Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy
Ursula Wiedermann1,
Beatrice Jahn-Schmid1,2,
Marianne Lindblad3,
Carola Rask3,
Jan Holmgren3,
Dietrich Kraft1 and
Christof Ebner1
1 Division of Immunopathology, Institute of General and Experimental Pathology, University of Vienna, Waehringer Guertel 1820, 1090 Vienna, Austria
2 Centre of Ultrastructural Research, University of Agriculture, Vienna, Austria,
3 Department of Microbiology and Immunology, University of Göteborg, Göteborg, Sweden
Correspondence to:
U. Wiedermann
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Abstract
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Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain Th1-based autoimmune diseases. We have established a model of aerosol sensitization leading to Th2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence Th2-like immune responses. Intranasal administration of OVACTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4, but not IFN-
, production were markedly decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-
. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigensin conjugated as in unconjugated formhad different effects on the Th2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTBand probably also other antigen-delivery systemsstrongly depend on the nature of the coupled antigenallergen.
Keywords: aerosol, BALB/c, cholera B subunit, intranasal, immunomodulation, Th2 response
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Introduction
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It is well established that mucosal administration of soluble antigens induces systemic immunological unresponsiveness, a phenomenon known as mucosal tolerance (1,2). However, it has been recognized that the effectiveness of mucosal tolerance often requires repeated and large amounts of antigen, and sometimes the suppressed immune responses are of short duration. On the other hand, molecules with known mucosa-binding properties are known to induce local and systemic immune responses when administered by the mucosal routes (3). Among these, cholera toxin (CT) is one of the most potent mucosal immunogens, which, when administered simultaneously with an antigen by the mucosal route, enhances immune responses to the co-administered antigen (4). In contrast to cholera holotoxin, mucosal administration of only the non-toxic, mucosa-binding B subunit of CT (CTB), physically coupled to an antigen, has been recently recognized to enhance peripheral tolerance induction (5). Sun et al. demonstrated that a single feeding of antigen conjugated to CTB led to suppression of T cell responses in vivo and in vitro at doses 15- to 500-fold lower than at those of corresponding regimes using unconjugated antigens (5). In line with this finding further studies have demonstrated the effectiveness of such antigenCTB formulations in preventing or treating certain Th1-based diseases, such as experimental autoimmune encephalomyelitis (6) or spontaneous autoimmune diabetes (7). Recently, a similar transmucosal carrier-delivery system, the enterotoxin B of Escherichia coli conjugated to antigen, has been successfully used for suppression not only of antigen-specific T cell responses but also of antigen-specific IgE production (8). So far, this is the only study providing evidence that such a mucosal antigen-delivery system might be a promising strategy to suppress immune responses, based on excessive Th2 cell activity.
Type I allergy is very common in areas of the temperate climate zone and the prevalence of the disease has constantly increased within the last years (9). This genetically determined immunodisorder, manifested by symptoms like allergic rhinitis, conjuctivitis or allergic asthma, is based on the excessive production of IgE antibodies against allergenic molecules (10). The pathway of IgE regulation is well described for mice and humans, and essentially explained by the reciprocal activity of IL-4 and IL-5, mediating Th2 responses, and IFN-
, which antagonizes these effects (1113).
We have established a murine model of aerosol sensitization leading to allergen-specific Th2-mediated immune responses i.e. high IgE/IgG1 versus low IgG2a antibody levels and positive type I skin tests in vivo, an immunological state comparable to that of human type I allergy. Using this model system we have recently reported that simultaneous inhalation of allergen and cholera holotoxin can modulate the allergic immune response in naive as well as in sensitized animals (14).
In the present study we investigated, whether mucosal delivery of antigenCTB conjugates could also be successfully used to suppress type I allergic immune responses. Two different antigens were used for this purpose: ovalbumin (OVA), a frequently experimentally used dietary antigen, and recombinant Bet v 1, the major allergen of birch pollen (BP) and a clinically important inhalant allergen (15,16).
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Methods
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Animals
Female, 7-week-old, BALB/c mice were obtained from Charles River (Sulzfeld, Germany). All experiments were approved by the Animal Experimentation Ethics Committee of the University of Vienna and the Ministry of Science and Research.
Antigens
Recombinant Bet v 1 (rBet v 1) was purchased from Biomay (Linz, Austria).
BP was obtained from Allergon (Engelholm, Sweden) and used for the preparation of a BP extract as previously described (14).
