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*
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115; and
Department of Otolaryngology, Okayama University Medical School, Okayama, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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following
in vitro stimulation with HSA or HSA-LNFPIII. In addition, these
activated nasal lymphocytes also showed a significant increase of B7-2
expression on B220-positive cells. Furthermore, not only intranasal but
also both i.p. and s.c. immunization with HSA-LNFPIII induced
significant production of HSA-specific Abs compared with the
immunization with HSA alone, suggesting that the activity of LNFPIII
was not restricted on particular route of immunization. These results
demonstrate that Lewis type carbohydrate LNFPIII can function as an
adjuvant by their ability to induce a Th2
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently demonstrated that Schistosoma mansoni egg Ag (SEA)3 induces a strong Th2-associated cytokine and Ab responses in BALB/c mice following intranasal immunization (7). Conversely, we also found that deglycosylation of SEA by periodate treatment significantly abrogates its ability to induce a Th2 response (8). These findings indicate that carbohydrates on SEA are required for in vivo induction of Th2 response in this model. Recent studies have demonstrated that lacto-N-fucopentaose III (LNFPIII) is the predominant carbohydrate component of SEA that activates B and B-1 cells to produce IL-10 and PGE2 (9, 10). LNFPIII also binds ICAM-1, P-, L-, and E-selectins and plays a critical role in embryonic development and differentiation (11). Moreover, LNFPIII is also expressed by cancer cells and considered as a risk factor for tumor metastasis (12). Nevertheless, this oligosaccharide is also found in human milk (13).
The purpose of this study was to investigate the role of LNFPIII as an adjuvant by its ability to induce a Th2-like response. To approach this question, we immunized BALB/c mice intranasally, i.p., and s.c. with a protein Ag human serum albumin (HSA) conjugated to LNFPIII (HSA-LNFPIII). Our results demonstrate that LNFPIII is a potent inducer of a Th2 response and can also act as an adjuvant by inducing Ab production against coupled protein Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven- to 9-wk-old female BALB/c and C57BL/6 mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were maintained in the specific pathogen-free facility at Harvard School of Public Health according to the guidelines for animal research.
Antigens
HSA (fraction V) and BSA (fraction V) were purchased from Sigma
(St. Louis, MO). LNFPIII, lacto-N-neotetraose (LNnT, a
nonfucosylated homologue of LNFIII), LNFPIII conjugated to HSA
(HSA-LNFPIII) and LNnT conjugated to HSA (HSA-LNnT) were purchased from
Accurate Chemical and Scientific (Westbury, NY). Both LNFPIII-HSA and
LNnT-HSA contain 
13 mol of carbohydrates conjugated to 1 mol of HSA.
Lacto-N-fucopentaose I (LNFPI) conjugated to HSA (HSA-LNFPI)
was also obtained from Accurate Chemical and Scientific. LNFPIII
conjugated to BSA (BSA-LNFPIII) was purchased from Dextra Laboratories
(Reading, U.K.).
Immunization
Groups of four mice were immunized intranasally with HSA, HSA-LNFPIII, HSA-LNnT, or HSA-LNFPI in Dulbecco’s PBS (Life Technologies, Grand Island, NY) at several protein concentrations, or Dulbecco’s PBS (Life Technologies) as a control in a total volume of 20 µl. Two weeks later, mice were boosted by intranasal immunization using the same dose of Ag used for priming. Later the mice were challenged daily intranasally using 1/10 dose of Ag from day 22 to 28. Serum samples were obtained by tail bleeds for Ab determination after the prime, boost, and sixth challenge at day 13, 21, and 28, respectively. Twelve hours after the last challenge, mice were sacrificed using carbon dioxide, and nasal lymphocytes were isolated by enzyme extraction with collagenase (Boehringer Mannheim, Indianapolis, IN) as described previously (14).
