Immunomodulators
Arthur Kavanaugh and David H Broide
CONTENTS
• Introduction 1643
• Methotrexate 1643
• Immunophilin-binding agents and calcineurin inhibitors:
cyclosporin, tacrolimus, and pimecrolimus 1645
• Intravenous immunoglobulin 1648
• DNA-based therapies 1649
• Mycobacterial vaccines 1653
• Cytokine-based therapy 1654
• Conclusions 1655
■ INTRODUCTION ■
Recognition of the importance of the Th2 lymphocyte as an orches-
trator of the immune and inflammatory response associated with
asthma has focused attention on the use of immunomodulator therapy.
Immunomodulators that have been successfully used in other immuno-
logically mediated diseases (e.g., methotrexate in rheumatoid arthritis;
cyclosporin in transplant rejection; intravenous immunoglobulin (IVIG)
in idiopathic thrombocytopenic purpura) are not currently routinely
used in the therapy of asthma or allergy, either because of the lack of
studies showing a consistent beneficial effect and/or the potential side
effects related to therapy. Second-generation DNA-based immunomod-
ulators have shown significant promise in animal models of asthma, but
whether this potential will be borne out in human studies is at present
unknown. In this chapter we review the safety and efficacy of currently
available (methotrexate, cyclosporin, IVIG) and investigational (DNA-
based, anti-cytokine) immunomodulators. Traditional allergen protein
immunotherapy (Chapter 95) and novel immunomodulation strategies
targeting adhesion molecules (Chapter 9) and chemokines (Chapter 11)
are covered in other chapters.
94
METHOTREXATE
SUMMARY OF IMPORTANT CONCEPTS
>> At present, immunomodulator therapy (other than traditional
allergen protein immunotherapy and anti-IgE) in the field of
asthma and allergy remains investigational
>> Double-blind studies of IVIG in asthma have not demonstrated
a therapeutic benefit
>> Anti-TNF, methotrexate, and cyclosporin have shown a mod-
est benefit in some early-phase studies in asthma
>> Anti-IL-4 and anti-IL-5 therapies have not proved effective in
the treatment of persistent asthma
>> Immunotherapy with cytosine phosphorothioate guanosine
DNA conjugated to a protein allergen has shown promise in
animal models of asthma and in small-scale human studies,
but further large-scale studies are needed to determine its
safety and effectiveness
■ METHOTREXATE ■
PHARMACOLOGY, STRUCTURE,
AND METABOLISM
Methotrexate can be administered orally or parenterally.1 Oral administra-
tion results in a bioavailability of approximately 85%, although this may
decrease at higher doses.1 Subcutaneous or intramuscular dosing results in
equivalent, near complete bioavailability. About half of absorbed methotrex-
ate is protein bound, and may be displaced by agents such as non-steroidal
anti-inflammatory drugs (NSAIDs), sulfonamides, and others. The elimi-
nation half-life of methotrexate is approximately 8 hours, and excretion
occurs predominantly via glomerular filtration. Indeed, renal function is a
critical factor in methotrexate pharmacokinetics, and hence its toxicity.1
Methotrexate may be considered as both drug and prodrug, as it is
rapidly taken up and metabolized to a series of polyglutamated deriva-
tives that persist intracellularly for longer periods of time.1 Methotrexate
polyglutamated intracellular metabolites interfere with the same metabolic
1643
F THERAPEUTICS
N N NH2
CH3
N N
H
NH2
HOOC N
COOH O
Methotrexate
N N NH2
H N N Skin rash
OH
HOOC N H
METHOTREXATE
COOH O
Folic acid
Fig. 94.1. Chemical structures of methotrexate and folic acid.
pathways as does the parent compound.1 Serum methotrexate concentra-
tions do not correlate with clinical efficacy in non-malignant conditions,
whereas levels of intracellular polyglutamated methotrexate do.1
Methotrexate and folic acid are structurally similar (Fig. 94.1). Meth-
otrexate and its polyglutamated metabolites avidly bind the active site of
the enzyme dihydrofolate reductase, thereby blocking the conversion of
dietary folic acid to its reduced form, tetrahydrofolate. Depletion of the
reduced form of folate leads to reduced synthesis of thymidine, thereby
impairing DNA synthesis. Methotrexate also inhibits other enzymes
relevant to nucleotide synthesis, including thymidylate synthetase.
Allelic variation in the genes encoding enzymes affecting methotrexate’s
uptake and metabolism, and also in various potential targets affecting
their sensitivity to methotrexate, has been described. There is substantial
interest in the potential for pharmacogenetic profiling as a means to
enhance methotrexate’s efficacy while minimizing its toxicity.
IMMUNOLOGIC EFFECTS
Inhibition of cell replication may be the key therapeutic mechanism of
methotrexate in neoplastic conditions. Lymphocytes divide rapidly in the
course of the immune response, and thus immunosuppression may be one
mechanism of action of methotrexate in various immunologic diseases.
However, several observations have suggested that other mechanisms may
be relevant in non-malignant conditions. For example, in contrast to its use
in the treatment of malignancies, the lower doses of methotrexate com-
monly used in rheumatoid arthritis (RA) are infrequently associated with
myelosuppression or clinical signs of systemic immunosuppression. In ad-
dition, supplementation with low doses of folic acid may obviate some of
the toxicity seen in RA patients receiving methotrexate without signifi-
cantly attenuating its clinical efficacy.1,2 In vivo studies have shown that
methotrexate can induce various immunomodulatory effects, including
effects on cellular immunity, humoral immunity, and inflammation.1,3,4
Two relevant mechanisms underlie the anti-inflammatory effect of
methotrexate at the doses typically used in non-malignant conditions:
(1) inhibition of the intermediate step in purine metabolism catalyzed
1644
Table 94.1 Adverse effects potentially associated with
methotrexate
Common
Oral ulcerations
‘Post-dose’ reactions (arthralgia, fatigue)
Nausea, diarrhea
Less common
Abnormal liver function tests
Uncommon
Pulmonary toxicity
Myelosuppression
Alopecia
Increased risk of infection
Hepatic fibrosis
Lymphoma (Epstein–Barr virus associated)
by amino-imidazole-carboxy-amido-ribonucleotide (AICAR) trans-
formylase, leading to the increased release of adenosine; and (2) in-
terference with transmethylation reactions, such as the methylation
of homocysteine to methionine.1 The precise mechanisms by which
AICAR inhibition results in increased extracellular concentrations of
adenosine remain to be defined, but through interactions with specific
receptors (A1, A2a, A2b, A3) on various cell types, adenosine exerts
potent and diverse antiinflammatory actions.
ADVERSE EVENTS
Methotrexate can be associated with a number of adverse effects
(Table 94.1). Because dosing regimens, indications, and relevant factors
such as renal function vary significantly among published reports, it is
difficult to assign exact figures to the expected prevalence of these adverse
effects. Many side effects, including myelosuppression, are dose depend-
ent and occur infrequently at the doses typically used in non-malignant
diseases. Some side effects, such as pulmonary reactions, are idiosyncratic.