Recombinant CTB was produced in a mutant strain of Vibrio cholerae deleted from the CTA subunit gene and transfected with a plasmid encoding CTB. CTB was purified by sequential precipitation and gel filtration chromatography (17,18). OVA (Sigma, St Louis, MO) and rBet v 1 were covalently bound to CTB at a molar ratio of 1:1 using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as bifunctional coupling reagent, according to the protocol described by Sun et al. (5). The resulting OVACTB and rBet v 1CTB conjugates were purified by gel filtration through a column of Sephacryl S-300. The conjugates were shown to retain GM1 binding capacity and serological activity of both OVA and rBet v 1 by means of solid-phase ELISA (19).
Nasal administration of CTB-coupled proteins
Mice (n = 6/group) were anesthetized and 40 µg/30 µl NaCl of the OVACTB (20 µg OVA conjugated to 20 µg CTB) or rBet v 1CTB (20 µg rBet v 1 coupled to 20 µg CTB) were intranasally applied. For control purposes corresponding amounts of unconjugated OVA or rBet v 1 or NaCl were intranasally applied to mice (n = 6/group). This procedure was performed at three occasions at 7 day intervals prior to sensitization (days 21, 14 and 7; Fig. 1
experimental design).

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Fig. 1. Experimental design. Two groups of control mice (n = 6/group) were either i.p. immunized with OVA/Al(OH)3 or with rBet v 1/Al(OH)3 and subsequently aerosolized with an 0.1% OVA or BP solution. Before sensitization 0.9% NaCl (sham-pretreatment) was intranasally applied. Four groups of mice (n = 6/group) were 3xintranasally treated with OVACTB, rBet v 1CTB, unconjugated OVA or unconjugated rBet v 1. Thereafter the mice were immunized with OVA or rBet v 1/BP as described for the control mice.
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Sensitization
Pretreated and sham-pretreated mice were once injected i.p. with 50 µg OVA or 1 µg rBet v 1 adsorbed to Al(OH)3(day 0). Fourteen days later aerosol exposure to a 0.1% OVA or BP solution was daily performed during a period of 2 times over 5 days (days 1418 and 2125) as previously described (14). The OVA and the BP solution were aerosolized using a nebulizer (nebulizer 646; Devilbiss, Sommerset, PA). The mice were exposed to 4 ml solution for 20 min/day (Fig. 1
, experimental design).
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Sampling
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Blood samples were taken before sensitization and 6 days after the last aerosol exposure. Serum was prepared and stored at 20° C until analyzed.
Bronchoalveolar lavage (BAL) was collected by lavaging the lungs with 1 ml PBS inserted through a small incision in the trachea of the sacrificed mice (14).
Immunoblots
Proteins were separated by SDSPAGE and transferred to nitrocellulose by electroblotting according to standard protocols (20,21). Sera from OVA and Bet v 1/BP immunized mice were incubated in 1/5 (IgE) and 1/100 (IgG1 and IgG2a) dilutions with nitrocellulose stripes coated with OVA or BP extract. Immunoblots were performed with rat anti-mouse IgE (1/250), IgG1 (1/500) or IgG2a (1/250; PharMingen, San Diego, CA) followed by radiolabeled sheep anti-rat Ig (1/1000; Amersham, Little Chalfont, UK).
Determination of antigen-specific antibody levels in serum and bronchial lavage
Microtiter plates (Nunc, Roskilde, Denmark) were coated with OVA (10 µg/ml carbonate buffer) and BP extract (50 µg/ml). Serum samples were diluted 1/1000 for IgG1, 1/500 for IgG2a and 1/10 for IgE; BAL was used undiluted. Rat anti-mouse IgG1, IgG2a, IgE and IgA antibodies (1 µg/ml; PharMingen) and thereafter peroxidase-conjugated mouse anti-rat IgG antibodies (1/1000; Jackson ImmunoResearch, PA) were used. Color development was performed as previously described (14).
Lymphocyte proliferation assay
At sacrifice, 7 days after the last aerosol exposure (day 32), spleens and lungs with associated lymph nodes were removed under sterile conditions. The organs were homogenized, erythrocytes were lysed, and the cells washed and resuspended in complete medium (RPMI, 10% FCS, 0.1 mg/ml gentamycin, 2 mM glutamine and 50 µM 2-mercaptoethanol). Suspensions of spleen cells as well as lung cells with associated lymph nodes were plated into 96-well round-bottom plates at a concentration of 2x105/200 µl/well and stimulated with and without concanavalin A (Con A; 0.5 µg/well), OVA (40 µg/well) or BP (5 µg/well) for 4 days. Thereafter the cultures were pulsed with 0.5 µCi/well [3H]thymidine (Amersham) for 16 h and the proliferative responses measured by scintillation counting.