In addition, groups of four mice were immunized i.p. or s.c. with 10 µg (protein) of these Ags in the presence or absence of alum (1 mg) and boosted 2 wk later via same immunization routes and similar Ag dose. One week following the boosting sensitization, serum was collected for Ab determination by ELISA.
Ag-specific IgM, IgG, IgG1, IgG2a, and IgA determination
Serum levels of Ag-specific Ab were determined by ELISA as described previously (7). In brief, ELISA plates (Corning Glass, Corning, NY) were coated with 100 ml of Ag (2 µg/ml) overnight at 4°C in carbonate-bicarbonate buffer (pH 9.6), then blocked with 10% PBS containing 10% FCS for 2 h at 37°C. Individual serum samples were then plated in duplicate in 2-fold serial dilution beginning at 1/100 and incubated for 2 h at 37°C. Plates were washed with PBS and 0.05% Tween 20 and bound Ab were detected by incubation with peroxidase-conjugated goat anti-mouse Ig for 1 h at 37°C. Optimum dilutions of Ab to mouse IgM, IgG, and IgG subclasses (Boehringer Mannheim) and IgA (BioSource International, Camarillo, CA) were 1/10,000, 1/10,000, 1/2,000, and 1/2,000 respectively. After addition of tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD), the reaction was stopped by addition of 50 µl phosphoric acid (5.0%) and the absorbance at 450 nm was read using an automated plate reader (Molecular Devices, Menlo Park, CA). Results were expressed as endpoint titers where the endpoint equals the final serum dilution yielding an absorbance twice background.
Serum total and Ag-specific IgE
Total and Ag-specific IgE in sera were determined by ELISA as described previously (7). Briefly, ELISA plates were coated with 100 µl rat anti-mouse IgE (5 µg/ml) in carbonate-bicarbonate buffer overnight at 4°C. Plates were blocked and washed as described above, then samples of serially diluted serum or mouse IgE standard were plated in duplicate and incubated for 2 h at 37°C. Biotinylated monoclonal rat anti-mouse IgE (PharMingen, San Diego, CA) (0.5 µg/ml) or biotinylated Ag (1 µg/ml) was then added to wells for 2 h at 37°C for detection of total and Ag-specific IgE, respectively. After washing the plates with PBS and 0.05% Tween 20, peroxidase-conjugated streptavidin (1/1000 dilution; Sigma) was added to the wells and incubated for 1 h at 37°C. The color was developed by adding TMB substrate and the reaction was stopped as described above. For biotinylation, HSA (1.8 mg/ml) in sodium bicarbonate buffer (pH 8.5) was incubated with biotin (long arm) N-hydroxy succumide ester (Vector Laboratories, Burlingame, CA) for 2 h at room temperature. The reaction was stopped by adding 5 µl of ethanolamine (Sigma) and dialyzed overnight with PBS containing 0.05% sodium azide.
Competitive inhibition ELISA
The competitive ELISA for Ag-specific IgG that we previously described was followed (15). Briefly, ELISA plates were coated with HSA-LNFPIII overnight at 4°C. Plates were washed with PBS/0.05% Tween 20, and blocked with 10% FCS/PBS at 37°C for 1 h. Serum samples were diluted 500-fold and mixed with different doses of HSA or monovalent LNFPIII as inhibitors. Following incubation for 15 min at room temperature, samples were added to the Ag-coated plate in duplicate and incubated for 2 h at 37°C. Plates were washed with PBS/0.05% Tween 20, and peroxidase-conjugated goat anti-mouse IgG was added to the wells for 1 h at 37°C. Levels of bound Ab were determined by addition of TMB substrate and stopped by addition of acid as described. Results were presented as the average OD at 450 nm ± SEM of the percent of response from sera of four individual mice with inhibitors added divided by the response without inhibitors.
