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,
*
Department of Medicine, and
The Sam and Rose Stein Institute for Research on Aging, University of California at San Diego, La Jolla, CA 92093;
Dynavax Technologies Corporation, San Diego, CA 92121; and
§
Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production and redirect the immune system
toward a Th1 response. Thus, systemic or mucosal administration of ISS
before allergen exposure could provide a novel form of active
immunotherapy in allergic diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More than a decade ago, Tokunaga and coworkers discovered that DNA
purified from mycobacteria induced the release of IFNs from splenocytes
(5). Fractionation of the mycobacterial DNA led to the isolation of
several short DNA sequences (containing CpG dinucleotide cores) that
mediated the immunostimulatory activity (6). Subsequent experiments
have shown that oligodeoxynucleotides (ODN) containing ISS (ISS-ODN)
induce the release of several additional cytokines, including
IFN-
,ß (5, 6) IL-6 (7), IL-12 (8, 9), and IL-18 (10) primarily
from monocytes (6, 10, 11), and IFN- from NK cells (6). The immune
response triggered by ISS is similar to the innate immune response
evoked by intracellular pathogens (10), triggering the release of
cytokines that bias the immune response toward development of an
Ag-specific Th1 effector and memory response (10). Cellular activation
by ISS DNA such as CpG is not mediated through binding to a cell
surface receptor, but requires cellular uptake by adsorptive
endocytosis (12). Studies suggest that CpG DNA, taken up by B cells and
monocytes by adsorptive endocytosis, is acidified in an intracellular
endosomal compartment (12). Endosomal acidification of DNA is coupled
to the rapid generation of reactive oxygen species that leads to
nuclear factor-
B activation and subsequent cytokine expression (12).
In this study, we have used a mouse model of eosinophilic airway
inflammation to investigate whether ISS could inhibit both the
generation of Th2 cytokines important to eosinophil proliferation and
survival (IL-5, GM-CSF, IL-3), as well as the subsequent airway
hyperreactivity in response to methacholine (MCh) challenge. The data
presented in this study indicate that administration of ISS-ODN
inhibits both airway hyperresponsiveness and airway eosinophilia by
exerting a significant inhibitory effect on the generation of
eosinophil-active cytokines (IL-5, GM-CSF, and IL-3) as well as the
subsequent bone marrow production of eosinophils. Moreover, while both
ISS and corticosteroids inhibited IL-5 generation, only ISS was able to
induce IFN-, a cytokine that importantly biases the immune system to
generate a Th1 (and not Th2) response to subsequently encountered
allergens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endotoxin-free (<1 ng/mg DNA) phosphorothioate ISS-ODN (5'-TGACTGTGAACGTTCGAGATGA-3') or phosphorothioate M-ODN (5'-TGACTGTGAAGGTTAGAGATGA-3') (Trilink, San Diego, CA), as previously described (10), were used in the in vivo and in vitro experiments described below.
Animals
Female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used when they reached 8–10 wk of age. All animal experimental protocols were approved by the University of California, San Diego, and the National Jewish Medical Research Center Animal Subjects Committees.
Determination of airway responsiveness to MCh in vivo
In experiments performed at the National Jewish Medical and Research Center, BALB/c mice were sensitized by the i.p. injection of OVA/alum on days 1 and 10, and subsequently received nebulized 1% OVA on days 22, 23, and 24. Airway responsiveness was assessed on day 26, 48 h after the final OVA inhalation, using a single chamber whole body plethysmograph obtained from Buxco (Troy, NY), as previously described (13). In this system, an unrestrained, spontaneously breathing mouse is placed into the main chamber of the plethysmograph, and pressure differences between this chamber and a reference chamber are recorded. The resulting box pressure signal is caused by volume and resultant pressure changes during the respiratory cycle of the mouse. A low pass filter in the wall of the main chamber allows thermal compensation. From these box pressure signals, the phases of the respiratory cycle, tidal volumes, and the enhanced pause (Penh) can be calculated. Penh is a dimensionless value that represents a function of the proportion of maximal expiratory to maximal inspiratory box pressure signals and of the timing of expiration. It correlates closely with pulmonary resistance measured by conventional two-chamber plethysmography in ventilated mice (13). Penh was used to monitor airway responsiveness in this study. In the plethysmograph, mice were exposed for 3 min to nebulized PBS and subsequently to increasing concentrations of nebulized MCh (Sigma, St. Louis, MO) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss). After each nebulization, recordings were taken for 3 min. The Penh values measured during each 3-min sequence were averaged and are expressed for each MCh concentration as the percentage of baseline Penh values following PBS exposure (13). To determine the effect of ISS on airway responsiveness in OVA-sensitized mice, 50 µg of either ISS-ODN or M-ODN was injected i.p. 24 h before each of three OVA inhalation challenges, and airway responsiveness was determined (5 days after the first dose of ISS). The number of bronchoalveolar lavage fluid (BALF) eosinophils was assessed in parallel.
Induction of pulmonary allergic eosinophilic inflammation
To investigate whether ISS inhibits airway eosinophilia, the OVA sensitization and challenge protocol of Corry et al. (14) were used. This protocol (14) differs in the number and route of OVA injections and inhalations compared with the protocol used to assess airway hyperresponsiveness. In these studies, mice were immunized s.c. on days 0, 7, 14, and 21 with 25 µg of OVA (OVA, grade V; Sigma) adsorbed to 1 mg of alum (Aldrich) in 200 µl normal saline. The OVA inhalation challenge (days 26 and 31) consisted of three 30-min inhalations (separated by 30-min rest intervals) of OVA at a concentration of 10 mg/ml in an inhalation chamber. The nebulizer (DeVILBISS UltraNeb-99; Sunrise Medical, Sommerset, PA) was set up to aerosolize 80–100 ml of protein solution in the 30-min inhalation time period. The outflow of the inhalation chamber was attached to a vacuum line and adjusted to a minimal suction rate (enough to prevent excess condensation from occurring in the chamber). Mice were sacrificed, and BALF, lungs, peripheral blood, and bone marrow were analyzed 24 h after the second OVA inhalation.
