompatibility complex
and the T-cell receptor, and via B7.2:CD28 interaction and results in Th2
deviation, activation and interleukin (IL)-2 secretion. T-cell receptor
stimulation in the absence of costimulation via CD28 induces clonal
anergy. NK: natural killer cell; IFN-c: interferon-gamma.
IgE inhibition
rhu IL-4 mutant proteins
STAT-6 inhibition
Anti-IL-5 monoclonal
GATA inhibition
Soluble IL-13Ra
Peptide immunotherapy
M. vaccae vaccination
CCR3 antagonist
CCR3 antisense
VLA4 inhibitor
ICAM-1 inhibitor
Monoclonal anti-IgE (E25)
CTLA4: cytotoxic T-lymphocyte associated protein-4; IL:
interleukin; STAT-6: signal transduction and activation of
transcription-6; IFN: interferon; SIT: specific immunotherapy;
M. vaccae: Mycobacterium vaccae; CCR3: eotaxin receptor;
VLA4: very late antigen-4; ICAM-1: intercellular adhesion
molecule-1; Ig: immunoglobulin; E25: nonanaphylactogenic
IgE monoclonal antibody.
Inhibition of T-cell costimulation: cytotoxic
T-lymphocyte associated protein-4-immunoglobulin
FK506 also have effects on other cells and markedly
reduce basophil histamine release [41]. In human studies,
cyclosporin A by inhalation provides significant inhibition of the allergen-induced late allergic reaction [42]. In
corticosteroid-dependent asthma, low-dose cyclosporin A
improved lung function [43] and allowed for a 62%
reduction in oral steroid dose requirement [44], but at
the expense of adverse effects which would not prove
tolerable in mild disease.
The folate antagonist, methotrexate, has well recognized
anti-inflammatory effects in rheumatoid arthritis and is
used as a steroid-sparing agent in asthma. Meta-analysis of
controlled trials confirms steroid-sparing effects while
maintaining lung function or symptoms [45±47]. In clinical practice, these immunomodulators are only modestly
effective in a proportion of patients. A number of compounds, which show immunomodulatory activity, are
currently under development. However, it is likely that
compounds that have nonspecific immunosuppressive Tcell activity may not be as effective as the more specific
Th2 cell inhibitors [48]. Suplatast tosilate, a compound
developed in Japan, selectively prevents the release of IL4 and IL-5 from Th2 cells and can reduce bronchial
eosinophilia in animal models of bronchial hyperresponsiveness (BHR) [49, 50]. It has been shown to improve
pulmonary function and symptom control, and allows for
a decrease in the dose of inhaled corticosteroids [51], and
has been launched for the treatment of asthma in Japan.
Although corticosteroids inhibit antigen uptake and
processing, they do not appear to impact on antigen
presentation [65]. Therefore, the potential for disruption
of antigen presentation by specific inhibition of costimulatory molecule interaction has been recognized and
studied in murine models. A recombinant fusion protein
consisting of the extracellular domain of CTLA4 linked
to the constant region of IgG1, known as CTLA-4-Ig,
binds B7 molecules with an affinity similar to that of
membrane CTLA4. It therefore, acts as a powerful
inhibitor of B7: CD28 mediated costimulation. T-cell
activation following B7:CD28 signalling may be blocked
using the soluble protein ligand CTLA4-Ig, and this
can substantially reduce BHR, bronchoalveolar lavage
(BAL) eosinophilia and specific IgE responses when
given either prior to sensitization or to allergen challenge [66]. CTLA4-Ig reduced IL-4 and IgE levels while
IFN-c and IgG2a were unchanged, suggesting downregulation of Th2 response without upregulation of the
Th1 response [67]. Similar outcomes are observed following monoclonal anti-B7.2 (anti-CD86) treatment in
allergen-challenged mice [68]. Thus, CTLA4-Ig is potentially a powerful immunomodulator with the potential
of suppressing Th2 based responses to allergens in
T-cell depletion: anti-CD4 monoclonal antibody
Modulation of T-helper 1/T-helper 2 differentiation
CD4+ T-cells appear central to the orchestration of
allergen-mediated airway disease. Depletion of T-cells in
murine sensitized and allergen-challenge models using
recombinant monoclonal antibodies results in a complete
ablation of airway hyperresponsiveness and airway
eosinophilia [52]. Conversely, antibody-mediated CD8+
T-cell depletion augments BHR and eosinophilic inflammation in the allergen-challenge model [53]. A preliminary clinical trial in asthma has evaluated the use of a
single dose of anti-CD4 monoclonal antibody in severe
corticosteroid dependent asthma [54]. This treatment led
to a reduction in circulating CD4+ T-cell numbers and
caused an improvement in morning and evening peak
expiratory flows but did not significantly impact on
asthma symptoms. Early studies have also suggested
some benefit of this approach in the CD4+ dependent
processes of multiple sclerosis [55, 56] and collagenarthritis [57].