In vitro culture of nasal lymphocytes
Nasal lymphocytes from naive or Ag-primed BALB/c mice were isolated by enzyme extraction with collagenase (14). Cell suspensions containing 2 x 106 lymphocytes per milliliter were cultured with or without Ag (5 µg/ml) for 48 h at 37°C in RPMI 1640 medium (Life Technologies) containing 10% FCS, 5 x 10-5 M 2-ME (Sigma), and 100 U/ml and 100 µg/ml penicillin/streptomycin (Sigma) in flat-bottom 48-well plates (Corning Glass). Cell supernatants were collected and stored at -80°C until assayed. Pelleted cells were resuspended then stained with mAbs coupled to FITC or PE for flow cytometry analysis.
Cytokine determination
Levels of IL-4, IL-5, IL-10, and IFN- in the above culture
supernatants were measured by captured ELISA as described previously
(8). Briefly, Maxisorp multititer 96-well plates (Nunc,
Roskilde, Denmark) were coated overnight at 4°C with 2 µg/ml of
capture Ab (rat anti-mouse IL-5, IL-10, and IFN- from PharMingen
and rat anti-mouse IL-4 from Endogen (Woburn, MA) in Tris-HCl
buffer at pH 9.0. The plates were blocked for 1 h at 37°C with
10% FCS in PBS after which murine recombinant cytokine standards IL-4
(0–860 pg/ml; Endogen), IL-5 (0–5,000 pg/ml; PharMingen), IL-10
(0–25,000 pg/ml; PharMingen), or IFN- (0–20,000 pg/ml; PharMingen)
and the culture supernatants were added in duplicate and incubated
overnight at 4°C. The plates were washed three times in PBS/Tween 20
and incubated for 1 h at 37°C with 1 µg/ml appropriate
biotinylated detection Ab. (rat anti-mouse IL-5, IL-10, and IFN-
from PharMingen, and rat anti-mouse IL-4 from Endogen). To detect
biotinylated Abs, streptavidin-linked alkaline phosphotase (1/2,000
dilution in 10% FCS in PBS; PharMingen) was added and incubated for 45
min in the dark at 37°C. After a final washing in PBS/Tween 20,
p-nitrophenylphophotase substrate (Sigma) in glycine buffer
was added to each well. The absorbance was read at 405 nm using an
automated microplate reader (Molecular Devices), and the concentration
of the samples was calculated using the standard curve.
Flow cytometry
Nasal lymphocytes (1–2 x 105) were incubated on ice for 15 min with the following FITC- or PE-conjugated mAbs (PharMingen): anti-CD45R/B220 (RA3-6B2), anti-B7-1 (16-10A1), and anti-B7-2 (GL-1) or isotype-matched controls. Then cells were washed with cold HBSS containing 0.05% sodium azide. Flow cytometry analysis was performed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA). Dead cells were excluded from analysis using propidium iodide (Molecular Probes, Eugene, OR) staining. Lymphocytes were gated according to forward and side scatter and at least 10,000 events were acquired and analyzed.
Statistical analysis
Data are expressed the mean ± SEM for each subject group. Statistical analysis was performed using the Student’s unpaired t test. Differences in Ab endpoint titers were determined using the Mann-Whitney U prime test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following intranasal immunization with HSA-LNFPIII, BALB/c mice
displayed a significant increase in HSA-specific IgG production as
compared with those immunized with HSA alone (Fig. 1
A). This effect of LNFPIII
was observed following primary immunization, and was further enhanced
with boosting. Furthermore, anti-HSA IgG response was largely IgG1
following the prime, and remained so after boosting. Small amounts of
HSA-specific IgG2a and IgG2b were seen after boosting, but IgM or IgA
were not detectable at any time point throughout the course of the
experiment. (Fig. 1B). In addition, BALB/c mice immunized
intranasally with HSA-LNFPIII also displayed significantly higher
levels of total IgE (p < 0.05) as well as
HSA-specific IgE (p < 0.005) as compared with
those immunized with HSA or saline (Fig. 1, C and
D). In contrast, immunization of the mice with HSA-LNnT, the
nonfucosylated homologue of HSA-LNFPIII, failed to increase total or
HSA-specific IgE and IgG above baseline (Fig. 1, A,
C, and D). C57BL/6 mice also produced significant
amounts of HSA-specific IgG and IgE following intranasal immunization
with HSA-LNFPIII, but not saline, HSA alone, or HSA-LNnT, although the
amounts were significantly lower than those produced in BALB/c mice
(Fig. 1, E and F). Moreover, HSA-LNFPI also
failed to induce a significant production of Ab against HSA in mice
when used as a sensitizing Ag (data not shown). In terms of Ag dose
capable for the induction of Ab production, HSA-LNFPIII was able to
induce a significant production of HSA-specific IgE and IgG in doses
1.0–10 µg (weight of HSA) as compared with 1000 µg of HSA alone
required to induce equivalent HSA-specific IgG response. Moreover, even
at such a high dose, HSA alone failed to induce a significant IgE
production (Fig. 2, A and
B).