Therapeutic intervention with ISS-ODN or corticosteroids
The therapeutic intervention protocol is summarized in Fig. 2
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ISS-ODN or M-ODN was injected i.p. (100 µg in 100 µl of sterile,
endotoxin-free PBS) or instilled intranasally (i.n.) (50 µg of
ODN/naris, in 20 µl PBS, under metofane anesthesia). The ODNs were
administered either twice (on days 25 and 30, 1 day before each OVA
inhalation challenge) or once (on day 25, 1 day before the first OVA
inhalation challenge; or once on day 30, 1 day before the second OVA
inhalation; or on day 31, 30 min before OVA inhalation). Intratracheal
(i.t.) administration of ISS-ODN or M-ODN (100 µg of ODN in 50 µl
PBS, under metofane anesthesia) was performed twice on days 25 and 30,
1 day before each OVA inhalation challenge.
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Eosinophil counts
Twenty-four hours after the second OVA inhalation challenge (day 32), mice were sacrificed by cervical dislocation, and eosinophil counts of various tissues were performed.
Lung. Lung tissues embedded in OCT in 10 x 50 x 50-mm tissue wells were cryosectioned at 10 µ and acetone fixed onto poly(L-lysine)-coated slides. Total eosinophil numbers were enumerated by detection of eosinophil peroxidase using DAB staining and microscopic examination, as described in this laboratory (15). A key code was established for mice groups, and color-coded slides were labeled to designate mouse numbers within groups (i.e., 1–4). Slides were incubated at room temperature for 1 min in the presence of cyanide buffer (10 mM potassium cyanide, pH 6), rinsed in PBS, and incubated for 10 min with the peroxidase substrate DAB (Vector Lab, Burlingame, CA). Slides were subsequently washed in PBS, counterstained with hematoxylin, air dried, and examined by light microscopy (x40 magnification) by a "blinded" examiner. Five random fields were selected and eosinophils were counted (cells staining brown) to determine total eosinophil number per microscope field.
BALF. The sacrificed mice had their tracheas surgically exposed and cannulated with 27-gauge silicon tubing attached to a 23-gauge needle on a 1-ml tuberculin syringe. Following instillation of 600 µl of sterile saline through the trachea into the lung, BALF was withdrawn and cytospun (3 min at 500 rpm) onto microscope slides. Eosinophil counts were performed as described above.
Peripheral blood. Blood was collected from the carotid artery. RBC were lysed using a 1:10 solution of 100 mM potassium carbonate, 1.5 M ammonium chloride. The remaining cells were cytospun (3 min at 500 rpm) onto microscope slides and air dried. Eosinophil counts were performed as described above.
Bone marrow. Bone marrow cells were flushed from femurs with 1 ml PBS and cytospun onto microscope slides, and separate slides were stained with Wright-Giemsa and DAB for cell differential counts.
Stimulation of splenocytes in vitro by ISS-ODN and anti-CD3 Abs
Ninety-six-well flat microtiter plates were coated with rat
anti-mouse anti-CD3 Abs (1 µg/ml; PharMingen, San Diego, CA)
at room temperature for 2 h and then washed with PBS. Splenocytes
(5 x 106/ml) from each mouse (female BALB/c,
n = 4) were incubated with ISS-ODN or M-ODN (10
µg/ml) at 37°C for 2 h and then added to anti-CD3-coated
plates (5 x 105 cells/well), in triplicate, with or
without neutralizing Abs to mouse IL-12, IFN-, and IFN-
(Biosource, Camarillo, CA). The optimal concentration of each
neutralizing Ab used was determined previously in pilot experiments
performed to neutralize the levels of IL-12, IFN-, and IFN-
produced by ISS-ODN-stimulated splenocytes (data not shown). Splenocyte
supernatants (24, 48, and 72 h poststimulation) were assayed in
duplicate to determine the level of each cytokine. All cytokines were
analyzed by ELISA.
Administration of ISS to OVA-sensitized, but not OVA inhalation-challenged mice in vivo
Female BALB/c mice were sensitized to develop a Th2 response to
OVA, as detailed above and in Fig. 2
, but without subsequent aerosol
OVA inhalation challenge. Eight weeks after the last OVA/alum
injection, mice were injected i.p. with 100 µg of ODN, 1 day (-1d)
or 3 days (-3d) before their sacrifice. The cytokine profiles of the
supernatants derived from splenocytes incubated for 72 h with 100
µg/µl of OVA were assayed by ELISA.
Cytokine assays
The levels of various cytokines (IL-3, IL-5, GM-CSF, and
IFN-) were measured in cell supernatants following either
anti-CD3 Ab or OVA Ag stimulation by ELISA (PharMingen), as was
previously described (10, 11, 16).
Eosinophil apoptosis assay
Eosinophils of >90% purity and >95% viability were purified from the blood of IL-5 transgenic mice using a Percoll gradient, as previously described in this laboratory (17). The eosinophils were then treated with ISS-ODN (1 µg/ml), M-ODN (1 µg/ml), or controls, including mouse rGM-CSF (1 ng/ml) (PharMingen) and anti-Fas Ab (1 µg/ml) (clone Jo2 from PharMingen), and analyzed at 2, 8, 18, and 32 h after treatment. Eosinophil apoptosis was measured by quantitating the number of apoptotic nuclei relative to healthy nuclei by cell permeabilization, propidium iodide staining, and FACS analysis, as described (18).
Statistical analysis
Statistical analysis was performed with ANOVA and Student’s t test, as previously described (10, 11). In studies of airway responsiveness, groups were compared by Tukey-Kramer HSD test. A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Airway responsiveness to MCh was increased significantly in mice following OVA sensitization and OVA inhalation challenge, as opposed to mice sensitized to OVA alone with PBS challenge (14 ± 1.8-fold increase in Penh values OVA versus PBS following inhalation of 50 mg/ml MCh). Mice sensitized to OVA without inhalation challenge, or mice OVA challenged without OVA sensitization showed minimal change in Penh in response to MCh (data not shown).
Administration of ISS-ODN i.p. before each inhalation challenge
significantly reduced the increase in MCh airway responsiveness in
OVA-sensitized and OVA-challenged mice by 69 ± 9.9% (Fig. 1). These changes in airway
responsiveness induced by ISS-ODN were accompanied by a significant
reduction in BALF eosinophilia (M-ODN-treated mice, 253 ±
207 x 103 BALF eosinophils versus ISS-ODN-treated
mice, 14 ± 12 x 103 BALF eosinophils)
(p < 0.05).