Measurement of cytokine production in spleen and lung cell cultures
For determination of IL-5, IL-4, IL-10 and IFN-
production spleen and lung cells were cultured with and without Con A (2.5 µg/well), OVA (100 µg/well) and BP (25 µg/well) in 48-well plates (Costar, Cambridge, MA, USA) at a concentration of 5x106/0.5 ml/well. After 24 and 48 h supernatants were taken and stored at 20° C until analyzed.
The levels of IL-5, IL-4, IL-10 and IFN-
were measured with mouse ELISA kits (Endogene, Cambridge, MA). The sensitivity of the IL-5 and IL-4 assay was <5 pg/ml and of the IL-10 and IFN-
ELISA <15 pg/ml.
Statistics
For statistical analysis the MannWhithney U-test was used.
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Results
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Sensitization
Mice, immunized i.p. with OVA or Bet v 1 adsorbed to Al(OH)3 and subsequently exposed to an OVA or BP solution via aerosol, displayed high allergen-specific IgG1/IgE versus low IgG2a antibody levels. Figure 2
(A) demonstrates the binding of serum IgE, IgG1 and IgG2a antibodies from OVA-sensitized mice to the 40 kDa molecule. Mouse sera from Bet v 1/BP immunized mice only bound to the 17 kDa Bet v 1 molecule (Fig. 2B
). Therefore, all tests performed with BP extract reflect the immune reactivity to Bet v 1 exclusively.

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Fig. 2. Immunoblot, performed with pooled sera of mice immunized according to the standard immunization protocol, i.e. 1xi.p. injection of Al(OH)3 adsorbed OVA or Bet v 1, followed by aerosol exposure to 0.1% OVA or BP solution. (pre) Nitrocellulose stripes incubated with pooled preimmune sera. (OVA) Binding of serum IgE, IgG1 and IgG2a from OVA sensitized mice to the 40 kDa OVA molecule. (BP) Binding of serum IgE, IgG1 and IgG2a of Bet v 1/BP immunized mice to Bet v 1, the 17 kDa major BP allergen. Additional IgG1 binding to the 34 kDa Bet v 1 dimer.
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Mucosal administration of OVACTB conjugate leads to suppression whereas application of rBet v 1CTB conjugates augments Th2-like immune responses in vivo
Antigen-specific antibody levels in serum and BAL were measured in mice treated 3 times intranasally with OVACTB or unconjugated OVA or 3 times intranasally pretreated with rBet v 1CTB conjugates or unconjugated rBet v 1 and compared with those of OVA- or Bet v 1-sensitized control mice.
As shown in Fig. 3
(A) mucosal pretreatment with OVACTB conjugates led to a significant reduction of OVA-specific IgE antibody levels but simultaneously to a drastic increase of anti-OVA IgG2a antibody levels. IgG1 anti-OVA antibody levels (Fig. 3A
) as well as IgA anti-OVA levels in BAL did not differ between the sensitized (IgA-OD: 0.279 ± 0.23) and the nasally pretreated mice (IgA-OD: 0.219 ± 219). Intranasal administration of equivalent doses of unconjugated OVA could not markedly decrease antigen-specific IgE nor significantly increase IgG2a antibody levels (Fig. 3B
).

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Fig. 3. Serum IgE, IgG1 and IgG2a antibody responses against OVA or Bet v 1 measured by ELISA. (A) White bars represent the mean OD of six control mice immunized with OVA or Bet v 1/BP according to the standard protocol. Hatched bars represent the mean OD of six mice, intranasally treated with OVACTB or rBet v 1CTB prior to sensitization. (B) White bars represent the mean OD of six control mice immunized with OVA or Bet v 1/BP. Black bars represent the mean OD of six mice, intranasally treated with unconjugated OVA or unconjugated rBet v 1 prior to sensitization. Error bars show SEM. *P < 0.05, **P < 0.01, MannWhitney U-test.
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In total contrast to the pretreatment with OVACTB conjugates, intranasal administration of rBet v 1CTB conjugate prior to sensitization led to enhanced antibody responses of all isotypes examined. In particular, IgE, IgG1 and IgG2a antibodies against BP/Bet v 1 were significantly higher in the conjugate pretreated group as compared to the sensitized animals (P < 0.01, Fig. 3A
). A similar increase in allergen-specific IgA antibody levels in BAL was noted in the conjugate pretreated animals (OD: 1.0 ± 0.7) compared to the controls (OD: 0.087 ± 0.05). To investigate, if the enhanced immune responses were due to CTB, unconjugated rBet v 1 was intranasally applied before sensitization. This pretreatment led to a strong reduction of the allergen-specific antibody responses of all isotypes as shown in Fig. 3
(B).