Many side effects are more commonly seen early in the treatment course,
or with changes in dose regimen. Many side effects can be attenuated
by decreasing methotrexate dosages or by the supplemental use of folic
acid.2 Although the administration of folic or folinic acid, particularly in
large doses, can reduce efficacy, there appears to be consensus that the use
of folic acid in low doses (e.g., approximately 1 mg/day) is a cost-effective
way to reduce adverse effects in patients taking methotrexate while at the
same time not substantially reducing efficacy. Moreover, supplemental
folic acid reverses the increase in serum homocysteine that is observed
with methotrexate therapy – a potentially important consideration for
patients with other risk factors for the development of atherosclerotic
disease. The potential hepatic toxicity of methotrexate may vary with
factors such as frequency of dosing (weekly versus daily), concomitant

alcohol use, and differential host susceptibility. Significant hepatic fibrosis
is rarely seen in patients who do not drink alcohol. Screening for other
factors that might predispose to liver injury, such as hepatitis, is now rou-
tine. The American College of Rheumatology have developed guidelines
for monitoring liver toxicity (Table 94.2).5 Guidelines call for the evalu-
ation of selected liver function tests at regular intervals during therapy. If
liver function tests do become elevated, this often reverses upon decreas-
ing the dose of methotrexate. Putative markers of active hepatic fibrosis,
for example serum concentrations of the aminoterminal propeptide of
type III collagen, are under investigation.6
Potential pulmonary toxicities related to methotrexate use have been
somewhat difficult to define clearly. The largest literature addressing this
issue comes from the use of methotrexate in patients with RA, 0.5–5%
of whom are reported to develop pulmonary toxicity.7 Much of the dif-
ficulty defining the condition or establishing the true incidence arises
from the lack of pathognomonic clinical, laboratory, or radiological fea-
tures of ‘methotrexate pneumonitis.’ Common clinical characteristics
may include shortness of breath, cough, and fever. Interstitial or alveolar
infiltrates on chest radiograph, and hypercellularity with lymphocytic
predominance on bronchoalveolar lavage, are considered consistent
with the diagnosis. Differentiation from pulmonary complications of
the underlying disease may be suggested by the acute or subacute on-
set of symptoms in a previously stable patient beginning methotrexate
relatively recently; half of affected patients started methotrexate in the
previous 8 months. However, differentiation from an infectious process
is difficult. Corticosteroids may shorten the duration or improve the
symptoms of methotrexate pneumonitis in some patients.
Because it is a teratogen, methotrexate is strictly contraindicated in
pregnancy (US Food and Drug Administration category X).
CLINICAL STUDIES IN ASTHMA AND ALLERGY
In the late 1980s, open-label observations suggested that methotrexate
may have a corticosteroid-sparing effect in corticosteroid-dependent
adults with asthma.8 This was subsequently assessed in controlled trials
in adults and children9–11 and in systematic reviews12,13 which concluded
that there is evidence to support the ability of methotrexate to act as a
corticosteroid-sparing agent in corticosteroid-dependent asthma; how-
ever, the magnitude of the dose reduction was relatively modest (mean
reduction of 4.1–4.4 mg in the daily oral dose of prednisone equivalent).
Although this may represent a reduction in total oral corticosteroid dose
by 20% or more, the clinical significance of this effect can be debated.
Methotrexate, typically given in a dose of 15 mg/week, was generally
well tolerated. Longer courses of therapy (e.g. 6 months or more) might
have been associated with a greater benefit.12
CURRENT RECOMMENDATIONS FOR USE
IN CLINICAL PRACTICE
Although it is widely used as an immunomodulatory therapy in autoim-
mune conditions, the use of methotrexate as a therapeutic agent for pa-
tients with allergic disease may be most appropriate in patients with severe
asthma that has been refractory to or dependent on oral corticosteroids.
In ‘corticosteroid-resistant’ asthma, methotrexate has been shown to have
a modest corticosteroid-sparing effect. The greatest guidance for the use
of methotrexate in non-malignant conditions comes from its use in RA.
Typical weekly doses for patients with normal renal function range from
Immunomodulators 94
Table 94.2 Recommendations for monitoring for
methotrexate hepatic safety in patients with rheumatoid
arthritis (RA).
A Baseline
1. Tests for all patients
a. Liver blood tests (aspartate aminotransferase [AST],
alanine aminotransferase [ALT], alkaline phosphatase,
albumin, bilirubin, serologic tests for
hepatitis B and C
b. Other tests (serum creatinine, complete blood count)
2. Pre-treatment liver biopsy only for patients with:
a. Prior excessive alcohol consumption
b. Persistently abnormal baseline AST
c. Chronic hepatitis B or C infection
B Monitor AST, ALT, albumin at 4–8-week intervals
IMMUNOPHILIN-BINDING AGENTS AND CALCINEURIN INHIBITORS
C Perform liver biopsy if:
1. Five of nine determinations of AST within a given 12-month
interval (6 of 12 if performed monthly) are abnormal
2. Albumin decreases below normal
D If results of liver biopsy are:
1. Roenigk grade I, II, or IIIa, resume methotrexate and
monitor
2. Roenigk grade IIIB or IV, discontinue methotrexate
E. Discontinue methotrexate in patients with persistent liver test
abnormalities who refuse liver biopsy
(Adapted from Kremer J, Alarcon G, Weinblatt M, et al. Clinical, laboratory,
radiographic and histopathologic features of methotrexate-associated lung
injury in patients with rheumatoid arthritis. Arthritis Rheum 1997; 40:1829,7
with permission. Copyright Wiley-Liss Inc, a subsidiary of John Wiley &
Sons, Inc.).
15 to 25 mg. Concurrent use of folic acid (1 mg/day) may obviate some
adverse effects. Regular monitoring of selected liver function tests, blood
cell counts, and renal function is useful to prevent adverse effects.
■ IMMUNOPHILIN-BINDING AGENTS
AND CALCINEURIN INHIBITORS:
CYCLOSPORIN, TACROLIMUS,
AND PIMECROLIMUS ■
PHARMACOLOGY AND METABOLISM
Cyclosporin A (CsA) is an 11 amino acid cyclic peptide initially isolated
in the 1970s from a soil organism (Tolypocladium inflatum) (Fig. 94.2).
It was found to have substantial immunosuppressive properties, par-
ticularly inhibition of helper T-cell function.14 CsA blocks T cells via
inhibition of calcineurin, which subsequently downregulates cytokines
such as IL-2 that are critical for T-cell activation. Tacrolimus (previously
referred to as FK506; Fig. 94.2), also initially isolated from a soil organ-
ism (Streptomyces tsukubanesis) in 1984, and pimecrolimus are structur-
ally distinct from CsA, but share its mechanisms of action.14–17 CsA
and tacrolimus have been introduced for clinical use in the setting of
transplantation and for autoimmune diseases. Topical formulations of
1645
F THERAPEUTICS

O O TCR
H
N N N O
N N
O OH O IP3
H
O N N
H H
O O
Calcium
N N C
N NH O I
O O Calcineurin Calcineurin
I
Cyclosporin A T

O P NF-AT NF-AT P
Dephosphorylation
IMMUNOPHILIN-BINDING AGENTS AND CALCINEURIN INHIBITORS

O O
O Cytokine
H OH
transcription
O
N

H Fig. 94.3. Mechanism of action of cyclosporin (cyclosporin A) and

tacrolimus. Antigen stimulation of the T-cell receptor (TCR) leads to the
O O generation of inositol triphosphate (IP3), increases in cytosolic calcium, and
OH
formation of an activated calcineurin. Calcineurin is a phosphatase which
O dephosphorylates the phosphorylated transcription factor for nuclear factor of
O
activated T cells (NF-AT), allowing NF-AT to translocate to the nucleus to
induce expression of cytokine genes. Cyclosporin (C) and tacrolimus (T) bind
to their respective immunophilins (I) to block the phosphatase action of cal-
O
cineurin and thus prevent the expression of cytokine genes induced by NF-AT.
Tacrolimus

Fig. 94.2. Chemical structures of cyclosporin (cyclosporin A) and
tacrolimus.
tacrolimus and pimecrolimus have been approved for use in patients
with moderate to severe atopic dermatitis. Another structurally similar
compound, sirolimus (previously referred to as rapamycin), has a distinct
mechanism of action and has not yet been introduced for clinical use.15
CsA and tacrolimus have variable pharmacokinetics and narrow
therapeutic windows, making close monitoring mandatory.16 Monitor-
ing of trough whole blood concentrations is standard for the use of these
drugs in solid organ transplantation. Monitoring of drug concentrations
is not standard in autoimmune conditions such as rheumatoid arthritis
and psoriasis, partly because of the lower doses used and because of the
lack of correlation of drug concentrations with relevant clinical outcomes
in non-transplant diseases.16 The microemulsion formulation of CsA has
more consistent bioavailability than with older preparations. Tacrolimus
has about 100 times more immunosuppressive activity in vitro than CsA;
therapeutic whole blood trough concentrations of tacrolimus are about
20 times lower than those for CsA.
CsA and tacrolimus are both available as intravenous and oral prepa-
rations. Typical intravenous doses are about one-third of the oral dose.