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To investigate the mechanism by which ISS inhibits airway
eosinophilia, the number and route of OVA injections and inhalations in
the mouse protocol were modified (depicted in Fig. 2). Both OVA protocols induce significant
BAL eosinophilia (47 ± 16% BAL eosinophils with protocol used to
assess airway responsiveness versus 42 ± 4% BAL eosinophils with
protocol depicted in Fig. 2). However, the OVA protocol depicted in
Fig. 2 induces an approximate eightfold greater absolute number of BAL
eosinophils/ml compared with the protocol used to assess airway
hyperresponsiveness (2075 x 103 BAL eosinophils
versus 253 x 103 BAL eosinophils). Using this
protocol (Fig. 2), OVA-sensitized and OVA inhalation-challenged mice
developed significant airway eosinophilia (Fig. 3) compared with control PBS-challenged
mice in the BALF (42 ± 4% versus 0% BALF eosinophils) and lungs
(67 ± 5 eosinophils/hpf versus 2 ± 1 eosinophils/hpf,
p < 0.05, Fig. 4). Even
with the stronger stimulus for eosinophil recruitment in this protocol,
ISS-ODN significantly inhibited eosinophil recruitment to BALF (91%
inhibition compared with M-ODN) (Fig. 4A) and lung tissue
(90% inhibition compared with M-ODN) (Fig. 4B). ISS-ODN
administered once 3 to 6 days before the termination of the experiment
was the optimal time point (of those examined) for ISS administration
in inhibiting eosinophil recruitment into BAL and lung (Fig. 4, and data not shown). These studies demonstrate the sustained 6-day
inhibitory effect of a single dose of ISS on subsequent
allergen-induced eosinophil recruitment.
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ISS did not affect the number of BAL neutrophils, lymphocytes, or mononuclear cells. However, as there is not a significant increase in these cell types in BAL in models of allergen-induced airway inflammation, this may not be the appropriate model to determine whether ISS effects these cell types.
Mucosal administration of ISS-ODN inhibits airway and lung eosinophilia
Not only systemic (i.p.) ISS-ODN administration, but also
mucosal (i.e., i.n. or i.t.). ISS-ODN administration had a similar
inhibitory effect on BALF and lung eosinophil accumulation (Fig. 6, A and B).
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ISS not only inhibited eosinophilia in the airway (by 91%) and
lung parenchyma (90%), but also inhibited blood eosinophilia (86%)
(Fig. 4C), suggesting that ISS was exerting a significant
effect on the bone marrow production of eosinophils (number of bone
marrow eosinophils inhibited 58%) (Fig. 4D). OVA challenge
increased the total number of nucleated cells in the bone marrow of
M-ODN-treated mice (19.8 ± 9.2 x 106 cells/ml)
(p = 0.04 versus ISS-ODN), whereas ISS-ODN
treatment of OVA-challenged mice (7.5 ± 1.8 x
106 cells/ml) reduced the total number of bone marrow cells
to a level similar to that noted in naive mice not treated with ODN
(4 ± 0.3 x 106 cells/ml). ISS inhibited the
total number of bone marrow eosinophils (M-ODN, 1812 ± 614
x 103 bone marrow eosinophils/ml versus ISS, 349 ±
101 x 103 bone marrow eosinophils/ml,
p = 0.05) and reduced the absolute number of peripheral
blood eosinophils (M-ODN, 2815 ± 995 x 103
eosinophils/ml versus ISS, 1390 ± 44 x 103
eosinophils/ml, p = 0.05), but did not affect the total
peripheral blood white blood cell count (M-ODN, 2.3 ± 0.4 x
104 white blood cells/µl versus ISS, 2.6 ± 0.3
x 104 white blood cells/µl).
The effect of ISS on reducing the number of eosinophils was not due to ISS directly inducing eosinophil apoptosis, as assessed in eosinophil apoptosis experiments in vitro. The percentage of apoptotic eosinophils after incubation for 18 h with ISS-ODN (51%) did not differ significantly from the percentage of apoptotic eosinophils after incubation with M-ODN (54%), or from the percentage of apoptotic eosinophils in untreated control cultures (55%). In contrast, positive and negative control experiments demonstrated that anti-Fas induced a significant degree of eosinophil apoptosis (87%), whereas GM-CSF protected eosinophils from apoptosis (5% apoptotic eosinophils).
Effect of ISS on the generation of IL-5
To investigate potential immunomodulatory mechanisms responsible
for the ISS-induced inhibition of airway eosinophilia, the effect of
ISS-ODN on IL-5 generation was evaluated. IL-5 induces eosinophil
proliferation, differentiation, and resistance to apoptosis (1, 4), and
genetically engineered elimination of IL-5 in mice in vivo dramatically
reduces eosinophil numbers, as shown by the lack of eosinophils in IL-5
knockout mice (19). We evaluated whether inhibition of IL-5 production
was responsible for ISS-ODN-mediated inhibition of bone marrow,
peripheral blood, and airway eosinophilia. Systemic (i.p.)
administration of ISS-ODN to OVA-sensitized and OVA-challenged mice
reduced IL-5 production by 84%, while simultaneously inducing a
30-fold increase in IFN- production by OVA-stimulated
CD4+ splenocytes (Fig. 5). The kinetics of ISS-induced
inhibition of IL-5 correlated with the inhibitory effect of ISS on
airway eosinophilia in vivo. Similar results, i.e., suppression of IL-5
and induction of IFN- by CD4+ splenocytes, were observed
with mucosal ISS-ODN administration (i.n. and i.t.) (Fig. 7).