It is likely that other T-cell subsets may also be
important in asthma. Recent investigations have highlighted the existence of CD8+ T-cells (i.e. TC2), which
secrete Th2 type cytokines and their influence on the
mediation of allergic airways disease is as yet to be
established [58±60]. Further uncertainty regarding the
approach of CD4 depletion relates to the induction of
CD4 lymphopenia and immunosuppression and the
resultant risk of opportunistic infection and neoplasia
[61±63]. Nondepleting CD4 antibodies have been
generated but are as yet untried in pulmonary disease
Specific attempts to alter Th1/Th2 balance by enhancing
Th1 and abrogation of Th2 responses have been the major
thrust of immune approaches to the prevention and treatment of allergies and asthma. Induction of Th1 responses
may have a direct suppressive effect on Th2 mediated
inflammatory processes (table 3). The most direct approaches include administration of cytokines that will induce
activation of Th1 pathways (e.g. IFN-c, IL-12 and IL-18)
or of blocking antibodies that inhibit the effect of Th2related cytokines (e.g. anti-IL-4, anti-IL-5, anti-IL-9, and
Table 3. ± Summary of T-helper 1 cytokine effects as
predicted by cytokine stimulation and ablation studies
(animal and human data included)
Th response BHR IgE
Th1 Th2
Blood Airway
IFN: interferon; IL: interleukin; BHR: bronchial hyperresponsiveness; IgE: immunoglobulin-E; TNF-a: tumour necrosis
Inhibition of allergy and asthma by
T-helper 1 related cytokines
Interferon gamma
IFN-c, released from CD4+(Th1) and CD8+(TC2) cells,
is a critical factor controlling the balance of Th1/Th2
development, and exerts an inhibitory effect on Th2 cells
[4]. IFN-c is also a powerful and relatively specific
inhibitor of IL-4-induced IgE and IgG4 synthesis by Bcells. A reduced production of IFN-c by the T-cells of
asthmatic patients is seen and correlates with disease
severity [69]. Intra-tracheal IFN-c in allergen-sensitized
and -challenged mice causes a dose-dependent reduction
in BAL IL-5 levels and airway T-cells and eosinophils
[70, 71]. Intraperitoneal IFN-c also reduced lung Th2
cytokine levels, attenuated allergen-induced BHR with
concomitant reduction in BAL eosinophilia, while an
IFN-c-blocking antibody led to an increase in airway
CD4+ T-cells and BHR [72]. The inhibition of eosinophil
recruitment appears to be dependent on the inhibition of
CD4+, but not on CD8+ T-cell accumulation within the
airways [70, 73]. Inhalation of IFN-c by nonasthmatic
humans increases epithelial lining and BAL fluid IFN-c
levels but does not affect serum IFN-c levels and
therefore, may avoid toxicity associated with systemic
administration [74, 75]. However, in one study of subcutaneous IFN-c therapy in steroid-dependent asthma no
effect on lung function or treatment requirement was
observed despite a significant reduction in circulating
eosinophil numbers [76].
IL-12 is produced by antigen-presenting cells and
enhances the growth of activated T- and natural killer
(NK)-cells, stimulating them to produce IFN-c [77, 78].
IL-12 also promotes the differentiation of IFN-c-producing T-cells and inhibits the differentiation of T-cells into
IL-4 secreting cells [77]. Thus, IL-12 can regulate Th1
cell differentiation while suppressing the expansion of
Th2 cell clones by early priming of undifferentiated Th0
cells for IFN-c secretion. IL-12 may play an important
role in inhibiting inappropriate IgE synthesis and allergic
inflammation as a result of allergen exposure. In murine
asthma models, administration of IL-12 leads to a
reduction in allergen specific IgE levels, ablation of
airway hyperresponsiveness and inhibition of eosinophil
recruitment [79±81]. However, further studies have
suggested that this protective role may only be conferred
when acting synergistically with IL-18 [82]. In therapeutic trials, IL-12 levels increased during corticosteroid
therapy [83] and during specific immunotherapy [84]. In
a phase-I trial of IL-12 in asthma, a significant reduction
of peripheral eosinophils and a trend towards a reduction
in airway eosinophils was observed without effect on
allergen-induced early or late phase responses [85]. IL-12
has proved a useful adjunct to cancer chemotherapy by
induction of a protective Th1 response [86], but significant toxicity including arrhythmias, liver function
abnormalities and flu-like illness will limit its potential
utility in asthma [87].
IL-18 (IFN-c-inducing factor) is a potent inducer of
IFN-c production by T-, NK- and B-cells and plays an
important part in the induction of Th1 responses [88, 89].