Pretreatment with OVACTB conjugates leads to suppression, whereas rBet v 1CTB conjugates cause enhancement of antigen-specific lympho-proliferative responses in vitro
Antigen-specific lympho-proliferative responses were measured in lung and spleen cell cultures of mice pretreated with antigenCTB conjugate or unconjugated antigen and compared with cultures derived from sensitized animals. Lympho-proliferative responses in lung cells of OVACTB pretreated mice were reduced by 65% [stimulation index (SI): 2 ± 1.0] compared to the sensitized mice (SI: 5.7 ± 4.7, P < 0.05). An even stronger reduction (81%) of the lympho-proliferative response was noted in the group of mice pretreated with unconjugated OVA (SI: 2.1 ± 2.3) compared to the sensitized mice (SI: 11.3 ± 4.4, P < 0.02). However, in spleen cell cultures no differences in the antigen-specific proliferative responses were observed between the groups (Fig. 4
).

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Fig. 4. Relative levels of stimulation indices of responder cells from lung and spleen cell cultures, stimulated with OVA or BP antigen. The white bars represent the proliferative response of sensitized mice, hatched bars of antigenCTB pretreated mice and black bars of mice pretreated with unconjugated antigen prior to sensitization. The stimulation index of the sensitized mice was set to 100% and the stimulation indices of the pretreated groups were expressed as relative values.
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In contrast, intranasal administration of rBet v 1CTB prior to sensitization with BP led to a 273% increase of lympho-proliferative responses of lung cell cultures (SI: 5.6 ± 5.2) and a 163% enhanced response of spleen cells (SI: 5.1 ± SD 3.5) as compared to the sensitized animals (SI: 1.5 ± SD: 0.7 respectively SI: 1.9 ± SD: 0.3) (Fig. 4
).
However, intranasal administration of unconjugated Bet v 1 resulted in a 3744% reduction of allergen-specific lympho-proliferative responses in lung (SI: 1.5 ± 0.2 ) and spleen cells (SI: 2.0 ± 0.3 ) as compared to the sensitized animals (SI: 2.4 ± 0.9 respectively SI: 3.6 ± 1.7) (Fig. 4
).
Pretreatment with OVACTB decreases production of IL-5 and IL-4, whereas rBet v 1CTB leads to increased cytokine production in vitro
In vitro production of IL-5, IL-4, IL-10 and IFN-
was measured in spleen and lung cell cultures of sensitized and pretreated animals. Intranasal administration of OVACTB conjugates prior to sensitization led to reduced IL-5 and IL-4 production in cultures of pooled spleen and lung cells compared to the sensitized controls (Table 1
). IFN-
and IL-10 levels of did not markedly differ between the groups. No significant differences in IL-5 and IFN-
production were observed in cell cultures of mice pretreated with unconjugated OVA compared to OVA sensitized mice (Table 1
).
In contrast, pretreatment with rBet v 1CTB led to a drastic increase of IL-5, IL-4, IFN-
and IL-10 production in spleen and lung cell cultures compared to the cytokine levels measured in cultures of the sensitized control animals (Table 1
). This augmented immune response in vitro was consistent with the enhanced allergen-specific antibody responses in vivo (Fig. 3
).
The opposite effects were obtained from mice intranasally treated with unconjugated Bet v 1 prior to sensitization. In line with the suppressed antibody production (Fig. 4
) also the in vitro production of IL-5, IL-4, IL-10 and IFN-
was markedly reduced compared to that of the sensitized animals (Table 1
).
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Discussion
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In the present study we demonstrated that the use of antigenCTB conjugates had opposite effects on Th2-like immune responses depending on the antigen coupled to CTB. Intranasal administration of OVACTB conjugates prior to sensitization suppressed, whereas administration of rBet v 1CTB conjugates stimulated immune responses in vivo and in vitro.
Previous reports have demonstrated that mucosal administration of soluble antigens conjugated to CTB is more effective than that of unconjugated antigens to induce peripheral tolerance in naive as well as sensitized animals. These studies, mainly performed in models of Th1- mediated diseases, consistently reported about the ability of antigenCTB conjugates to reduce delayed-type hypersensitivity reactions or T cell responses in vitro (5,6,7,22).