In transplantation, doses of CsA are typically in the range of 5–8 mg/
kg/day, whereas for tacrolimus a typical dose would be 0.3 mg/kg/day. In
transplantation, dosing is guided by whole blood drug concentrations.
Steady-state whole blood levels of 400 ng/mL are often associated with
trough concentrations of 300 ng/mL – a reasonable therapeutic target.
In non-transplant conditions the doses are typically lower. These agents
are lipophilic, and are highly bound to lipoproteins in the circulation.
Topical tacrolimus and pimecrolimus have minimal systemic absorp-
tion, with low serum levels being detectable in approximately 20–25%
of treated patients.17
CsA and tacrolimus are eliminated primarily via biotransformation in
the cytochrome P450 system (CYP3A) in the gut wall and liver. Impair-
ment of hepatic function reduces elimination of metabolites, whereas re-
nal failure has an insignificant effect. A number of other medications are
also metabolized by this same pathway, and therefore drug–drug interac-
tions may significantly affect concentrations of CsA and tacrolimus.
IMMUNOLOGIC EFFECTS
CsA, tacrolimus, and pimecrolimus exert their more prominent immu-
nomodulatory effects on T-cell function. After being taken up by the cell,
these agents bind to cytosolic proteins called immunophilins: CsA binds
to cyclophilin, and tacrolimus to FK-binding protein (FKBP). The com-
bination of drug and immunophilin binds to and inhibits calcineurin,
a calcium–calmodulin-dependent serine/threonine phosphatase that
activates various transcriptional regulatory factors (Fig. 94.3).15,18 A
key target of calcineurin is nuclear factor of activated T cells (NF-AT).
Upon activation, NF-AT activates the transcription of genes encoding

1646

cytokines critical for T-cell function, particularly IL-2. Other T-cell
cytokines blocked by these agents include IL-3, IL-4, IL-5, GM-CSF,
and IFN-γ. Because helper T cells play a central role in the initiation and
propagation of immune reactions, inhibition of helper T-cell function
also exerts secondary modulatory effects on various other components
of the immune and inflammatory responses. In addition, CsA and tac-
rolimus exert direct immunomodulatory effects on other cell types, for
example blocking degranulation and inhibition of the transcriptional
activation of genes encoding IL-3 and IL-5 by mast cells and basophils.18
Other changes that have been observed with immunophilin-binding
agents include downregulation of the expression of the high-affinity
receptor for IgE.
ADVERSE EVENTS
Potential adverse effects occurring with oral and parenteral CsA and
tacrolimus are similar (Table 94.3). One of the most common and sig-
nificant adverse effects is nephrotoxicity. Whereas nephrotoxicity is seen
similarly with both agents, hypertension, which can be a contributing
factor in renal dysfunction, may occur less frequently with tacrolimus.19
Both an acute, reversible nephrotoxicity, related to alterations in intrare-
nal hemodynamics, as well as a chronic irreversible renal dysfunction can
be seen. The arteriolar vasoconstriction relevant to the acute decrease in
renal function seen with these agents may be mitigated by calcium chan-
nel blocking agents. Hypertension can be an insidious problem, even at
lower doses, and may limit long-term use of these drugs.
A variety of adverse effects may occur with CsA and tacrolimus that,
although not life-threatening, can be bothersome enough to preclude
long-term use of these drugs. Neurologic symptoms, including headache,
Table 94.3 Adverse effects potentially associated with
cyclosporin and tacrolimus
Common
Renal insufficiency (acute and chronic)
Hypertension
Hypertrichosis
Headache
Gingival hypertrophy
Hyperuricemia
Less common
Paresthesias
Tremor
Nausea
Glucose intolerance
Uncommon
Increased risk of infection
Increased risk of lymphoproliferative diseases
Cholestasis
Immunomodulators 94
tremor, and dysesthesias, have been reported in up to 20% of transplant
patients. Hypertrichosis, with hair growth typically on the face, arms, and
shoulders, has been reported in up to 50% of transplant patients, as has
gingival hyperplasia. These effects are observed less commonly among
non-transplant patients receiving lower doses. Glucose intolerance,
which is often compounded by the concurrent use of corticosteroids,
may relate to a direct effect of these drugs on pancreatic islet cell func-
tion, and seems to be more of a problem with tacrolimus than with CsA.
Cholestasis and increases in hepatic transaminase concentrations have
also been observed.
As with any agents that suppress the immune response, potential
side effects related to the interference with normal immunosurveillance,
namely increased susceptibility to infection and malignancy, are a con-
cern with CsA and tacrolimus. In the setting of allograft transplanta-
tion, where the situation is made more difficult because of the concur-
rent use of other immunosuppressants, cases of opportunistic infections
have been observed. In addition, cases of lymphoproliferative disease
IMMUNOPHILIN-BINDING AGENTS AND CALCINEURIN INHIBITORS
and other malignancies have been reported among transplant patients.
In non-transplant recipients, such as patients with autoimmune diseases,
there does not appear to be an increased incidence of infection or malig-
nancy related to the use of these agents.20
Topical tacrolimus and pimecrolimus are generally well tolerated, with
the most common side effects being local burning upon application.17
In 2006, the US Food and Drug Administration mandated a ‘black box’
warning on the product label for topical tacrolimus and pimecrolimus
concerning a potential risk for treatment-related malignancy. Analysis
of clinical trial and pharmacovigilance data, however, would not seem
to support an increased risk of malignancy related to the use of these
agents.17
CLINICAL STUDIES IN ASTHMA AND ALLERGY
To date, the calcineurin inhibitors have been studied mainly in two allergic
conditions: asthma and atopic dermatitis. In a blinded, 12-week crossover
study, treatment with CsA, at an initial dose of 5mg/kg/day, resulted in an
improvement in FEV1 of 12% compared to placebo.21 No attempt at cor-
ticosteroid sparing was investigated, but fewer increases in corticosteroid
dose were needed among the CsA-treated patients.21 In a follow-up study,
39 patients with corticosteroid-dependent asthma experienced a 25% re-
duction in corticosteroid dose while on CsA.22 The potential utility of CsA
in asthma would appear to be supported by studies showing that it can
attenuate the allergen-induced late-phase reaction.23 Inhibition of the late-
phase asthmatic response is associated with attenuation of allergen-induced
increases in IL-5, GM-CSF, eotaxin, and eosinophilia in the airway.
Not all studies of CsA have had a positive outcome. In one study24
neither improvements in lung function nor any corticosteroid-sparing
effect could be demonstrated; hence CsA remains an investigational
treatment in asthma. Novel routes of administration, such as nebuliza-
tion, are under investigation.
T cells appear to have an important role in the initiation and mainte-
nance of atopic dermatitis (AD), providing a rationale for the use of CsA
and tacrolimus in this condition. In one double-blind, placebo-controlled,
8-week crossover study CsA significantly improved the extent and
severity of AD.25 Of note, despite the short duration of the study, at the
dose of CsA used (5 mg/kg/day), 20 of the 33 patients developed adverse
effects, compared to 8 of 33 receiving placebo. Other studies have sug-
gested that CsA may have a beneficial effect in AD, but concerns about
1647
F THERAPEUTICS
toxicity remain. Because much of the relevant immune reaction driving
AD takes place locally in the dermis, topical calcineurin inhibitor admin-
istration could theoretically minimize adverse effects while maintaining
its therapeutic effect. A topical preparation of CsA was not effective,26
presumably related to poor absorption. In contrast, tacrolimus and pime-
crolimus have been successfully developed as local immunomodulatory
agents for the treatment of AD.17,27 Trials with both agents have shown
notable improvements in signs and symptoms, with minimal toxicity
(see Chapter 62).
CURRENT RECOMMENDATIONS FOR USE
IN CLINICAL PRACTICE
Recommendations for the use of calcineurin inhibitors in clinical practice
come from evidence accumulated to date in allergic diseases and extrapo-
lation from other diseases. CsA remains an investigational agent in the
INTRAVENOUS IMMUNOGLOBULIN
treatment of asthma, in which its use is based on the wider experience
with this agent in autoimmune diseases such as RA and psoriasis. In those
conditions, therapy with CsA is often started with a dose of the micro-
emulsion formulation of about 2.5 mg/kg/day, given in divided doses.