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, A and B).
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To determine whether HSA-LNFPIII-immunized mice produce Ab against
LNFPIII, we performed competitive inhibition ELISA as described before.
HSA could completely inhibit, in a dose-dependent fashion, IgG binding
to HSA-LNFPIII-coated plates. In contrast, LNFPIII itself could not
inhibit IgG binding at all (Fig. 4A). Furthermore, IgG from
mice immunized with HSA-LNFPIII bound to HSA, HSA-LNnT, and
HSA-LNFPIII, but not BSA-LNFPIII, indicating that immunization with
HSA-LNFPIII induced Ab response against HSA (data not shown).
Similarly, IgE from mice immunized with HSA-LNFPIII did not react with
biotinylated BSA-LNFPIII or biotinylated Lewisx, which is
the fucose-containing triasaccharide of LNFPIII (9),
indicating that this carbohydrate was not an epitope for IgE (Fig. 4B).
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Mice were primed, boosted, and challenged with saline, HSA, or
HSA-LNFPIII as described in Materials and Methods. Nasal
lymphocytes were isolated 12 h after final challenge, and cultured
with or without Ag for 48 h. Cytokine levels in these supernatants
were determined by captured ELISA. Following in vitro stimulation with
HSA, nasal lymphocytes from with HSA-LNFPIII-immunized mice produced
significantly more IL-4 (p < 0.05), IL-5
(p < 0.05), and IL-10
(p < 0.01) as compared with those from saline-
and HSA-immunized mice. Interestingly, nasal lymphocytes from mice
sensitized with HSA alone or saline could produce IL-5 and IL-10 in
response to HSA-LNFPIII in vitro; however, the amounts were
significantly less than those from mice sensitized with HSA-LNFPIII. In
contrast, Ag-stimulated nasal lymphocytes from all three groups of mice
failed to produce IFN- (Fig. 6).
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Next, we examined whether immunization with HSA-LNFPIII altered
expression of costimulatory molecules necessary for T cell activation
and differentiation (16). Flow cytometric analysis showed
that Ag-stimulated nasal lymphocytes from mice immunized with
HSA-LNFPIII showed significantly higher (p <
0.005) expression of B7-2 on B220+ cells as
compared with cells from mice immunized with HSA alone or saline (Fig. 7, D–F). However,
expression of B7-1 was similar among all groups tested (Fig. 7, A–C).
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Further, we investigated whether the adjuvant activity of LNFPIII
on the induction of Th2-type responses was dependent on the route of
immunization. HSA-LNFPIII induced a significant HSA-specific IgG and
IgE production in BALB/c mice when administered i.p. (Fig. 8, A and B).
Furthermore, levels of Abs in HSA-LNFPIII-immunized mice were
comparable to those immunized with HSA adsorbed with alum, the
classical Th2-inducing adjuvant (17) (Fig. 8, A
and B). Similar results were observed when mice were
immunized s.c. although the levels of IgE were lower as compared with
those observed after i.p. immunization (Fig. 8, C and
D). Furthermore, mice immunized i.p. or s.c. with
HSA-LNFPIII adsorbed with alum displayed an increased production of
both specific IgG and IgE as compared with mice immunized with either
HSA adsorbed with alum or HSA-LNFPIII by same routes.