IL-18 receptors are expressed selectively on murine Th1
but not Th2 cells. Recombinant human IL-18 potently
induces IFN-c production by mitogen-stimulated peripheral blood mononuclear cells and enhances NK-cell
cytotoxicity [90], while increasing NK-cell granulocytemacrophage colony-stimulating factor (GM-CSF) release
and CD8+ T-cell IL-10 production [91]. IL-18 and IL-12
have synergistic effects on Th1 development, which may
be due to reciprocal upregulation of their receptors. IL-18
may be important in the control of allergen-induced BHR
by vaccination. Vaccination using heat-killed Listeria
monocytogenes caused a marked inhibition of allergeninduced BHR and airway inflammation, associated with
conversion to the Th1 phenotype in mice [92, 93]. This
effect was IL-12-dependent and associated with a
marked upregulation of IL-18 messenger ribonucleic
acid (mRNA) expression. These studies also demonstrated that administration of an adjuvant after allergen
exposure, was able to reverse established BHR. The
direct administration of IL-18 in the murine asthma model
provides more confusing results; although IL-18 appears
to play a protective role when administered with allergen
challenge, administration with sensitization paradoxically
increased IgE and IL-5 levels and promoted BAL
eosinophilia [94, 95]. Thus, IL-18 appears to be involved
in vaccination-mediated inhibition of Th2 responses and
may have properties directly driving BHR in mice.
Inhibition of T-helper 2 related cytokines
IL-4 has a central role in Th cell development as a potent
inducer of Th2 maturation from the naive T-lymphocyte
[96, 97]. Additionally, IL-4 induces the isotype-switch
necessary for IgE synthesis [98], upregulates IgE receptors [99, 100] and vascular cell adhesion molecule-1
(VCAM-1) expression on vascular endothelium, thus
facilitating endothelial passage and accumulation of
eosinophils [101]. IL-4 is therefore, an attractive target
to inhibit, resulting in downregulation of Th2 activation.
Anti-IL-4 monoclonal antibody treatment of mice prior to
allergic sensitization markedly reduces IgE synthesis
[102], but does not appear to inhibit airway eosinophilia
or BHR [103]. A recombinant soluble IL-4 receptor (sIL4R) has been designed as a mimic of the cell-surface
receptor, which thus binds and sequestrates free IL-4,
but because these soluble receptors lack transmembrane
and cytoplasmic domains, they act as an IL-4 receptor
blocker. In murine studies, sIL-4R reduces allergenspecific IgE responses, airway hyperresponsiveness,
VCAM-1 expression and eosinophil accumulation
[104]. Preliminary human asthma studies with single
doses of sIL-4R have shown acceptable tolerance,
improvement in lung function and a reduction in rescue
b2-agonist requirement [105]. Additionally, a trend towards a reduction in serum eosinophilic cationic protein
(ECP) and exhaled nitric oxide (NO) levels suggests suppression of inflammation by this agent. A recombinant
mutant human protein (BAY 16-9996) binds the IL-4
receptor a- but not cc-chain, and therefore, antagonises
receptor transduction. This protein substantially reversed
allergen-induced BHR with a reduction in airway
inflammation in a primate model [106].
Another approach to inhibition of IL-4 production is to
target the control of transcription factors of the IL-4 gene.
Signal transduction and activation of transcription-6
(STAT-6) responsive elements are found in the promoter
region of IL-4 inducible genes and this transcription
factor is expressed at abnormally high levels in the
epithelium of severe asthmatics [107]. STAT-6 knockout
mice demonstrate a defect in IL-4 and IL-13 mediated
signal transduction [108]. The potential utility of STAT-6
targeted therapies is highlighted by the ability of STAT-6
directed antisense oligonucleotides to markedly downregulate germline Ce mRNA levels, reflecting inhibition
of IL-4-dependent IgE isotype switching [109, 110].
Interleukin 5
Eosinophil mobilization and trafficking, their maturation and maintenance are largely promoted by the Th2
cytokine, IL-5, making it an attractive therapeutic target in
eosinophilic conditions such as asthma and rhinitis.
Ablation of the effects of IL-5 has been accomplished
with antisense oligonucleotides in rodent models where a
reduction in IL-5 protein levels is followed by a reduction
in allergen-induced eosinophilia and BHR [111]. Importantly, this suppression of lung eosinophilia was maintained for 17 days following a single intravenous
Monoclonal antibodies used to block IL-5 in mouse,
guinea-pig, rabbit and monkey studies provided a sustained reduction in antigen-induced airway eosinophilia
but had little effect on antigen-induced BHR [112±114].
Two humanized forms of anti-IL-5 (SB-240563 and Sch
55700) are now available with the potential for clinical
studies [115, 116]. In mild asthmatics, SB-240563 had a
marked inhibitory effect on both airway and peripheral
blood eosinophil levels and on allergen-induced sputum
eosinophilia following single subcutaneous administration [117]. However, as with the studies in mouse, rabbit
and monkey, no significant effect on bronchial responsiveness or the late phase response to allergen-challenge
was observed. Phase-I trials in severe persistent asthma
have provided similar results [118]. IL-5 blocking antibodies prevent the mobilization and subsequent trafficking of eosinophils to the lung in allergic asthma, but are
unable to influence BHR or the late phase response.