Doses are typically titrated upwards in increments of 0.5 mg/kg/day at
about 8-week intervals. Long-term doses higher than 4 mg/kg/day are
not typically used outside the transplant setting. The utility of monitoring
trough concentrations of CsA in non-transplant patients remains unde-
fined. Longitudinal monitoring of renal function and blood pressure is
required for patients using CsA or tacrolimus chronically. If the baseline
creatinine increases by 30% or more, the dose should be reduced.28 Simi-
larly, significant increases in blood pressure necessitate CsA dose reduc-
tion or the introduction of antihypertensive agents (i.e. calcium channel
blocking agents such as felodipine, amlodipine), with attention given to
potential drug interactions. Patients should be monitored for signs and
symptoms of infections, neurologic side effects, and other adverse effects.
The use of topical tacrolimus and pimecrolimus for atopic dermatitis
has increased, and the clinical indications are reviewed in Chapter 62. At
present, the greatest experience with this agent has been in patients with
severe or refractory AD.
■ INTRAVENOUS
IMMUNOGLOBULIN ■
CLINICAL PHARMACOLOGY
In healthy individuals, following IVIG infusion there is a biphasic
plasma IgG disappearance curve, with the initial phase representing
distribution between body compartments and early catabolism, and the
second phase representing catabolism.29 The half-life of IVIG ranges
from 14 to 24 days in healthy individuals and is more prolonged (26–35
days) in patients with humoral immunodeficiencies.29
IMMUNOLOGIC EFFECTS
IVIG has a variety of immunomodulating properties29 which are of po-
tential benefit in immunologically mediated diseases such as immune
thrombocytopenic purpura and Kawasaki syndrome. These immunologic
effects include blockade of Fc receptors, anti-cytokine effects, downregu-
lation of T- and B-cell function, inhibition of complement activation,
1648
enhanced clearance of endogenous IgG, anti-idiotype suppression, and
neutralization of super antigens.30 Which, if any, of these effects may
be useful in the treatment of asthma or allergy is currently not known.
In vitro studies have shown that IVIG inhibits cytokine-dependent
lymphocyte proliferation, as well as cytokine (IL-2, IL-4) production by
T lymphocytes.
Immunoglobulin E
Inhibition of IgE production by B cells is one postulated mechanism
by which IVIG may exert an immunomodulating effect in asthma.31,32
This effect of IVIG is postulated to occur through co-ligation of the
B cell Fcγ RIIB receptor and the B-cell antigen receptor, with resultant
negative signaling in B cells.32 IVIG could thus provide an off signal to
the B cell to inhibit B-cell proliferation and immunoglobulin produc-
tion. In vitro, IVIG inhibits IgE production by B cells.31 In open-label
studies IVIG has been shown to reduce the immediate skin reactivity to
allergen.33 Although there is some experimental evidence to suggest that
IVIG may reduce IgE levels in vivo,33 a reduction in IgE is not noted in
the majority of studies which have investigated this immunomodulatory
property of IVIG.34,35
Sinopulmonary infections
IVIG might theoretically improve asthma by reducing the incidence of
sinopulmonary infections. Evidence for this has not adequately been ad-
dressed in current studies with IVIG.
ADVERSE EVENTS
IVIG therapy is generally safe and well tolerated. Side effects such as nau-
sea, headache, backache, chills, and flushing occur in approximately 5–10%
of IVIG infusions (Table 94.4)36 and usually resolve with interruption of
the infusion and resumption at a slower rate. More serious adverse events
(anaphylaxis, aseptic meningitis, renal failure) related to IVIG therapy are
fortunately uncommon. IgA deficiency should be suspected in individu-
als who develop anaphylaxis to IVIG.37 An explanation for why subjects
who have no IgA may develop anaphylaxis following IVIG infusion is
Table 94.4 Adverse effects potentially associated with IVIG
Common
Nausea
Headache
Chills
Backache
Flushing
Uncommon
Renal insufficiency
Aseptic meningitis
Anaphylaxis

suggested from the observation that IgA-deficient individuals may de-
velop anti-IgA IgE antibodies, as their immune system does not recognize
infused IgA as a self protein. In subjects who develop a severe headache
related to IVIG therapy the development of aseptic meningitis should be
considered.38 These subjects will often have associated photophobia and a
stiff neck, and their CSF will show a neutrophilic or a mixed cell pleocyto-
sis.38 The induction of renal insufficiency by IVIG has also been reported
and attributed to hyperosmolar renal damage induced by the breakdown
of sucrose, which is used as a stabilizer in IVIG preparations.39–41
As IVIG is derived from the pooled plasma of 3000–60 000 individu-
als,36,42 the theoretic transmission of infectious agents requires contin-
ued vigilance. All IVIG donors are screened for HIV, hepatitis B, and
hepatitis C. To date there have been no reported cases of transmission of
HIV or hepatitis B following IVIG infusion.42 However, in the USA in
1993–1994 there were outbreaks of hepatitis C associated with certain
brands of IVIG, particularly those produced by chromatography.43–45
Following the introduction of specific viral inactivation steps, no new
cases of hepatitis C related to IVIG have been reported in the USA.46
The theoretic potential for the transmission of Creutzfeld–Jakob disease
(CJD) has also been reviewed by the FDA, NIH, and CDC, who suggest
that the risk of transmission of CJD by blood products, if it exists, is con-
siderably lower than the risk for harm to public health from withdrawal of
the use of blood products.42,46 Although CJD transmission has never been
documented with transfusion of these products, the prolonged incubation
period and the lack of screening tests for CJD warrant continued research
and surveillance for the potential transmission of this rare condition.
CLINICAL STUDIES IN ASTHMA
Open-label high-dose IVIG
Three open-label studies in the USA of high-dose IVIG (2 g/kg) in
subjects with moderately severe asthma requiring daily or alternate-day
prednisone have been performed.33,47,48 Overall, these studies demon-
strate that IVIG (2 g/kg administered monthly for 6–9 months) has
an oral corticosteroid-sparing effect, as in two studies subjects were
able to significantly reduce their daily oral corticosteroid dosage (from
21 mg to 10 mg48 and from 31 mg to 5 mg)47 or in one study their alter-
nate-day oral corticosteroid dosage (from 32 mg to 11 mg).33 Symptom
scores, glucocorticoid bursts for exacerbations of asthma, and hospitali-
zations for asthma were all significantly reduced in subjects who received
IVIG.33,47,48 No change in methacholine responsiveness or response to
exercise challenge was noted in subjects who received IVIG in these
studies.33,47,48 Although the open-label IVIG studies suggest that IVIG
has a corticosteroid-sparing effect, the lack of a double-blind placebo-
controlled study design, and the small number of subjects studied (8–11
subjects in each study received IVIG)33,47,48 necessitate that these studies
be interpreted and validated in the context of additional double-blind
placebo-controlled studies with IVIG which have been performed.34,35
Double-blind studies of IVIG
Double-blind placebo-controlled studies of IVIG have been performed
in the USA and Europe.34,35 In the USA multicenter study,34 subjects
with corticosteroid-dependent asthma were randomized to receive
monthly infusions of either IVIG (2 g/kg or 1 g/kg) or placebo for
7 months. At entry into the study the mean prednisone dose in the group
Immunomodulators 94
receiving IVIG was 16 mg/day, their mean FEV1 87% of predicted, and
their mean age 16, characteristics that were not significantly different
from those of the placebo group. The primary outcome measured in the
study was the mean daily prednisone dose requirements (adjusted for
weight) determined during the observation month preceding the insti-
tution of IVIG therapy, compared to the mean daily prednisone dose
requirements determined during month 7 of the study. The mean daily
prednisone dose requirements declined by 33% in the placebo group,
and this reduction was not significantly different from the reduction in
prednisone in the 2 g/kg IVIG group (33% reduction in prednisone), or
the 1 g/kg IVIG group (39% reduction in prednisone). There was also
no difference between the placebo and IVIG groups in other asthma
outcomes measured, including FEV1, emergency department visits,
hospitalizations, or missing days from work or school. The mean levels
of IgE did not change significantly from baseline after the institution of
IVIG therapy. The study was terminated prematurely as 20% (3/15) of
the subjects randomized to the 2 g/kg IVIG group were hospitalized
DNA-BASED THERAPIES
with symptoms of headache, nausea, vomiting, photophobia, and men-
ingismus, consistent with aseptic meningitis.34
IVIG was also evaluated in a European double-blind placebo-
controlled study in moderately severe asthmatics (mean FEV1 77% pre-
dicted) who had a mean age of 14 years.35 After a 2-month stabilization
phase, during which inhalation and oral corticosteroids were tapered to a
minimum dose allowing effective control of asthma symptoms (mean dose
of inhaled corticosteroid of 1350 μg/day; 31% of subjects on oral corti-
costeroids), subjects were randomized to receive IVIG (1g/kg) or placebo
monthly for 3 months. The addition of IVIG to a stable dose of inhaled
and/or oral corticosteroid did not result in a significant improvement in
asthma symptoms, FEV1, or airway hyperreactivity to histamine at the
end of the study period. IVIG had no significant effect on IgE levels.