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Finally we sought to determine whether the priming with
LNFPIII-conjugated protein would lead to an increase in Ab production
against protein following the subsequent immunization with
nonconjugated protein. BALB/c mice primed with HSA-LNFPIII and
subsequently boosted and challenged with HSA alone produced
significantly higher amounts of both HSA-specific IgG and IgE following
the nasal challenge as compared with those following the primary
immunization. In addition, these mice produced significantly higher
amounts of specific IgG and IgE following the challenge as compared
with mice immunized with HSA alone throughout the study (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have demonstrated that SEA are potent inducers of a
Th2-like response that is associated with an increased Th2-type
cytokine and IgE production, and eosinophilia (18, 19).
LNFPIII is one of the oligosaccharides that is present on SEA and is
also found in human milk (13, 20). We recently
demonstrated that the carbohydrates present on SEA not only play a
critical role in induction of Th2 response but LNFPIII itself induces
IL-10 and PGE2 production by B cells in vitro (9). In the
present study, BALB/c mice immunization with HSA-LNFPIII produced
significantly higher levels of total IgE and HSA-specific IgE and IgG1
as compared with those immunized with HSA alone. Moreover, nasal
lymphocytes from HSA-LNFPIII-immunized mice produced significantly more
IL-4, IL-5, and IL-10, but not IFN-, in response to re-stimulation
with HSA-LNFPIII or HSA. Taken together, these findings suggest that
LNFPIII when conjugated to a protein Ag acts as an adjuvant and induces
Ag-specific Th2-type Ab and cytokine productions in murine intranasal
immunization model. The ability of nasal lymphocytes from
HSA-LNFPIII-immunized mice to produce significant amount of Th2
cytokines in response to in vitro antigenic stimulation also suggests
that that these nasal lymphocytes may be the effector cells responsible
for induction of Th2 type response in this model. Interestingly,
adjuvant activity of LNFPIII via intranasal route was also comparable
to alum administered via i.p. route, which is the classical
Th2-inducing adjuvant but difficult to use in mucosal immunization.
Moreover, HSA-LNFPIII adsorbed with alum significantly enhanced Ab
production indicating that these two adjuvants have a synergic effect.
Nevertheless, we also found that HSA-LNFPIII induced a significant
production of HSA-specific Th2-associated isotypes following i.p. as
well as s.c. immunization, indicating that the adjuvant activity of
LNFPIII is not dependent on the route of immunization.
Nasal lymphocytes from mice immunized with HSA produced significantly higher amounts of IL-5 and IL-10, but not IL-4, in response to HSA-LNFPIII as compared with HSA alone. And interestingly, the lymphocytes from mice immunized with saline also produced IL-5 and IL-10 in response to HSA-LNFPIII, suggesting that HSA-LNFPIII has an ability to enhance both IL-5 and IL-10 production in vitro. At least two possibilities can be thought. One, HSA peptides are produced more effectively from HSA-LNFPIII by APC as compared with HSA alone. Second, LNFPIII directly activate naive cells (e.g. macrophages, T cells, and B cells) to produce these cytokines. In fact, we have previously reported that LNFPIII induce IL-10 production by B cells (9, 10). In addition, these results may arise the possibility that LNFPIII or its related carbohydrates may lead to a nonspecific immune responses including polyclonal Ig synthesis, and the investigations are currently undertaken.
There are several mechanisms that may explain the adjuvant activity of
LNFPIII. First, LNFIII may simply enhance Ag uptake by APC through
binding to one of several APC receptors specific for carbohydrates.
This can be explained by the results presented here that only the
conjugated LNFIII but not free form of the sugar acts as an adjuvant.