Currently, studies are in progress to determine whether
anti-IL-5 blocking antibodies can improve the control of
asthma, despite their apparent lack of protection against
Recent studies on IL-5 signalling (fig. 3) suggest that
various IL-5-dependent functions are mediated by distinct and separate secondary messenger systems [118].
Thus, targetting such pathways may lead to inhibition
of IL-5 effector functions. The anti-apoptotic effects of
IL-5 on eosinophils are dependent on the activation of
lyn-, Janus kinase-2 (Jak2) and Raf-1 kinase, of these,
only Raf-1 is necessary for eosinophil activation and
degranulation [119, 120]. Lyn-kinase dependent signalling may be specifically inhibited by a peptide inhibitor,
Gene transcription
Fig. 3. ± Binding of IL-5 activates the IL-5 receptor and triggers a
cascade of secondary messengers. The secondary signalling pathway is a
linear system in which cell surface associated receptor protein tyrosine
kinases (e.g. Jak-1/2 and Lyn) activate the signal transducers and
activators of transcription (STAT1 and STAT5) and Ras family of serine/
threonine protein kinases (e.g. Raf-1) respectively. Ras/Raf activation
may function as a relay switch, positioned upstream from a further
cytoplasmic cascade of kinases that include the mitogen-activated
protein kinases (MAPK) and mitogen-activated protien kinases/
extracellular regulated protein kinase (MEK). Activated MAPKs in
turn regulate the activities of nuclear transcription factors such as
activator protein-1 (AP-1, of which c-jun and c-fos are important
resulting in inhibition of lyn-dependent IL-5 signalling
without affecting Jak2 dependent IL-5 signalling. This
inhibitor blocks allergen-driven airway eosinophilia
[121]. The transcription factor GATA-3 is critical for
IL-5 expression in Th2 cells, and increased GATA-3 gene
expression in association with IL-5 mRNA positivity
has been shown in airway cells isolated from patients
with asthma. These findings support a causal association
between augmented GATA-3 expression and dysregulated IL-5 expression and asthma [122, 123]. Specific
inhibition of GATA-3 expression using dominant negative GATA-3 transgenic mice led to a reduction in Th2
cytokine expression and marked attenuation of airway
eosinophilia, mucus production, and IgE synthesis [124].
These outcomes could also potentially be achieved using
antisense oligonucleotide technology.
IL-9 has been identified in airway tissue in asthma and is
known to induce BHR, elevated serum IgE, mucin gene
transcription and epithelial CC chemokine release [125±
128], and thus is a broadly attractive therapeutic target
in asthma [129]. One mechanism by which IL-9 may
induce airway eosinophilia is by enhancing IL-5 receptor
expression, thereby increasing the differentiation and
survival of eosinophils [130]. Specific blockade of IL-9
activity has been accomplished in the mouse by intratracheal instillation of monoclonal anti-IL-9 antibody
[131]. Anti-IL-9 significantly inhibited BHR, airway
eosinophilia, serum IgE, airway inflammatory cell infiltration and mucin production induced by allergen, demonstrating a surprisingly broad inflammatory effect
for this cytokine. As yet there are no reported clinical
trials adopting IL-9 as a therapeutic target.
This cytokine shares 70% of its sequence homology
with IL-4 and despite a degree of functional redundancy
due to sharing of the IL-4Ra subunit, a specific role in the
development of asthma has been demonstrated in murine
models [132±134]. Notably IL-13 does not share the IL-4
influence on specific induction of the Th2 phenotype
from naive Th0 cells. By using a soluble form of the IL13Ra chain of the IL-13 receptor, known to bind IL-13
exclusively, these groups demonstrated a significant reduction in airway hyperreactivity, airway eosinophilia
and mucus hyperplasia. Curiously, when administered
after the initial allergen sensitization however, this agent
had no effect on serum IgE levels [133, 134]. Notably, an
IL-13 blocking antibody reduced BHR independently of
IL-5 [135].
T-helper 1/T-helper 2 modulation by vaccination
An indirect way of modulating Th1/Th2 balance has
been to boost innate immunity by the use of vaccines,
particularly for redirection of the Th2 response in favour of
the Th1 response. Several approaches are currently under
investigation, and these may even raise the possibility of
preventing the development of asthma and allergic diseases. The beneficial effects of specific immunotherapy may
result from increasing Th1 immune responses while the
development of peptide immunotherapy may lead to a
more effective and safer treatment.