In contrast to the open-label studies of IVIG, the double-blind
placebo-controlled studies of IVIG34,35 do not demonstrate a significant
improvement in asthma symptoms, emergency department visits, hospi-
talizations, FEV1, airway hyperreactivity measurements, or reductions in
IgE levels. In addition, the high-dose IVIG in one study was associated
with a significant risk for adverse events such as aseptic meningitis.
CURRENT RECOMMENDATIONS
FOR USE IN CLINICAL PRACTICE
Although the results of the open-label studies with IVIG in asthma
are encouraging in terms of their oral corticosteroid-sparing effect, the
placebo-controlled studies do not confirm that IVIG has a corticoster-
oid-sparing effect. The number of subjects randomized to IVIG groups
in the double-blind studies (9–16 subjects per IVIG group) are small,
but similar numbers of subjects have been studied in the open-label
studies (8–11 subjects per IVIG group). At present IVIG is an experi-
mental therapy for asthma. It should be evaluated further in the context
of randomized, placebo-controlled clinical trials.
■ DNA-BASED THERAPIES ■
Several types of DNA-based therapy are currently being evaluated in the
treatment of allergy and asthma. Although these therapies are similar in
that they all utilize oligodeoxynucleotides, they differ in structural DNA
sequences and their mechanism of action. The DNA-based therapies
1649
F THERAPEUTICS
Promoter
Antibiotic
resistance
Antigen
coding
cDNA
CpG
DNA-BASED THERAPIES
Plasmid backbone Transcription unit
Th1 adjuvant Antigen synthesis
Fig. 94.4. Deoxyribonucleic acid (DNA) vaccine. DNA vaccines are
circular, extrachromosomal pieces of plasmid DNA that can be modified to
carry genes of interest in the antigen coding region (e.g. dust mite allergen
for allergen immunotherapy). The transcription unit includes a promoter
which initiates the transcription of the antigen coding sequence. The plasmid
backbone contains CpG (cytosine guanosine) motifs which bias the immune
response to a Th1 response. Antibiotic resistance sequences allow for
selection of the plasmid during preparation of the DNA vaccine.
currently being evaluated include DNA vaccines encoding the expression of
allergens, antisense oligonucleotides which inhibit the translation of specific
host cellular mRNA, and immunostimulatory DNA sequences containing
a cytosine phosphorothioate-linked guanosine DNA motif (CpG DNA)
which bias the immune response away from a Th2 or pro-allergic response.
DNA VACCINES
DNA vaccines are circular, extrachromosomal pieces of plasmid DNA
that can be modified to carry genes of interest (i.e. dust mite allergen for
allergen immunotherapy)61 (Fig. 94.4).
Immune response
There are several potential mechanisms by which the antigen encoded by
the injected plasmid DNA vaccine is processed and presented to elicit
an immune response. The subcutaneously injected plasmid DNA could
be taken up by dendritic cells or somatic cells (skin and/or muscle) that
then transcribe and express the encoded antigen or allergen and elicit
an immune response.49 Alternatively, a non-antigen-presenting cell (i.e.
muscle cell) injected with plasmid DNA could express and transfer the
protein (cross-priming) to a professional antigen-presenting cell.49 The
preponderance of evidence suggests that the primary immune response
after DNA vaccination is mediated by dendritic cells.49 During cross-
priming, antigen or peptides (both MHC class I and II) generated by
plasmid DNA injected into somatic cells can be taken up by professional
antigen-presenting cells such as dendritic cells to prime T-cell responses.
Thus, somatic cells (i.e. myocytes and keratinocytes) may serve as a
significant reservoir of antigen available for cross-priming, as these cells
1650
are the predominant cells transfected after DNA inoculation via muscle
or skin injection, respectively. Cross-priming may serve as a mechanism
to augment or maintain the immune response.49 DNA vaccination has
generally been associated with the induction of a Th1 as opposed to a Th2
immune response. The presence of immunostimulatory DNA sequences
containing a CpG motif in the plasmid backbone of the DNA vaccine are
considered to be important in generating the Th1 immune response.50
Mouse models of allergy and asthma
The use of DNA vaccines encoding an allergen to modify the Th2 im-
mune response to that allergen has been studied extensively in mice.50
Both humoral and cellular immune responses to encoded allergens can
be generated following intramuscular or intradermal injection of plas-
mid DNA encoding a specific antigen.50 Furthermore, studies using a
rat model of asthma have demonstrated that the IgE response, histamine
release into bronchoalveolar lavage fluid, and bronchial hyperreactivity
following challenge with aerosolized dust mite allergen Der p5 were
inhibited in rats immunized with a plasmid DNA encoding the Der p 5
allergen.51 The response to the DNA vaccine was antigen specific, as the
rats immunized with plasmid DNA encoding the Der p 5 allergen were
protected against Der p 5 allergen challenge, but not against a different
allergen, i.e., ovalbumin challenge.
DNA vaccines also prevent anaphylactic reactions to peanut allergen (Ara
h 1) in mouse models of anaphylaxis.52 In these studies the DNA vaccine
could be administered orally using chitin particles containing the DNA
plasmid encoding Ara h 1. The orally administered DNA vaccine remained
functional, as assessed by its ability to inhibit Th2 immune responses
as well as anaphylaxis following challenge with Ara h 1.52 Additional
studies in mice have demonstrated that DNA vaccines can significantly
reduce mortality associated with anaphylaxis.53 Moreover, these vaccines
can prevent allergic responses to birch pollen54 and latex55 allergens.
Potential use in human allergy and asthma
DNA vaccines in humans have been evaluated in infectious disease and
in oncology, in which no serious adverse events related to injection of
plasmid DNA have been reported.49 Although DNA vaccines alone
can elicit humoral and cellular immune responses to many antigens, the
immune response may be suboptimal for protection against infectious
disease.49 At present there are no published studies of the human im-
mune response to DNA vaccines encoding allergens. Several potential
safety concerns regarding DNA vaccines will need to be addressed in
all studies, including the theoretical potential for integration of the in-
jected plasmid DNA into the host genome (with the theoretical risk of
increasing the risk for malignancy), as well as the potential for triggering
autoimmune responses to injected DNA. To date there is no evidence
that plasmids integrate into the host genome.49
IMMUNOSTIMULATORY DNA THERAPY
Immune response
An alternative method of DNA-based immunization that has re-
ceived significant attention recently is the use of CpG-rich (cytosine
phosphorothioate-linked guanosine DNA) immunostimulatory DNA

A
5
Purine Purine C G Pyr Pyr
CpG
B DNA also significantly inhibits blood eosinophilia, suggesting that CpG
CH3
C G
CpG
Fig. 94.5. Cytosine–guanosine (CpG) DNA. A CpG DNA hexamer
sequences that have an optimal Th1 adjuvant effect in mice include the
motif 5′-purine–purine–CpG–pyrimidine–pyrimidine-3′, e.g. AACGTT or
GACGTC. These sequences are non-coding. B Cytosine (C) methylation
(CH3) abolishes the Th1 adjuvant effect of CpG DNA sequences.