Less data exist about the suppressive effects on Th2-like immune responses. A recent study showed that CTB conjugated to the Leishmania antigen LACK or OVA selectively reduced Th1 responses (IFN-
production), whereas Th2 responses (IL-4/IL-5 production) remained unaffected by this treatment. It was therefore suggested that the selective tolerance of Th1 cells was due to a common effect of CTB rather than an antigen-specific effect of the proteins conjugated to CTB. The authors concluded that such therapeutical strategies might therefore preferentially be used for treatment of Th1-based immunopathological situations (22). However, another study recently reported that a similar antigen delivery systemthe bacterial product LTB coupled to OVAcould suppress both Th1 and Th2 responses, as demonstrated in a decreased delayed-type hypersensitivity reaction as well as suppressed OVA-specific IgE antibody responses (8).
In the present study we used our murine model of aerosol sensitization to investigate the potential of two different antigens, the dietary antigen OVA and the inhalant allergen Bet v 1, coupled to CTB for their potential to affect Th2-like immune responses. Our present data demonstrated that intranasal pretreatment with OVACTB conjugates led to a significant reduction of OVA-specific IgE antibody levels. We also noted a significant increase in antigen-specific IgG2a antibody production, indicating the capacity to drive the immune response towards a Th1-like response (Fig. 3A
). The reciprocal activities of IL-5/IL-4crucial for induction of Th2 responsesand IFN-
known to control the production of IgG2a in the murine systemis well documented (12,23). In accordance with the antibody responses the in vitro synthesis of IL-5/IL-4, but not of IFN-
, was markedly decreased in lung and spleen cell cultures of OVACTB pretreated mice (Table 1
). This immunomodulating effect was due to the CTB component of the conjugate, since intranasal application of comparable amounts of unconjugated OVA could not reduce IgE or enhance IgG2a antibody levels (Fig. 3B
), nor significantly modulate the cytokine production profile (Table 1
). Our data indicates that Th2 responses can be influenced by intranasal administration of OVACTB. This is in contrast to the study of McSorely et al., reporting that pretreatment with OVACTB conjugates via the same route failed to affect Th2 immune responses (22).
Notably, using rBet v 1CTB conjugates under identical experimental conditions resulted in a general stimulation of allergen-specific antibody production of all isotypes in serum (Fig. 3A
) and a concomitant increase of IgA in BAL. T cell proliferative responses and the cytokine production in vitro (IL-5, IL-4, IFN-
and IL-10) were also significantly enhanced in cell cultures (Fig. 4
and Table 1
). In contrast to Bet v 1 coupled to CTB, intranasal application of unconjugated Bet v 1 led to a significant reduction of antibody responses of all isotypes (Fig. 3B
), T cell proliferation (Fig. 3B
) as well as cytokine production in vitro (Table 1
). These observations demonstrated that, in the case of Bet v 1, CTB acted as an efficient adjuvant.
It has been demonstrated that the method of conjugation can influence the effect of such a transmucosal delivery system in terms of suppression or stimulation of the immune responses. These different effects have been explained by possible alterations of the antigen structure due to different methods of conjugation (8,24). Since we used the same method of conjugation for both antigens, our study indicates that the nature of the antigen per se influenced the quality of the immune response. It has been suggested that certain allergens have intrinsic abilitiesin addition to acting as an immunogento intervene with regulatory processes involved in the development of allergy. Such intrinsic activities can be due to enzymatic properties (2527) and/or certain molecular/structural features of the allergen. In the latter respect, Bet v 1 shows conformational epitopes, which can be destroyed by producing two parts of the molecule (28), whereas OVA is known to possess only continuous epitopes (29). These structural differences of the two molecules might have been responsible for the different outcome of the treatment with conjugated as well as unconjugated Bet v 1 compared to OVA.
In conclusion, considering CTB as a potential antigen-delivery system in Th2-based immunopathological conditionssuch as type I allergythe above described formulation might only be successfully applicable with certain allergens. Consequently, the effects of novel adjuvants/antigen-delivery systems cannot be generalized based on experiments performed with model antigens, but need to be individually evaluated.
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Acknowledgments
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The excellent technical assistance of Ms Renate Steiner-Göltl is very much appreciated. The study was supported by grants from the Austrian Science Foundation (P 12889, S 7206-MOB), ALK, Copenhagen, Denmark and the Swedish Medical Research Council.
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Abbreviations
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BAL | bronchoalveolar lavage |
BP | birch pollen |
Bet v 1 | major birch pollen allergen |
Con A | concanavalin A |
CT | cholera toxin |
CTB | cholera toxin B subunit |
OVA | ovalbumin |
r | recombinant |
SI | stimulation index |
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Notes
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Transmitting editor: A. Radbruch
Received 22 December 1998,
accepted 25 March 1999.
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