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We also monitored the effects of ISS-ODN on OVA/alum-sensitized,
but nonchallenged mice. These OVA-sensitized mice provide an in vivo
model to study whether ISS-ODN can inhibit the ability of an atopic
mouse to release eosinophil active cytokines (IL-5, GM-CSF, IL-3). We
postulated that administration of ISS-ODN alone (in the absence of Ag
challenge) would stimulate the in vivo release of innate cytokines
(IFN-,ß, IFN-, and IL-12), and that these cytokines would
inhibit the secretion by T cells of eosinophil active cytokines (IL-5,
GM-CSF, IL-3) without the induction of a subsequent Ag-specific Th1
response (no OVA Ag was administered by inhalation). In these studies,
we evaluated OVA-stimulated spleen cell (derived from ISS-ODN-treated
OVA-sensitized mice) production of IL-5, IL-3 (Table I), and GM-CSF (Table II), all of which are similar in their
ability to induce eosinophil proliferation, and block eosinophil
apoptosis through their activation of a common ß-chain shared by
these cytokine receptors (4). As shown in Table I, i.p. injection of
ISS-ODN (1 day or 3 days before the in vitro cytokine assay) reduced
secretion of IL-5 (83% inhibition group C versus A) and IL-3 (76%
inhibition, group C versus A) by OVA-stimulated splenocytes without
inducing any OVA-specific IFN- production. The experiments in Table I (OVA sensitization with no OVA challenge) and Fig. 5 (OVA
sensitization and OVA challenge) demonstrate that ISS can inhibit IL-5,
GM-CSF, and IL-3 in OVA-sensitized mice in the presence or absence of
OVA challenge. In contrast, ISS-treated mice require reexposure to OVA
Ag to generate an OVA-specific Th1 response (induction of IFN-)
(Figs. 5 and 7).
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/ß,
IFN-, or IL-12 (Table III). In vitro
incubation with ISS-ODN significantly inhibited anti-CD3-induced T
cell production of IL-5 (47%), GM-CSF (49%), and IL-3 (47%) (Tables
I and II). This inhibitory effect of ISS-ODN on eosinophil active
cytokine release was partially mediated by the innate cytokines (IFNs
and IL-12), as was shown in the related Ab neutralization studies
(Table III). The in vitro stimulation of splenocytes with ISS-ODN and
anti-CD3 Abs enhanced IFN- levels by threefold (data not shown).
ISS stimulation neither inhibited IL-4 secretion nor increased IL-2
levels elicited by anti-CD3 stimulation (data not shown).
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) following a single ISS
administration as compared with 7 days of corticosteroids
A single i.p. or i.n. ISS-ODN administration was as effective as
seven daily doses of dexamethasone (5 mg/kg s.c.) in the suppression of
airway eosinophilia (Figs. 4 and 6). A single dose of dexamethasone was
not effective in inhibiting eosinophilic airway inflammation
(data not shown), whereas two doses of dexamethasone had a
partial effect on inhibiting eosinophil recruitment into the lung
(Figs. 4 and 5). While both ISS and dexamethasone inhibited IL-5
generation (Figs. 5 and
7), only ISS was able to induce IFN-
(Figs. 5 and 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(a cytokine that importantly biases the immune system to
generate a Th1 and not a Th2 response to subsequently encountered
allergens). Thus, systemic or mucosal administration of ISS before
allergen exposure provides a novel form of active immunotherapy in
allergic diseases.
The inhibitory effects of ISS are likely to be mediated by the innate
cytokines derived from monocytes/macrophages and NK cells stimulated by
ISS (6, 9, 10). In studies utilizing purified populations of mouse bone
marrow-derived macrophages, we have demonstrated recently that ISS
induces these macrophages to release IL-12, but not
IFN-.4 In additional
studies,4 stimulation of cultured splenocytes derived from
SCID mice with ISS induced the release of both IL-12 and IFN-. As
purified populations of BALB/c-derived macrophages stimulated with ISS
did not generate IFN-,4 these studies with SCID mice
suggest that NK cells may be the predominant source of IFN- in SCID
mice stimulated with ISS. These4 and other studies (10, 21)
suggest that ISS is able to stimulate both macrophages and NK cells to
produce cytokines that modulate T cell cytokine production.
ISS-ODN administration, via activation of innate immunity, could
inhibit pulmonary eosinophilia through at least three different, but
additive mechanisms (Fig. 8). The first
mechanism by which ISS can inhibit pulmonary eosinophilia is through an
effect on T cell-derived cytokines important to the bone marrow
generation of eosinophils. ISS exerts this inhibitory effect indirectly
by stimulating monocytes/macrophages and NK cells to generate IL-12 and
IFNs that subsequently inhibit T cell generation of IL-5, GM-CSF, and
IL-3. The greater inhibition by ISS of peripheral blood eosinophilia
(86% inhibition) compared with inhibition of bone marrow eosinophilia
(58% inhibition) suggests that ISS may have also inhibited release of
eosinophils from the bone marrow. In this regard, IL-5 is known to
induce release of eosinophils from the bone marrow (22), and inhibition
of IL-5 generation by ISS could thus prevent bone marrow release of
eosinophils. A second mechanism by which ISS-induced generation of IFNs
and IL-12 could inhibit pulmonary eosinophilia is through an effect on
eosinophil recruitment, as has previously been demonstrated with IL-12
(23, 24), IFN- (25), and IFN- (26) in models of allergic
inflammation and parasitic infection. A third eosinophil-inhibitory
mechanism induced by ISS is the generation of an allergen-specific Th1
as opposed to a Th2 response. This would be important for long-term
protection and immunologic memory. This ISS-induced OVA-specific Th1
response would generate IFN-, which further inhibits eosinophil
accumulation by biasing naive T cells encountering Ag in an IFN-
milieu to generate Th1 as opposed to Th2 responses to newly encountered
allergens. The first two inhibitory mechanisms affecting pulmonary
eosinophilia are most likely mediated by the innate immune response,
and are therefore primarily immediate and Ag nonspecific in nature. In
contrast, the third effect is mediated by an adaptive immune response
and requires a longer period of time for differentiation and maturation
of Ag-specific Th1 cells from naive CD4+ T cells (Fig. 8).
Furthermore, while the first two mechanisms lead to a dramatic, but
probably temporary reduction in eosinophil recruitment, the third
mechanism is involved in the generation of immunologic memory that may
prevent Th2 cell responses and eosinophil recruitment into the target
organ (i.e., the lung) from developing following subsequent airway
allergen challenge.
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) and attenuated preexisting OVA-specific Th2
responses (i.e., reduction of IL-5, Figs. 5 and 7). The administration
of ISS 1 day before, or together with the final OVA inhalation
challenge was sufficient to inhibit pulmonary eosinophil recruitment as
well as the generation of eosinophil active cytokines 24 h later.
However, this method of ISS administration did not result in
OVA-specific Th1 responses, probably because Ag-specific IFN-
production generally requires more than 24–48 h.