sequences as inhibitors of Th2 responses to antigen. Interest in this
approach began when studies demonstrated that the CpG DNA motifs
in Mycobacterium bovis BCG DNA induced interferon-γ, a cytokine
produced by Th1 cells.56 In contrast, the immunostimulatory effect of
vertebrate DNA is significantly lower than that of bacterial DNA. The
reduced immunostimulatory effect of vertebrate DNA is probably re-
lated to a combination of the lower frequency of CpG DNA motifs in
vertebrate compared to bacterial DNA, as well as a high frequency of
cytosine methylation in vertebrate compared to bacterial DNA, which
abolishes the immunostimulatory effect of vertebrate CpG DNA se-
quences.57 The DNA hexamer sequences generating the optimal Th1
adjuvant effect in mice include the motif 5′-purine–purine–CpG–
pyrimidine–pyridimidine-3′, e.g. AACGTT or GACGTC (Fig. 94.5),57
which activates the secretion of IFN-γ. CpG DNA has an indirect
effect on the adaptive immune response by activating the innate im-
mune system (e.g., plasmacytoid dendritic cells) to upregulate cytokine
expression (IL-12, IL-18, IFN-γ, IFN-α, IFN-β), upregulate MHC
molecules, and upregulate co-stimulatory molecules.57 These effects on
the innate immune system lead to a cytokine milieu (IFN-γ+, IL-12+)
which biases the T-lymphocyte immune response towards a Th1 re-
sponse to newly encountered antigens.
Studies have demonstrated an important role for a Toll receptor
TLR9 (Toll receptor 9)58 in mediating signaling of CpG DNA, as the
deletion of genes encoding TLR-9 in mice results in mice that are un-
able to respond to CpG DNA.58 As TLR9 in humans is expressed pri-
marily by plasmacytoid dendritic cells and B cells, CpG DNA exerts its
immunomodulatory effects in humans predominantly through its effects
on plasmacytoid dendritic cells, which subsequently influence the adap-
tive immune response. Toll-like receptors (expressed on a variety of in-
nate immune cells) belong to a family of pattern recognition receptors
that are postulated to allow the innate immune system to sense different
classes of pathogen.57
Immunomodulators 94
Mouse models of allergy and asthma
3

Several studies have demonstrated that CpG DNA can inhibit Th2
cytokine responses, as well as eosinophilic inflammation and airway hy-
perreactivity to methacholine in mouse models of asthma.59 In addi-
tion to inhibiting eosinophilia of the airway and lung parenchyma, CpG
DNA exerts a significant effect on the bone marrow production and/or
the release of eosinophils.59 The inhibition of the bone marrow produc-
tion of eosinophils is associated with a significant inhibition of Th2 cell-
derived cytokine production (IL-4, IL-5).
The cellular mechanism of action of CpG DNA in vivo may be more
complex than that initially hypothesized based on results from in vitro
studies. Although CpG DNA induces a strong Th1 response, the anti-
allergic effect of CpG DNA may not be mediated by this mechanism in
vivo. In support of this hypothesis are studies demonstrating that adap-
tive transfer of antigen-specific Th1 cells does not protect against eosi-
DNA-BASED THERAPIES
nophilic airway inflammation in a mouse model of asthma,60 and studies
demonstrating that CpG DNA can mediate its anti-allergic effect in
vivo independent of the Th1 cytokines interferon-γ and IL-12.61 Thus,
the ability of CpG DNA to induce a Th1 response and interferon-γ ex-
pression may not be essential to its inhibitory effect on allergic inflam-
mation in vivo.
Potential use in human allergy and asthma
CpG-based therapeutics are currently undergoing evaluation in human
clinical trials in allergy and asthma, infectious disease, and cancer.57 For
therapeutic use, CpG DNA oligodeoxynucleotides are typically syn-
thesized with a phosphorothiate linkage (rather than using the native
phosphodiester linkage) to prevent nuclease digestion and increase the
half-life of CpG. Subcutaneous administration of CpG DNA in healthy
human volunteers results in high concentrations in the draining lymph
node and induces high levels of serum cytokines.57 In contrast, intrave-
nous administration of the same CpG DNA results in a significant dilu-
tion in the blood, binding of CpG DNA to serum proteins, and failure
to induce a measurable serum cytokine response.57 Thus, the regional
lymph nodes play an important role in the immune response to subcu-
taneously administered CpG DNA. The most common side effect noted
with subcutaneous injection of CpG DNA is a mild local injection site
reaction. Some individuals may develop transient ‘flu-like’ symptoms of
headache, fever, myalgia, and nausea.57 Although induction of autoim-
munity has not been noted, the duration of therapy with CpG DNA
in the majority of studies has been less than 6 months; hence further
studies are needed to address this issue.
At present there are limited published results of CpG DNA in sub-
jects with allergy and asthma. In a phase I study of CpG DNA admin-
istered weekly by inhalation for 4 weeks to humans with mild asthma
(FEV1 87% of predicted), the nebulized CpG DNA induced expression
of IFN-γ in sputum cells, but did not attenuate the physiologic response
to allergen inhalation challenge (early- or late-phase FEV1), sputum
eosinophils, or Th2 cytokines in sputum.62 Nebulized CpG DNA was
well tolerated and no significant adverse events were noted. Further
studies are needed to determine whether different doses of CpG DNA,
different routes of CpG administration, or administration of CpG DNA
in clinical asthma (as opposed to a model of allergen-induced asthma),
demonstrate similar or different results.
1651
F THERAPEUTICS
CYTOSINE PHOSPHOROTHIOATE GUANOSINE
DNA CONJUGATED TO PROTEIN ALLERGEN
Recently, interest has developed in the use of allergen protein conjugated
to CpG DNA for the treatment of allergic diseases, as this approach has
certain theoretical advantages compared to unconjugated CpG DNA.
The primary advantage of allergen protein conjugated CpG DNA is that
physically linking CpG DNA to antigens would increase the likelihood
of their delivery to the same antigen-presenting cell (APC), resulting in
an amplified, antigen-specific Th1 immune response,63 which is much less
likely to occur when CpG DNA and antigen are administered separately.
The same APC would therefore process the antigen and present the an-
tigen to Th cells, whereas CpG DNA would induce the APC to release
IL-12, thus biasing the well-targeted Th cell to differentiate towards a
Th1 phenotype. Furthermore, theoretically, the CpG DNA allergen pro-
DNA-BASED THERAPIES
tein conjugate might be less allergenic than the CpG DNA allergen pro-
tein mixture, as steric hindrance or electrostatic blockade of IgE-binding
epitopes could occur with CpG DNA coating the allergen.63
Immune response
The immune response to Amb a 1, the major short ragweed allergen,
conjugated to CpG DNA has been investigated in mice, rabbits, mon-
keys, and humans.63 Administration of subcutaneous injections of a
ragweed protein CpG DNA conjugate to ragweed-allergic individuals
induced their peripheral blood mononuclear cells to express a Th1 rather
than a Th2 cytokine profile.64 Individuals with ragweed-induced allergic
rhinitis who have received immunotherapy with a ragweed protein CpG
DNA conjugate respond to ragweed nasal challenge by increasing nasal
mucosa Th1 cytokine production, as well as by decreasing Th2 cytokine
production and nasal mucosa eosinophilia.65
Mouse models of allergy and asthma
In a mouse model of asthma, concomitant administration of antigen
conjugated to CpG DNA intratracheally prior to allergen challenge re-
duced airway eosinophilia and airway hyperreactivity to methacholine,
downregulated the Th2 immune response, and enhanced Th1 cytokine
expression.66 The allergen protein conjugated CpG DNA was found to
be 100 times more efficient than the unconjugated mixture in reducing
airway eosinophilia and bronchial hyperreactivity, with this inhibitory
effect observed for at least 2 months. CpG DNA allergen protein con-
jugates therefore offer promise as therapy for asthma and other allergic
disorders, although investigation of its immune modulating properties in
humans requires further study.