Mycobacterium species
The potential benefit of Bacille Calmette-Guerin (BCG)
vaccination in atopic diseases was first raised by a study in
Japanese school children, in whom an association between
BCG vaccination and a diminished incidence of atopy and
allergic disease was observed, suggesting a role for early
mycobacterial exposure in the subsequent development of
atopic responsiveness [34]. Experimental models have
supported this concept using the nonpathogenic mycobacterial products of Mycobacterium bovis and Mycobacterium vaccae. Mice vaccinated with BCG prior to
allergen sensitization had increased IFN-c and decreased
IL-4 and IL-5 expression along with reduced levels of
airway T-cells and eosinophilia and bronchial reactivity
[136, 137]. Mycobacterium vaccae is ubiquitously present in the soil as a saprophyte and can evoke a strong
production of IFN-c. A suppression of Th2 activation has
been demonstrated using heat-killed M. vaccae in mice
[138±140], and these studies have opened the way to
clinical studies in human asthma [138].
Deoxyribonucleic acid vaccines:
cytosine-guanosine repeat motifs
The cytosine-guanosine dinucleotide repeat, when
present in a particular base context, is known as a CpG
motif and has been recognized as an important procaryotic
immunomodulatory effector whose role is probably that of
a warning or priming agent against bacterial infection
[141]. This motif is expressed at a very low frequency in
vertebrates, in a nonfunctional methylated form, and is
without function. Rodent studies highlight a potential
function of synthetic CpG motifs in vertebrates, where a
potent effect on lymphocyte function has been found
[142]. CpG vaccination directly induces antigen-presenting cells and B-lymphocytes to release IL-12, IL-18 and
tumour necrosis factor-alpha (TNF-a), effectively suppressing Th2 responses by inducing the Th1 phenotype.
These CpG oligodinucleotides (ODN) are effective in
preventing the development of eosinophilic airway inflammation, allergen-induced elevation of serum IgE and
BHR in murine asthma models [142]. These ODN not
only effectively prevent Th2 type responses by pre-administration and co-administration with allergen-challenge
but also reduce established inflammation [142±144].
Further, these motifs show a large degree of flexibility
towards the dose and the route of administration, being
active following transmucosal [145], inhaled and parenteral administration [142]. As an added potential, a
deoxyribonucleic acid (DNA) vaccine containing this
motif may provide a more effective adjuvant than the
widely used, alum. The clinical efficacy and safety of
such CpG motifs needs evaluation [146] and such trials
are currently being considered.
Potential drawbacks of manipulating
T-helper 1/T-helper 2 balance
The mounting evidence for Th2 cell activation in
allergic asthma suggests downregulation or ablation of the
Th2 response to be an appropriate aim in treating asthma
and allergic disorders. However, the consequences of such
a strategy are as yet unclear. Two potential outcomes need
consideration; first the consequences of Th2 ablation and
second, those of allowing a Th1 dominated immune activation. Parasite exposure is known to induce Th2 type
cytokine synthesis [147±149], and mice with Th2 cytokine deletions demonstrate impaired parasite clearance
[150±152]. Although early Th1 expression may be a more
important response to acute phase parasitaemia, subsequent parasite clearance appears to be more Th2 cytokine
dependent [152, 153]. Autoimmune diseases including
experimental allergic encephalomyelitis, myasthenia gravis and hypothyroidism may be derived from an exuberance of Th1 activity [154±156], raising the concern
that increased Th1 cell activity may induce these autoimmune diseases. Several reports note associations between IFN-c, IFN-b and IL-12 exposure and the
development or exacerbation of autoimmune processes
[154, 157±159]. Th1 cells may contribute to asthmatic
inflammation. In adoptive transfer studies of Th1 cells
in mice, a prolonged enhancement of cell-mediated
immune responses was observed [160], and passive
transfer of Th1 cells with Th2 cells induces enhanced
tissue eosinophilia compared to when Th2 cells alone
were administered [161±163]. However, in other studies
in mice and rats, transfer of allergen-specific Th1 cells
[164] inhibited the effects of allergen-specific Th2 cells,
namely BAL eosinophilia and BHR in rats, without itself
inducing any inflammation [165]. Allergen-specific Tc1
cells also diminished allergen-induced BHR and BAL
eosinophilia [163]. Nevertheless, IFN-c produced by Th1
cells may activate epithelial cells to induce the production
of pro-inflammatory cytokines and chemokines [166±
Two further observations appear important to the
contribution of Th1 type processes to asthma. First, the
prominent expression of the Th1 dependent transcription
factor, STAT1, in stable asthma [169] and second, the
association between respiratory viral infections and
asthma [170, 171]. Viral infections induce Th1 expression
with upregulation of TNF-a and VCAM1 expression
supporting a role for Th1 mediated processes in asthma
exacerbation [169, 172]. Therefore, caution is necessary
in studies that aim towards altering the balance of the
Th1/Th2 in the treatment of asthma and allergic diseases.