The activation of the innate immune response by ISS-ODN could initially
prevent the development of asthmatic symptoms by preventing early
eosinophil accumulation, and later even bias the immune system to
generate an allergen-specific Th1 response to any allergen subsequently
encountered by the host in his own natural environment (Fig. 8). Thus,
therapy with ISS has potential beneficial immunomodulatory effects on
allergic inflammation not noted with corticosteroids. For example,
therapy with corticosteroids, while effective in inhibiting IL-5
generation, did not induce IFN- (Fig. 5). The potential benefit of
ISS therapy in comparison with corticosteroids in allergic disease
would be the ability of ISS to alter the cytokine mileu of the host
(IFN-) to favor generation of Th1 responses to subsequently
encountered allergens.
A recent study demonstrated an inverse epidemiologic association between exposure to mycobacteria and the prevalence of atopic disorders, suggesting that the relatively recent decline in infections (e.g., tuberculosis) in developed countries is a factor underlying the increasing severity and prevalence of allergic diseases (27). Furthermore, infection of mice with Mycobacterium bovisBacillus Calmette Guerin has been shown to suppress allergen-induced airway eosinophilia (28). As previously mentioned, the ISS were initially identified and isolated from mycobacterial DNA (5, 6) and they appear at the expected frequency in many pathogenic bacteria and viruses, while they are underrepresented in the vertebrate genome (7). Thus, the natural exposure to ISS-enriched DNA from normal intracellular pathogens, through infection, may play a role in shaping the immune response away from an allergic phenotype and allergic responses following allergen challenge. As shown in this study, ISS-ODN can provide this immunomodulatory effect without the risk of inducing active infection.
In summary, ISS-ODN administration provides an alternative to the current practice of allergen protein desensitization, which has relatively low efficacy and high potential for significant side effects, including anaphylaxis (29, 30). ISS-ODN delivery via the systemic or mucosal route (i.n. or i.t.) in allergic patients may inhibit the allergic inflammatory responses in asthmatic lungs after natural allergen exposure. The future application of ISS-ODN therapy to human allergic diseases will depend on how well the inhibitory effects of ISS-ODN noted in murine models of experimental allergic airway inflammation translate to related human diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. Broide, Department of Medicine, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0635.
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; DAB, diaminobenzidine; i.n., intranasal; i.t., intratracheal; MCh, methacholine; ISS-ODN, immunostimulatory sequence-oligodeoxynucleotide; M-ODN, mutated-oligodeoxynucleotide.
4 E. Martin-Orozco, H. Kobayashi, M.-D. Nguyen, J. Van Uden, R. S. Kornbluth, and E. Raz. Activation of APCs by immunostimulatory DNA sequences. Submitted for publication.
Received for publication April 16, 1998. Accepted for publication August 25, 1998.
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D. Lam, N. Ng, S. Lee, G. Batzer, and A. A. Horner Airway House Dust Extract Exposures Modify Allergen-Induced Airway Hypersensitivity Responses by TLR4-Dependent and Independent Pathways J. Immunol., August 15, 2008; 181(4): 2925 - 2932. [Abstract] [Full Text] [PDF]
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X. Han, Y. Fan, S. Wang, L. Jiao, H. Qiu, and X. Yang NK Cells Contribute to Intracellular Bacterial Infection-Mediated Inhibition of Allergic Responses J. Immunol., April 1, 2008; 180(7): 4621 - 4628. [Abstract] [Full Text] [PDF]
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 S. Ashino, D. Wakita, Y. Zhang, K. Chamoto, H. Kitamura, and T. Nishimura CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen-specific Th2-cell migration into lung Int. Immunol., February 1, 2008; 20(2): 259 - 266. [Abstract] [Full Text] [PDF]
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 J. N. Kline Eat Dirt: CpG DNA and Immunomodulation of Asthma Proceedings of the ATS, July 1, 2007; 4(3): 283 - 288. [Abstract] [Full Text] [PDF]
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 P. Camateros, M. Tamaoka, M. Hassan, R. Marino, J. Moisan, D. Marion, M.-C. Guiot, J. G. Martin, and D. Radzioch Chronic Asthma-induced Airway Remodeling Is Prevented by Toll-like Receptor-7/8 Ligand S28463 Am. J. Respir. Crit. Care Med., June 15, 2007; 175(12): 1241 - 1249. [Abstract] [Full Text] [PDF]
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 M. Zhang, T. Angata, J. Y. Cho, M. Miller, D. H. Broide, and A. Varki Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils Blood, May 15, 2007; 109(10): 4280 - 4287. [Abstract] [Full Text] [PDF]
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D. H. Lim, J. Y. Cho, M. Miller, K. McElwain, S. McElwain, and D. H. Broide Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice. Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L265 - L271. [Abstract] [Full Text] [PDF]
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J. E. Korf, G. Pynaert, K. Tournoy, T. Boonefaes, A. Van Oosterhout, D. Ginneberge, A. Haegeman, J. A. Verschoor, P. De Baetselier, and J. Grooten Macrophage Reprogramming by Mycolic Acid Promotes a Tolerogenic Response in Experimental Asthma Am. J. Respir. Crit. Care Med., July 15, 2006; 174(2): 152 - 160. [Abstract] [Full Text] [PDF]
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G. M. Gauvreau, E. M. Hessel, L.-P. Boulet, R. L. Coffman, and P. M. O'Byrne Immunostimulatory Sequences Regulate Interferon-inducible Genes but not Allergic Airway Responses Am. J. Respir. Crit. Care Med., July 1, 2006; 174(1): 15 - 20. [Abstract] [Full Text] [PDF]
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X. Han, S. Wang, Y. Fan, J. Yang, L. Jiao, H. Qiu, and X. Yang Chlamydia Infection Induces ICOS Ligand-Expressing and IL-10-Producing Dendritic Cells That Can Inhibit Airway Inflammation and Mucus Overproduction Elicited by Allergen Challenge in BALB/c Mice J. Immunol., May 1, 2006; 176(9): 5232 - 5239. [Abstract] [Full Text] [PDF]