Potential use in human allergy and asthma
In human studies a ragweed protein CpG DNA conjugate induces sig-
nificantly less basophil histamine release than does ragweed alone.63 In
a phase II clinical study of 25 individuals with allergic rhinitis due to
ragweed, a short-course of immunotherapy with six weekly injections of
a ragweed protein CpG DNA conjugate significantly reduced rhinitis
symptom scores during the ragweed season, and these symptom improve-
ments persisted through the second ragweed season without additional
immunotherapy.67 Other than mild local injection site reactions the
1652
ragweed protein CpG DNA conjugate has not been associated with
any significant side effects.64,65,67 The ability to reach a target dose of
12 μg of Amb a 1, the major ragweed allergen, without inducing a sys-
temic allergic reaction using only six escalating dose injections of the
conjugate is a significant advantage of the conjugate compared to stand-
ard immunotherapy, which requires many more injections to safely reach
the maintenance dose. As some, but not all, studies have shown a clinical
benefit of the ragweed protein CpG DNA conjugate in subjects with
ragweed-induced allergic rhinitis, further large-scale studies are needed
to determine both its effectiveness and its long-term safety.
ANTISENSE OLIGODEOXYNUCLEOTIDE THERAPY
The goal of antisense oligodeoxynucleotide (ODN) therapy is to selec-
tively inhibit the expression of a specific gene product by preventing the
translation of mRNA into protein68 (Fig. 94.6). Antisense ODN therapy
acts by sequence-specific hybridization to mRNA, inhibiting the expres-
sion of that specific gene product while not affecting the expression of
other genes. Antisense ODN therapy prevents the translation of the
RNA message into protein and promotes the degradation of the message
by ribonucleases. In order for antisense ODNs to be synthesized the cod-
ing sequence of the gene to be inhibited must be known. Antisense com-
pounds are generally about 20 base pairs of oligodeoxynucleotides that
have a sequence complementary to a portion of the targeted mRNA. The
Normal Antisense
DNA DNA
mRNA mRNA
5
3
mRNA
3
5
Antisense
5
3
mRNA
Protein mRNA
degradaiton
Reduced translation
of mRNA to protein
Fig. 94.6. Antisense oligodeoxynucleotides. Transcription of genes
follows the sequence of DNA, mRNA, and translation into protein.
Antisense oligonucleotides are designed to have complementary sequences
to target and bind to specific known mRNA sequences. This binding blocks
access of the mRNA/antisense hybrid to the ribosome and translation of the
mRNA to protein. An enzyme Rnase H degrades the RNA strand of the
mRNA/antisense hybrid.

poor stability of oligonucleotides in vivo has been improved by modify-
ing the phosphodiester backbone to a sulfur-containing phosphorothio-
ate backbone, which enhances the stability of oligonucleotides to resist
enzymatic degradation.
Animal models of allergy and asthma
Several gene products important to asthma and allergic inflammation
have been targeted with antisense ODN in animal models of asthma.
These include cell surface receptors (adenosine A1, IL-5 receptor), cy-
tokines (IL-4, stem cell factor), intracellular signaling molecules (Syk
protein tyrosine kinases), and transcription factors (GATA-3).68–70
The feasibility of using antisense therapy in asthma was first sug-
gested in studies using antisense targeted to the adenosine A1 receptor
in a rabbit model of asthma.68 Pretreatment of rabbits with inhaled
adenosine A1 receptor antisense resulted in an increase in the dose of
aerosolized adenosine required to reduce the dynamic compliance of
the lung (a measure of bronchoconstriction) by 50%.68 Airway smooth
muscle derived from rabbits treated with inhaled adenosine A1 re-
ceptor antisense demonstrated an approximately 75% decrease in the
adenosine A1 receptor density, but did not affect the adenosine A2
receptor density, indicating the specificity of the targeted antisense
therapy.68
GATA-3 is a transcription factor which is preferentially expressed
in Th2 cells and regulates the expression of Th2 cytokine genes im-
portant to allergic inflammation. Studies with antisense targeted to
GATA-3 in mouse models of asthma have demonstrated that GATA-3
antisense inhibits eosinophilic airway inflammation, airway hyperre-
activity, and cytokine expression, including IL-4 and IL-5.70 Target-
ing a transcription factor that regulates the expression of several Th2
cytokine genes is theoretically advantageous over targeting a single
cytokine, although this approach has the potential for increased side
effects.
Interestingly, antisense therapy to ameliorate allergic inflammation
has also been targeted at grass pollen allergens to reduce the generation
of pollen as opposed to targeting the allergic patient. The potential of
this approach has been demonstrated in studies of antisense-mediated
silencing of a gene encoding Lol p 5, a major ryegrass pollen allergen.71
A pollen-specific promoter was used to drive the antisense expression
of Lol p 5 in ryegrass plants. Approximately two-thirds of the IgE re-
activity of ryegrass pollen has been attributed to Lol p 5. The transgenic
ryegrass pollen showed significantly reduced allergenicity, as reflected
by low IgE-binding capacity of the transgenic pollen compared to that
of control pollen.71 The transgenic ryegrass plants in which Lol p 5
gene expression is perturbed showed normal fertile pollen development,
indicating that genetic engineering of hypoallergenic grass plants is
possible.
ALTERNATIVES TO ANTISENSE: SMALL
INTERFERING RNAs (siRNA)
In 2006 the Nobel Prize in Physiology or Medicine was awarded to two
scientists, Andrew Fire and Craig Mello, for their far-reaching discovery
in 1998 about how genes are controlled within living cells. They discov-
ered an unexpected system of gene regulation in living cells that resulted
in a subsequent explosive phase of research in a field known variously as
RNA interference or gene silencing. RNA interference may potentially
Immunomodulators 94
lead to a new class of drugs that switch off unwanted expression of
individual genes in disease.72 Two gene-silencing drugs using the RNA
interference strategy have been designed to treat macular degeneration
and are already in clinical trials.72
Post-transcriptional inhibition of gene expression at the mRNA level
can therefore be accomplished not only by antisense-based therapy but
also by small interfering RNAs (siRNAs).72 siRNAs are small double-
stranded RNAs (about 21 nucleotides) designed to have complemen-
tarity to a specific single-stranded mRNA, and mediate destruction
of the specific target mRNA.72 A pivotal point in mRNA silencing is
the selection of the active strand of the siRNA duplex. The translation
of the ability of siRNA to silence genes in cell culture in vitro and in
animal models in vivo to human application still requires significant
further development. In particular, improving targeting siRNAs in vivo
without their degradation, as well as assessing the risks and benefits of
such an approach in humans, remains problematic. siRNAs have been
delivered by inhalation to the airway in mice and shown to inhibit genes
MYCOBACTERIAL VACCINES
such as heme oxygenase-1 in the lung, but not in other organs.73 Phase I
human studies have begun to evaluate siRNA in the treatment of ocular
neovascularization,72 but no human studies have yet been reported in
allergy or asthma.
Potential use in human allergy and asthma
The potential for antisense to be utilized in human disease has best been
applied to cytomegalovirus (CMV) infection in subjects with AIDS.
Vitravene is an antisense therapy that inhibits CMV replication and
is approved for local therapy of CMV retinitis in patients with AIDS,
providing conceptual proof of principle that antisense therapy can be
used to treat human disease.74 At present there is no published infor-
mation on the use of antisense therapy in the treatment of human al-
lergic disease or asthma.
■ MYCOBACTERIAL VACCINES ■
Epidemiologic studies suggest an inverse correlation between rates
of tuberculosis and the prevalence of asthma in children. In Japanese
schoolchildren the rate of current symptoms of asthma was reduced by
approximately one-third in positive tuberculin responders. Based on
epidemiologic evidence that mycobacterial exposure inhibits the devel-
opment of allergy and asthma, animal model and subsequent clinical
studies with heat-killed Mycobacterium vaccae or heat-inactivated bacil-
lus Calmette–Guérin (BCG) have been investigated as a potential im-
munomodulatory therapy for asthma and allergy.