Specific immunotherapy
Specific immunotherapy (SIT) is a treatment aimed at
the induction of specific unresponsiveness, or anergy, in
peripheral T-cells to peptide epitopes. Peripheral T-cells
following SIT are characterized by reduced cytokine
production and attenuated proliferative responses to specific allergens [173]. This process is initiated by an
autocrine effect of IL-10 produced by antigen-specific Tcells [174±176]. IL-10 induces anergy by inhibition of
CD28-costimulatory molecule signalling [177, 178] and
also by anti-inflammatory effects on basophils, mast cells
and eosinophils [4]. There are reduced levels of IL-4 and
IL-5 and increased IFN-c production, indicating a shift of
the T-cells towards an increased Th1 response at the
expense of Th2 responses [179, 180]. This treatment has
been successfully used in asthma and allergic rhinitis and
for venom allergy [181, 182], but is usually most useful in
subjects allergic to single allergens such as grass pollens,
and in subjects with mild forms of allergic diseases [183,
184]. Recently, cloning of epitopes of allergens has
provided a higher level of epitope purity than previously
available, and there has been a greater understanding of
the specific mechanisms of tolerance induction. These
factors may lead to a wider application of SIT in atopic
disease, using better characterized peptides which are
more effective but with a lesser risk of side-effects, in
particular anaphylaxis.
specific T-lymphocytes. Such constructs have been demonstrated to induce IgE-independent late-phase bronchoconstrictor responses in cat-sensitive mild asthmatics
[185]. Ex vivo studies subsequently demonstrated a
dose-dependent reduction in T-cell IL-4 production
following cat peptide, not observed following whole
cat hair extract [186]. In vitro cat-peptide studies also
showed a reduction in IL-2, IL-4, IL-5 and IFN-c release
from peripheral blood T-cells and a reduction in CD40
ligand (CD40L) expression, resulting in a diminished
ability of these cells to give help to B-cells for the
production of IgE [187, 188]. In a six-week trial of cat
peptide immunotherapy in asthma, a reduction in IL-4
levels was again observed and was accompanied by an
improvement in the provocative dose causing a 20% fall
in (PD20) forced expiratory volume in one second (FEV1)
[187], although there was no effect on IFN-c or IgE,
suggesting the possible induction of a Th2 to Th0 change.
The possibility of selective induction of clonal anergy is
invited by this approach and phase I and II studies of cat
peptide immunotherapy are currently being launched.
Anti-immunoglobulin-E antibody
Studies of the recombinant human anti-IgE antibody
(rhu MAb-E25, Xolair) are now in phase IV in asthma and
allergic rhinitis. E25 binds to free IgE at the high affinity
receptor-binding site (FceR1), preventing the crosslinking
of IgE bound to effector mast cells and basophils. This
strategy ensures that the antibody is nonanaphylactogenic.
The antibody may be delivered by subcutaneous injection
on a 2±4 weekly schedule, dosing based on weight and
serum IgE levels. Tolerance and adverse effect profiles are
thus far acceptable with no reports of serum sickness,
hypersensitivity reaction or the development of anti-E25
antibodies. E25 induces a dose-dependent reduction in
serum free IgE associated with a reduction in basophil
FceR1 expression and histamine release [189, 190]. A
significant improvement in both early [191] and late
phase [192] bronchoconstrictor responses to allergenchallenge and a reduction in methacholine-induced
hyperresponsiveness, with a reduction in sputum eosinophil numbers have been reported following E25
administration. Results of several clinical trials show
subjective improvement and significant effects on
corticosteroid reduction in moderate to severe asthma
[193], and allergic rhinitis [194]. This treatment should
become available in the near future, and its initial role
may be in severe atopic asthma, although it is not known
whether E25 is also effective in nonatopic asthma.
Peptide immunotherapy
Peptide immunotherapy can be considered as a
refinement of SIT, with the specific intent of inhibition
of T-cell activation independent of IgE-mediated inflammatory processes. Synthetic MHC-restricted oligopeptides
mimicking recognized epitopes of the major cat allergen
dI (Fel dI), modified by single amino acid substitution
have been produced and are noted to have variable effects
on T-cell proliferation and cytokine production by Fel dI
Inhibition of eosinophil activation and
IL-5 is involved in the maturation and mobilization of
eosinophils and cooperates with chemoattractant cytokines
(chemokines) in chemoattraction and tissue activation of
Chemokine receptor antagonists
Chemokines are inducible pro-inflammatory proteins
whose key function is leukocyte chemoattraction and
activation. Three families are currently described, distinguished on a structural basis into C, CC and CXC families
[195]. Chemokine effects are mediated by chemokine
receptors and differential cell receptor profiles allow cellspecific attraction [196±198]. Thus, the CC chemokine
eotaxin has an eosinophil selective role in eosinophil
recruitment due to selective expression of the eotaxin
receptor CCR3 on the eosinophil [199, 200]. Chemokines
are however, also involved in the recruitment of monocytes, dendritic cells, T-lymphocytes, NK cells, Blymphocytes and basophils.