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J. Moisan, P. Camateros, T. Thuraisingam, D. Marion, H. Koohsari, P. Martin, M. L. Boghdady, A. Ding, M. Gaestel, M. C. Guiot, et al. TLR7 ligand prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2-independent pathway Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L987 - L995. [Abstract] [Full Text] [PDF]
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 M Peters, M Kauth, J Schwarze, C Korner-Rettberg, J Riedler, D Nowak, C Braun-Fahrlander, E von Mutius, A Bufe, O Holst, et al. Inhalation of stable dust extract prevents allergen induced airway inflammation and hyperresponsiveness Thorax, February 1, 2006; 61(2): 134 - 139. [Abstract] [Full Text] [PDF]
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 E. M. Hessel, M. Chu, J. O. Lizcano, B. Chang, N. Herman, S. A. Kell, M. Wills-Karp, and R. L. Coffman Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction J. Exp. Med., December 5, 2005; 202(11): 1563 - 1573. [Abstract] [Full Text] [PDF]
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 F Obermeier, U G Strauch, N Dunger, N Grunwald, H C Rath, H Herfarth, J Scholmerich, and W Falk In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis Gut, October 1, 2005; 54(10): 1428 - 1436. [Abstract] [Full Text] [PDF]
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T. Hayashi, X. Gong, C. Rossetto, C. Shen, K. Takabayashi, V. Redecke, H. Spiegelberg, D. Broide, and E. Raz Induction and Inhibition of the Th2 Phenotype Spread: Implications for Childhood Asthma J. Immunol., May 1, 2005; 174(9): 5864 - 5873. [Abstract] [Full Text] [PDF]
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 F. Tayyari, T. C. Sutton, H. E. Manson, and R. G. Hegele CpG-oligodeoxynucleotides inhibit RSV-enhanced allergic sensitisation in guinea pigs Eur. Respir. J., February 1, 2005; 25(2): 295 - 302. [Abstract] [Full Text] [PDF]
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H. Revets, G. Pynaert, J. Grooten, and P. De Baetselier Lipoprotein I, a TLR2/4 Ligand Modulates Th2-Driven Allergic Immune Responses J. Immunol., January 15, 2005; 174(2): 1097 - 1103. [Abstract] [Full Text] [PDF]
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C. J. Youn, M. Miller, K. J. Baek, J. W. Han, J. Nayar, S. Y. Lee, K. McElwain, S. McElwain, E. Raz, and D. H. Broide Immunostimulatory DNA Reverses Established Allergen-Induced Airway Remodeling J. Immunol., December 15, 2004; 173(12): 7556 - 7564. [Abstract] [Full Text] [PDF]
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M. V. Fanucchi, E. S. Schelegle, G. L. Baker, M. J. Evans, R. J. McDonald, L. J. Gershwin, E. Raz, D. M. Hyde, C. G. Plopper, and L. A. Miller Immunostimulatory Oligonucleotides Attenuate Airways Remodeling in Allergic Monkeys Am. J. Respir. Crit. Care Med., December 1, 2004; 170(11): 1153 - 1157. [Abstract] [Full Text] [PDF]
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H. J. de Heer, H. Hammad, T. Soullie, D. Hijdra, N. Vos, M. A.M. Willart, H. C. Hoogsteden, and B. N. Lambrecht Essential Role of Lung Plasmacytoid Dendritic Cells in Preventing Asthmatic Reactions to Harmless Inhaled Antigen J. Exp. Med., July 6, 2004; 200(1): 89 - 98. [Abstract] [Full Text] [PDF]
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 R. A. Joachim, V. Sagach, D. Quarcoo, Q. T. Dinh, P. C. Arck, and B. F. Klapp Neurokinin-1 Receptor Mediates Stress-Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness in Mice Psychosom Med, July 1, 2004; 66(4): 564 - 571. [Abstract] [Full Text] [PDF]
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S. Basu and M. J. Fenton Toll-like receptors: function and roles in lung disease Am J Physiol Lung Cell Mol Physiol, May 1, 2004; 286(5): L887 - L892. [Abstract] [Full Text] [PDF]
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 J. Y. Cho, M. Miller, K. J. Baek, J. W. Han, J. Nayar, M. Rodriguez, S. Y. Lee, K. McElwain, S. McElwain, E. Raz, et al. Immunostimulatory DNA Inhibits Transforming Growth Factor-{beta} Expression and Airway Remodeling Am. J. Respir. Cell Mol. Biol., May 1, 2004; 30(5): 651 - 661. [Abstract] [Full Text] [PDF]
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V. Redecke, H. Hacker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, and E. Raz Cutting Edge: Activation of Toll-Like Receptor 2 Induces a Th2 Immune Response and Promotes Experimental Asthma J. Immunol., March 1, 2004; 172(5): 2739 - 2743. [Abstract] [Full Text] [PDF]
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O. Duramad, K. L. Fearon, J. H. Chan, H. Kanzler, J. D. Marshall, R. L. Coffman, and F. J. Barrat IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences Blood, December 15, 2003; 102(13): 4487 - 4492. [Abstract] [Full Text] [PDF]
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R. K. Ikeda, M. Miller, J. Nayar, L. Walker, J. Y. Cho, K. McElwain, S. McElwain, E. Raz, and D. H. Broide Accumulation of Peribronchial Mast Cells in a Mouse Model of Ovalbumin Allergen Induced Chronic Airway Inflammation: Modulation by Immunostimulatory DNA Sequences J. Immunol., November 1, 2003; 171(9): 4860 - 4867. [Abstract] [Full Text] [PDF]
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P. P. Ho, P. Fontoura, P. J. Ruiz, L. Steinman, and H. Garren An Immunomodulatory GpG Oligonucleotide for the Treatment of Autoimmunity via the Innate and Adaptive Immune Systems J. Immunol., November 1, 2003; 171(9): 4920 - 4926. [Abstract] [Full Text] [PDF]
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V. Smart, P. S. Foster, M. E. Rothenberg, T. J. V. Higgins, and S. P. Hogan A Plant-Based Allergy Vaccine Suppresses Experimental Asthma Via an IFN-{gamma} and CD4+CD45RBlow T Cell-Dependent Mechanism J. Immunol., August 15, 2003; 171(4): 2116 - 2126. [Abstract] [Full Text] [PDF]
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E. S. Silverman and J. M. Drazen Immunostimulatory DNA for Asthma: Better than Eating Dirt Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 645 - 647. [Full Text] [PDF]