ANIMAL MODELS OF ALLERGY AND ASTHMA
Intranasal infection of mice with Mycobacterium bovis–BCG 1–3 months
prior to allergen challenge significantly inhibits the development of air-
way eosinophilia in a mouse model of allergen-induced asthma.75 The
mycobacterium-induced inhibition of airway eosinophila is associated
with inhibition of the Th2 cytokine IL-5, but not with inhibition of
IgE. The mycobacterium-induced inhibition of airway eosinophilia was
significantly reduced in interferon-γ receptor-deficient mice, suggesting
an important role for the Th1 cytokine IFN-γ in mediating the anti-
eosinophilic effect of the mycobacterial infection.
1653
F THERAPEUTICS
POTENTIAL USE IN HUMAN ALLERGY
AND ASTHMA
Two different Mycobacterium vaccae vaccines (heat-killed or delipidated
deglycolipidated) have been investigated in a double-blind study of
moderately severe atopic asthmatics (mean FEV1 76%; mean inhaled
corticosteroid dose 500–600 μg of inhaled beclomethasone daily) with
stable symptoms.76 Outcomes measured included the effect on asthma
severity (change in morning peak flow) and immunologic response
3 months later. This study demonstrated that there was no clinical benefit
or immunologic difference (eosinophil, IgE, T-cell proliferation, cytokine
response) in subjects who received two intradermal doses of either of
the two Mycobacterium vaccae vaccines tested compared to those who re-
ceived placebo.76 Additional ongoing studies with Mycobacterium vaccae
in asthma and allergy will determine whether the lack of benefit noted
in this particular study relates to a lack of effectiveness of Mycobacterium
CYTOKINE-BASED THERAPY
vaccae immunization, or alternatively, relates to study design issues (route
of administration, study population, outcome measure, sample size, etc).
HEAT-INACTIVATED BCG THERAPY IN HUMAN
ALLERGY AND ASTHMA
The effect on asthma severity of administering four weekly injections
of heat-inactivated BCG has been investigated in a placebo-controlled
study of 3 months’ duration in moderately severe asthmatics who were
Mantoux skin test negative.77 The heat-inactivated BCG did not im-
prove any of the markers of asthma severity (FEV1, peak flow, asthma
exacerbations, asthma symptom scores, β-agonist use), and also had
no effect on blood eosinophil and serum IgE levels. Recruitment for
the trial was halted early and the number of injections was reduced in
some patients owing to excessive local injection site reactions to BCG.77
In addition to the lack of efficacy of repeated heat-inactivated BCG in-
jections, the occurrence of severe local injection site reactions limits the
therapeutic application of this approach in asthma.
■ CYTOKINE-BASED THERAPY ■
Cytokines play a key role in regulating the initiation, perpetuation,
and resolution of allergic inflammation (Chapter 10). Based on stud-
ies demonstrating that several cytokines are expressed in the airway in
asthmatics,78 novel therapeutics have been developed to target individual
cytokines in patients with asthma to determine whether any of these
individual cytokines may play an important role in disease pathogenesis.
Pre-clinical studies in animal models of asthma have demonstrated that
targeting individual cytokines such as TNF, IL-4, or IL-5 reduces levels
of eosinophilic airway inflammation and airway responsiveness.
TNF
To date, the principle that one can target a single cytokine and have a
significant impact on human disease expression has been best validated
in the treatment of autoimmune disease such as rheumatoid arthritis
(RA) with inhibitors of the cytokine TNF-α.79 Currently there are three
TNF inhibitors used in clinical practice: infliximab (a chimeric mono-
clonal antibody), etanercept (a soluble TNF receptor/Fc fusion protein),
and adalimumab (a human monoclonal antibody).79 TNF inhibitors
1654
have also been shown to be highly effective in treating a number of other
systemic inflammatory autoimmune diseases, including ankylosing
spondylitis, psoriatic arthritis, psoriasis, and Crohn’s disease.79
The success of TNF-α inhibitors in RA represents a proof-of-concept
that inhibition of a single cytokine can be effective in treating a dis-
ease in which multiple cytokines are expressed, if that cytokine serves
a key or central role. Most RA patients respond to TNF-α inhibitors
with a reduction in signs and symptoms, improved quality of life, and
preservation of functional status.79 Radiographs demonstrate objective
evidence that TNF-α inhibitors also significantly slow disease progres-
sion in RA to an extent not seen with any previous agents. Interestingly,
although initial experience with TNF inhibitors was most commonly in
RA patients with chronic refractory disease, more recent studies have
demonstrated even better clinical outcomes when TNF inhibitors were
used earlier in the disease course.80 As TNF inhibitors do not induce
immunologic tolerance, maintenance of clinical efficacy almost always
requires continued therapy, certainly for patients with long-standing RA.
Importantly, 2–4-year and longer studies of anti-TNF-α therapy in RA
suggest that the clinical response is well maintained. Nevertheless, some
patients fail to respond to anti-TNF therapy and others eventually lose
their response. Interestingly, patients failing one TNF inhibitor may re-
spond to therapy with another.
Inhibition of TNF has also been studied in patients with asthma,81–84
although results have not been as clear cut nor as dramatic in all asthma
studies as in autoimmune conditions. Examples of studies in asthma
which have shown a benefit to TNF blockade include studies of the
soluble TNF receptor etanercept in 10 patients with severe asthma, which
noted improvements in FEV1, airway responsiveness to methacholine,
and asthma-related quality of life.81 In another study of 38 symptomatic
subjects with moderate asthma, infliximab did not demonstrate significant
clinical efficacy in terms of the primary endpoint of lung function.83 How-
ever, there was a significant reduction in the number of moderate asthma
exacerbations in the infliximab group compared to placebo.83 It is possible
that only a subset of severe asthmatics respond to anti-TNF therapy, and
that levels of expression of membrane-bound TNF-α by peripheral blood
monocytes in such subjects with asthma may predict responses.81
IL-4
IL-4 mediates several important proinflammatory functions in allergic
inflammation, including induction of the IgE isotype switch, induction of
vascular cell adhesion molecule-1, promotion of eosinophil transmigration
across the endothelium, stimulation of mucus production, and promotion
of Th2 lymphocyte differentiation. The therapeutic potential of a recom-
binant soluble IL-4 receptor (IL-4R) as an IL-4 antagonist has been stud-
ied in asthmatics. In two small studies, treatment with the IL-4 receptor
antagonist improved asthma symptom scores and pulmonary function,
reduced β2-agonist rescue use, as well as lowering levels of exhaled nitric
oxide.85,86 However, subsequently two large phase III studies in moderate-
to-severe asthma failed to reveal efficacy, possibly due to dose limitations
and the short duration of action of the IL-4 receptor antagonist.
IL-5
As IL-5 is a key regulator of eosinophil proliferation, studies have inves-
tigated whether targeting IL-5 would reduce eosinophilic inflammation
and improve asthma outcomes. In asthmatics, therapy with an anti-IL-5

antibody has a dramatic effect in reducing blood eosinophilia (~ 90%),
but interestingly does not inhibit the late-phase lower airway response
to inhaled allergen,87 nor improve measures of clinical asthma,88 thereby
raising questions about the previously held notion of the pivotal roles of
IL-5 and eosinophils in allergic asthma. Subsequent studies have dem-
onstrated that anti-IL-5 is less effective at inhibiting eosinophils in the
lower airways (~50%) than in the blood (~90%), indicating that partial
depletion of tissue eosinophils might be insufficient for clinical efficacy
in a complex disease such as asthma. Interestingly, anti-IL-5 reduces
levels of airway remodeling in asthma through inhibition of eosinophil
expression of TGF-β,89 and improves eosnophilia and clinical responses
in patients with eosinophilic esophagitis.90
■ CONCLUSIONS ■
Immunomodulatory therapy has made a significant therapeutic impact
in immunologically mediated diseases such as rheumatoid arthritis, but
the search for safe and effective immunomodulator therapy contin-
ues in immunologically mediated diseases such as asthma and allergy.
At present, however, immunomodulator therapy (other than traditional
allergen protein immunotherapy and anti-IgE) remains investigational
in the field of asthma and allergic disorders.
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