Antagonism of chemokine receptor signalling has been
accomplished by modification of endogenous agents to
create high-affinity receptor antagonists. These agents,
which include met-regulated upon activation normal T-cell
expressed and secreted (RANTES) and met-Ckb7 [201±
203], are potent CCR3 specific antagonists which inhibit
CCR3 receptor signalling and consequent eosinophil
migration. To date several small-molecular weight CCR3
antagonists have been developed and await clinical
evaluation. One potential strategy combines the inhibition
of both IL-5 and eotaxin, a double-pronged approach that
would reduce both mobilization, chemoattraction and
activation of eosinophils more effectively given the
cooperation between IL-5 and eotaxin demonstrated in in
vivo models [204, 205]. Such a combined approach may
lead to clinical improvement.
Blocking adhesion molecules and integrins
Eosinophil migration to sites of allergic inflammation is
specifically mediated by interactions between endothelium, epithelium and eosinophil adhesion molecules including the a4-integrin very late antigen-4 (VLA-4) [206].
a4-Integrins are also implicated in human lymphocyte
coactivation [204]. An a4-integrin monoclonal antibody
inhibited inflammation and BHR in a sensitized mouse
model [207], and also eosinophilic inflammation and
BHR induced by eotaxin in IL-5 transgenic mice [204].
Peptido-mimetic small molecule VLA-4 inhibitors,
administered prior to allergen challenge, also prevent
increases in VLA-4+ leukocytes (eosinophils, lymphocytes and macrophages) in lung tissue while significantly
inhibiting early and late phase allergic responses [208].
Similar approaches inhibiting interactions between leukocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) have also proven
effective in blocking eosinophil adhesion and transmigration [209].
Inhibition of pro-inflammatory cytokines and
Several pro-inflammatory cytokines such as TNF-a
[210] and IL-1b [211] have been shown to be overexpressed in asthma, and exposure to environmental
endotoxin such as that present in house dust [212] may
lead to further activation of a panel of such pro-
inflammatory cytokines. Activation of the transcription
factor, NFkB, which can regulate the production of a
range of pro-inflammatory cytokines including IL-1, IL6, IL-8 and TNF-a but also adhesion molecules ICAM-1
and VCAM-1, has been shown in the airway epithelium
and macrophages of patients with mild asthma [213].
Similarly, increased expression of the transcription factor,
AP-1, has also been reported [214].
Tumour necrosis factor-a
TNF-a increases airway responsiveness in BrownNorway rats [215] and in humans, together with an
increase in sputum neutrophils [216]. TNF-a also
potently stimulates airway epithelial cells to produce
cytokines including regulated upon activation, normal Tcell expressed and secreted (RANTES), IL-8 and GMCSF, and increases the expression of the adhesion molecule, ICAM-1. TNF-a has a further synergistic effect with
IL-4 and IFN-c to increase VCAM-1 expression on
endothelial cells [217, 218]. These factors indicate that
TNF-a may be an important mediator in initiating chronic
inflammation in the airways. Several approaches to
inhibition of TNF-a synthesis and effects are now under
investigation in asthma, including monoclonal antibodies
to TNF-a and soluble TNF-a receptors. An anti-TNF-a
antibody has retarded the progression of severe rheumatoid arthritis with significant clinical amelioration [219],
but has not been tried in asthma. This antibody may have
therapeutic effects in severe asthma. Inhibitors of TNF-a
converting enzyme (TACE), or of the cysteine protease
caspase-1 (IL-1 converting enzyme, ICE) are also
potential anti-inflammatory compounds that may be used
in asthma, and are currently under development. Recent
attention has also been focused on intracellular signalling
cascades such as inhibitors of p38 MAP kinase which
inhibit the synthesis of pro-inflammatory cytokines in
vivo [220, 221]. In this mouse model, inhibition of p38
kinase activity markedly reduced cytokine-associated
inflammation and inhibited lipopolysaccharid (LPS)induced TNF-a production while allergen-induced airway eosinophilia was all but abolished.
IL-1 co-induces CD4+ T-cell proliferation and IL-2
secretion following interaction of T-cells with antigen
presenting cells, and is an important growth factor for
antigen primed Th2 cells [222]. IL-1 also potently induces
the synthesis and release of multiple pro-inflammatory
cytokines and chemokines. The IL-1 receptor antagonist
(IL-1Ra) polypeptide shows significant homology with
IL-1a and IL-1b and binds the IL-1 receptor. IL-1Ra has
been isolated from multiple tissues including alveolar
macrophages [223], and inhibits most of the activities of
IL-1 [224]. IL1-Ra blocks Th2 but not Th1 clonal
proliferation in vitro and in the guinea pig reduces
allergen induced BHR and pulmonary eosinophilia [225].
Manipulation of this endogenous control mechanism may
therefore, impact on asthma and needs to be evaluated in
the clinical setting.