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R. K. Ikeda, J. Nayar, J. Y. Cho, M. Miller, M. Rodriguez, E. Raz, and D. H. Broide Resolution of Airway Inflammation following Ovalbumin Inhalation: Comparison of ISS DNA and Corticosteroids Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 655 - 663. [Abstract] [Full Text] [PDF]
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K. Takabayashi, L. Libet, D. Chisholm, J. Zubeldia, and A. A. Horner Intranasal Immunotherapy Is More Effective Than Intradermal Immunotherapy for the Induction of Airway Allergen Tolerance in Th2-Sensitized Mice J. Immunol., April 1, 2003; 170(7): 3898 - 3905. [Abstract] [Full Text] [PDF]
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 H. A. Boushey New and Exploratory Therapies for Asthma Chest, March 1, 2003; 123(2007): 439S - 445S. [Full Text] [PDF]
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Y. Wang and A. M. Krieg Synergy between CpG- or non-CpG DNA and specific antigen for B cell activation Int. Immunol., February 1, 2003; 15(2): 223 - 231. [Abstract] [Full Text] [PDF]
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B. K. Choudhury, J. S. Wild, R. Alam, D. M. Klinman, I. Boldogh, N. Dharajiya, W. J. Mileski, and S. Sur In Vivo Role of p38 Mitogen-Activated Protein Kinase in Mediating the Anti-inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma J. Immunol., November 15, 2002; 169(10): 5955 - 5961. [Abstract] [Full Text] [PDF]
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 K. Kitagaki, V. V. Jain, T. R. Businga, I. Hussain, and J. N. Kline Immunomodulatory Effects of CpG Oligodeoxynucleotides on Established Th2 Responses Clin. Vaccine Immunol., November 1, 2002; 9(6): 1260 - 1269. [Abstract] [Full Text] [PDF]
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Y.-H. Rha, C. Taube, A. Haczku, A. Joetham, K. Takeda, C. Duez, M. Siegel, M. K. Aydintug, W. K. Born, A. Dakhama, et al. Effect of Microbial Heat Shock Proteins on Airway Inflammation and Hyperresponsiveness J. Immunol., November 1, 2002; 169(9): 5300 - 5307. [Abstract] [Full Text] [PDF]
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 R. B. Pyles, D. Higgins, C. Chalk, A. Zalar, J. Eiden, C. Brown, G. Van Nest, and L. R. Stanberry Use of Immunostimulatory Sequence-Containing Oligonucleotides as Topical Therapy for Genital Herpes Simplex Virus Type 2 Infection J. Virol., October 11, 2002; 76(22): 11387 - 11396. [Abstract] [Full Text] [PDF]
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E. W. Gelfand Mice Are a Good Model of Human Airway Disease Am. J. Respir. Crit. Care Med., July 1, 2002; 166(1): 5 - 6. [Full Text] [PDF]
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J. N. Kline, K. Kitagaki, T. R. Businga, and V. V. Jain Treatment of established asthma in a murine model using CpG oligodeoxynucleotides Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L170 - L179. [Abstract] [Full Text] [PDF]
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 M. Zheng, D. M. Klinman, M. Gierynska, and B. T. Rouse DNA containing CpG motifs induces angiogenesis PNAS, June 25, 2002; 99(13): 8944 - 8949. [Abstract] [Full Text] [PDF]
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D. H. Broide, M. Miller, D. Castaneda, J. Nayar, J. Y. Cho, M. Roman, L. G. Ellies, and P. Sriramarao Core 2 oligosaccharides mediate eosinophil and neutrophil peritoneal but not lung recruitment Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L259 - L266. [Abstract] [Full Text] [PDF]
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H. Renz and U. Herz The bidirectional capacity of bacterial antigens to modulate allergy and asthma Eur. Respir. J., January 1, 2002; 19(1): 158 - 171. [Abstract] [Full Text] [PDF]
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Z. K. Ballas, A. M. Krieg, T. Warren, W. Rasmussen, H. L. Davis, M. Waldschmidt, and G. J. Weiner Divergent Therapeutic and Immunologic Effects of Oligodeoxynucleotides with Distinct CpG Motifs J. Immunol., November 1, 2001; 167(9): 4878 - 4886. [Abstract] [Full Text] [PDF]
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M. Hertz, S. Mahalingam, I. Dalum, S. Klysner, J. Mattes, A. Neisig, S. Mouritsen, P. S. Foster, and A. Gautam Active Vaccination Against IL-5 Bypasses Immunological Tolerance and Ameliorates Experimental Asthma J. Immunol., October 1, 2001; 167(7): 3792 - 3799. [Abstract] [Full Text] [PDF]
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I. Gursel, M. Gursel, K. J. Ishii, and D. M. Klinman Sterically Stabilized Cationic Liposomes Improve the Uptake and Immunostimulatory Activity of CpG Oligonucleotides J. Immunol., September 15, 2001; 167(6): 3324 - 3328. [Abstract] [Full Text] [PDF]
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K. H. Baek, S. J. Ha, and Y. C. Sung A Novel Function of Phosphorothioate Oligodeoxynucleotides as Chemoattractants for Primary Macrophages J. Immunol., September 1, 2001; 167(5): 2847 - 2854. [Abstract] [Full Text] [PDF]
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B. E. Britigan, T. S. Lewis, M. Waldschmidt, M. L. McCormick, and A. M. Krieg Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells J. Immunol., September 1, 2001; 167(5): 2921 - 2928. [Abstract] [Full Text] [PDF]
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M. Miller, K.-L. P. Sung, W. A. Muller, J. Y. Cho, M. Roman, D. Castaneda, J. Nayar, T. Condon, J. Kim, P. Sriramarao, et al. Eosinophil Tissue Recruitment to Sites of Allergic Inflammation in the Lung Is Platelet Endothelial Cell Adhesion Molecule Independent J. Immunol., August 15, 2001; 167(4): 2292 - 2297. [Abstract] [Full Text] [PDF]
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S. D. Hurst, B. W. P. Seymour, T. Muchamuel, V. P. Kurup, and R. L. Coffman Modulation of Inhaled Antigen-Induced IgE Tolerance by Ongoing Th2 Responses in the Lung J. Immunol., April 15, 2001; 166(8): 4922 - 4930. [Abstract] [Full Text] [PDF]
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B. Bohle, L. Orel, D. Kraft, and C. Ebner Oligodeoxynucleotides Containing CpG Motifs Induce Low Levels of TNF-{{alpha}} in Human B Lymphocytes: Possible Adjuvants for Th1 Responses J. Immunol., March 15, 2001; 166(6): 3743 - 3748. [Abstract] [Full Text] [PDF]
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D. H. Broide, G. Stachnick, D. Castaneda, J. Nayar, and P. Sriramarao Inhibition of Eosinophilic Inflammation in Allergen-Challenged TNF Receptor p55/p75- and TNF Receptor p55-Deficient Mice Am. J. Respir. Cell Mol. Biol., March 1, 2001; 24(3): 304 - 311. [Abstract] [Full Text] [PDF]
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S. Jilek, C. Barbey, F. Spertini, and B. Corthesy Antigen-Independent Suppression of the Allergic Immune Response to Bee Venom Phospholipase A2 by DNA Vaccination in CBA/J Mice J. Immunol., March 1, 2001; 166(5): 3612 - 3621. [Abstract] [Full Text] [PDF]
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D. Verthelyi, K. J. Ishii, M. Gursel, F. Takeshita, and D. M. Klinman Human Peripheral Blood Cells Differentially Recognize and Respond to Two Distinct CpG Motifs J. Immunol., February 15, 2001; 166(4): 2372 - 2377. [Abstract] [Full Text] [PDF]
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F.-G. Zhu and J. S. Marshall CpG-containing oligodeoxynucleotides induce TNF-{alpha} and IL-6 production but not degranulation from murine bone marrow-derived mast cells J. Leukoc. Biol., February 1, 2001; 69(2): 253 - 262. [Abstract] [Full Text]
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H. T. Maecker, G. Hansen, D. M. Walter, R. H. DeKruyff, S. Levy, and D. T. Umetsu Vaccination with Allergen-IL-18 Fusion DNA Protects Against, and Reverses Established, Airway Hyperreactivity in a Murine Asthma Model J. Immunol., January 15, 2001; 166(2): 959 - 965. [Abstract] [Full Text] [PDF]
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Z. Peng, H. Wang, X. Mao, K. T. HayGlass, and F. E. R. Simons CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice Int. Immunol., January 1, 2001; 13(1): 3 - 11. [Abstract] [Full Text] [PDF]
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T.-J. Huang, P. A. MacAry, P. Eynott, A. Moussavi, K. C. Daniel, P. W. Askenase, D. M. Kemeny, and K. F. Chung Allergen-Specific Th1 Cells Counteract Efferent Th2 Cell-Dependent Bronchial Hyperresponsiveness and Eosinophilic Inflammation Partly Via IFN-{{gamma}} J. Immunol., January 1, 2001; 166(1): 207 - 217. [Abstract] [Full Text] [PDF]
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D. Serebrisky, A. A. Teper, C.-K. Huang, S.-Y. Lee, T.-F. Zhang, B. H. Schofield, M. Kattan, H. A. Sampson, and X.-M. Li CpG Oligodeoxynucleotides Can Reverse Th2-Associated Allergic Airway Responses and Alter the B7.1/B7.2 Expression in a Murine Model of Asthma J. Immunol., November 15, 2000; 165(10): 5906 - 5912. [Abstract] [Full Text] [PDF]
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 D. Miyazaki, G. Liu, L. Clark, and S. J. Ono Prevention of Acute Allergic Conjunctivitis and Late-Phase Inflammation with Immunostimulatory DNA Sequences Invest. Ophthalmol. Vis. Sci., November 1, 2000; 41(12): 3850 - 3855. [Abstract] [Full Text]
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D. P. Sester, S. Naik, S. J. Beasley, D. A. Hume, and K. J. Stacey Phosphorothioate Backbone Modification Modulates Macrophage Activation by CpG DNA J. Immunol., October 15, 2000; 165(8): 4165 - 4173. [Abstract] [Full Text] [PDF]
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S. FUJIEDA, S. IHO, Y. KIMURA, H. YAMAMOTO, H. IGAWA, and H. SAITO Synthetic Oligodeoxynucleotides Inhibit IgE Induction in Human Lymphocytes Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 232 - 239. [Abstract] [Full Text]
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H. Shirota, K. Sano, T. Kikuchi, G. Tamura, and K. Shirato Regulation of Murine Airway Eosinophilia and Th2 Cells by Antigen-Conjugated CpG Oligodeoxynucleotides as a Novel Antigen-Specific Immunomodulator J. Immunol., June 1, 2000; 164(11): 5575 - 5582. [Abstract] [Full Text] [PDF]
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M. Castro, D. D. Chaplin, M. J. Walter, and M. J. Holtzman Could Asthma Be Worsened by Stimulating the T-helper Type 1 Immune Response? Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 143 - 146. [Full Text]
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H. Shirota, K. Sano, T. Kikuchi, G. Tamura, and K. Shirato Regulation of T-helper Type 2 Cell and Airway Eosinophilia by Transmucosal Coadministration of Antigen and Oligodeoxynucleotides Containing CpG Motifs Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 176 - 182. [Abstract] [Full Text]
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Y. Shibata, L. A. Foster, J. F. Bradfield, and Q. N. Myrvik Oral Administration of Chitin Down-Regulates Serum IgE Levels and Lung Eosinophilia in the Allergic Mouse J. Immunol., February 1, 2000; 164(3): 1314 - 1321. [Abstract] [Full Text] [PDF]
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G. Hansen, V. P. Yeung, G. Berry, D. T. Umetsu, and R. H. DeKruyff Vaccination with Heat-Killed Listeria as Adjuvant Reverses Established Allergen-Induced Airway Hyperreactivity and Inflammation: Role of CD8+ T Cells and IL-18 J. Immunol., January 1, 2000; 164(1): 223 - 230. [Abstract] [Full Text] [PDF]
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P. Parronchi, F. Brugnolo, F. Annunziato, C. Manuelli, S. Sampognaro, C. Mavilia, S. Romagnani, and E. Maggi Phosphorothioate Oligodeoxynucleotides Promote the In Vitro Development of Human Allergen-Specific CD4+ T Cells into Th1 Effectors J. Immunol., December 1, 1999; 163(11): 5946 - 5953. [Abstract] [Full Text] [PDF]
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G. Hartmann, G. J. Weiner, and A. M. Krieg CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells PNAS, August 3, 1999; 96(16): 9305 - 9310. [Abstract] [Full Text] [PDF]
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S. Sur, J. S. Wild, B. K. Choudhury, N. Sur, R. Alam, and D. M. Klinman Long Term Prevention of Allergic Lung Inflammation in a Mouse Model of Asthma by CpG Oligodeoxynucleotides J. Immunol., May 15, 1999; 162(10): 6284 - 6293. [Abstract] [Full Text] [PDF]
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