Although IL-10 is a Th2-derived cytokine, it has antiinflammatory properties that could be used to control
asthmatic inflammation. IL-10 is derived largely from
mononuclear cells, alveolar macrophages and both naive
and committed T-cells. It reduces MHC and costimulatory
molecule expression, reduces pro-inflammatory cytokine
release and increases IL-1Ra expression [4, 226]. T-cell
and macrophage IL-10 synthesis is significantly reduced
in asthmatic subjects compared with nonasthmatics [227,
228] but IL-10 expression in alveolar macrophages is
increased by corticosteroid therapy [229], suggesting in
vivo relevance of this cytokine. A polymorphism in the
promoter sequence of IL-10 has been associated with
attenuated IL-10 expression and has been identified at
increased frequency in severe asthma [230], while
promoter polymorphisms have been demonstrated in
asthma probands and are associated with elevation of IgE
levels [231]. This latter observation is of particular
interest given a proposed suppressive effect of IL-10 on
IL-4-induced IgE isotype switching by B-cells [232]. IL10 administration to mice reduces airway eosinophilia
and allergen-induced late responses [233]. When given to
normal volunteers, IL-10 reduced circulating CD3, CD4
and CD8 lymphocyte numbers and the proliferative
response and reduced TNF-a and IL-1 production [234].
Further studies in human asthma are awaited.
Use of antisense oligonucleotides
Oligonucleotide sequences forming a complementary
copy of normal, or sense, messenger (mRNA) are able to
bind to specific mRNA, and this dimer formation prevents
mRNA translation and protein synthesis. The effectiveness
of this technique was demonstrated in a rabbit model of
asthma where aerosol inhalation of adenosine-A1 receptor
antisense protected the animals from subsequent challenge
with either adenosine or dust-mite allergen, with effects
persisting up to 7 days [235, 236]. In a model in which the
late allergic reaction could be transferred in the rat by
ovalbumin sensitized T-cells [237], IL-4 antisense-treated
T-cells caused a significant reduction in the late allergic
reaction, airway eosinophilia, and IL-4 and IL-5 expression. However, when IL-5 antisense treated T-cells were
administered, only BAL IL-5 expression was reduced
suggesting a key role for IL-4 in CD4+ T-cell mediated
late allergic responses. In murine models, an IL-5 antisense oligonucleotide reduced IL-5 protein levels, airway
eosinophilia and allergen-induced late allergic responses
[111]. Similar approaches targetting CCR3 and the cytokine receptor a-subunits of IL-4, IL-13 and GM-CSF
have also been reported with promising results [238±
240]. Antisense based therapies are currently in clinical
use in the treatment of CMV retinitis and for advanced
cancer [241±243] and are undergoing further safety and
tolerability evaluation.
where mucosal transfer of IL-12 [248] and IFN-c [249]
significantly reduced Th2 cytokine expression and inhibited BHR. Transfer of the glucocorticoid receptor to the
epithelial cell line A549, lead to repression of transcription factor mediated gene transcription and may provide a
therapeutic approach in steroid-resistant asthma [250].
Gene transfer to the lungs in man is still in a developmental phase, and the efficiency of gene transfer using
liposomal transfer technology is thus far suboptimal, as
judged from the experience of the transfer of the cystic
fibrosis transmembrane regulator DNA to patients with
cystic fibrosis [251]. Use of the more efficient transfer
system, the adenovirus vector, may be detrimental in
inducing lung damage and inflammation, and is no longer
a viable option in man. The potential genes that may be
delivered to the airway epithelium of patients with asthma
for therapeutic effects are numerous. Candidates for gene
transfer might include Th1 cytokines IL-12 or IFN-c, or
of the anti-inflammatory cytokine, IL-10, or of IL-1Ra, or
of the corticosteroid receptor may be contemplated, but
gene therapy approaches for the treatment of asthma
remain in the distant future.
Recent advances in the techniques for the synthesis and
manufacture of monoclonal antibodies, synthetic peptides
and peptidomimetic small molecules has increased the
potential for the creation of specific inhibitors of immune
processes in allergic inflammation. These agents have
enabled specific intervention in the inflammatory cascade
and allow a clearer understanding of the roles of specific
agents in this cascade, while at the same time providing the
possibility of therapeutic intervention in asthma. In the first
instance, it is likely that these strategies will be aimed at
those with severe difficult-to-treat asthma as it is here that
the failings of current therapies are most evident. Whether
new therapies may provide remission of disease with shortterm treatment cannot be predicted from the current
understanding of asthma. While preliminary data for many
of these agents appears most promising, these agents will
have to endure rigorous evaluation of efficacy, long-term
safety and cost-effectiveness. Several agents targeted to
specific immunological or cytokine pathways may become
available and may be more effective in certain types of
asthma. Genetic pharmacological profiling may be needed
to identify the best responders to particular types of
specific drugs [252]. The future of asthma therapy looks
bright, but identifying targets is only a first step. The
development of these new approaches is very exciting but
the task remains daunting.
Use of gene therapy
The technique of gene therapy entails the specific
augmentation of gene expression by transfer of single
genes using viral vectors or liposome transfer [244±247].
Gene therapy has been explored in asthma models in mice
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