For example, macrophages and dendritic cells can express lectin-like
receptors such as mannose receptor or mannose-binding lectin receptor,
which bind to carbohydrates expressed on pathogens (21, 22). Our result that HSA linked to LNFIII but not nonfucosylated
LNnT induced Th2 type Ab and cytokine production in intranasal
immunization seems to be consistent with the function of this receptor,
and an investigation of the interaction between LNFIII and mannose
receptor is now underway. A second explanation is that LNFIII activates
cells through binding to P-selectin although this sugar is a weak
ligand for this molecule (3). Third, LNFIII binds to an as
yet uncharacterized receptor specific for 
1–3 linked fucose. In
agreement with this hypothesis is our observation that
Lewisx, but not other fucose-containing Lewis-family sugars
are capable of direct binding to T and B cells as well as macrophages
(our unpublished data). Alternatively, this sugar-protein conjugation
may be involved in a selective cleavage of peptides by cathepsins in
lysosome, which are known to induce selective immune responses
(23).
In the present study, intranasal immunization of mice with HSA
conjugated with LNnT (HSA-LNnT), the nonfucosylated homologue of
LNFPIII, failed to increase total or HSA-specific IgE and IgG above
baseline, suggesting that the fucose residue of LNFPIII is required for
such an adjuvant effect. However, LNFI, another homologue of LNFIII
that contains fucose 1–2 linked to galactose, also failed the
significant Ab production. In addition, we previously found that
carbohydrate-containing 1–3 fucose on phospholipase A2 of honey bee
venom had no effect of adjuvant activity on Th2-type Ab production
(15). Collectively, these findings indicate that the
composition, type of conjugation, and subsequent tertiary structures of
linked oligosaccharides might be important for adjuvant activity of
fucose residue.
The lack of IgM component in the serum anti-HSA is another feature
of LNFPIII conjugates on humoral immune responses. In addition,
HSA-specific IgG production was produced even after a single
immunization (Fig. 1
, A and B). Although the
precise mechanisms remain to be elucidated, one possibility can be
arisen that LNFPIII conjugate may directly stimulate a memory pool of B
cells.
Costimulatory molecules are known to be important for the activation, proliferation and cytokine production of Ag-specific T cells (16). Among these, the B7 family CD80 (B7-1) and CD86 (B7-2) and their counter-receptors on T cells, CD28, and CD152 (CTLA4) are the most extensively characterized and appear to play a role in Th1/Th2 differentiation and/or maintenance (23, 24). In allergic patients who have already been primed with allergens, we and others have found that CD86 is selectively up-regulated in peripheral blood B cells in response to allergen (25). Furthermore, Van Neerven et al. (26) reported that allergen-specific T cell proliferation and cytokine expression require CD28-CD86 costimulation. Our results that B220+ nasal lymphocytes from mice immunized with HSA-LNFPIII display a significant increase in expression of B7-2, but not B7-1 indicate that LNFPIII activates B cells led to the up-regulation of B7-2 expression that may favor Th2 development.
In summary, we found that lactosamine sugar LNFPIII when conjugated to
a protein Ag is capable of enhancing protein-specific Th2 responses in
vivo. Moreover, LNFPIII functioned in a dramatic fashion compared with
reports on another carbohydrate adjuvant, pullulan (polymer of
-glucose), which suppressed Ag specific IgE while enhancing
Ag-specific IgG (27). In addition, route of immunization
did not alter adjuvant activity of LNFPIII, and coadministration of
this carbohydrate together with alum synergistically enhanced adjuvant
activity of alum. These results suggest that LNFPIII can be used as a
novel adjuvant to enhance Th2-type immune responses in vaccines.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mitsuhiro Okano, Department of Otolaryngology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail address: mokano{at}cc.okayama-u.ac.jp
3 Abbreviations used in this paper: SEA, Schistosoma mansoni egg Ag; LNFPIII, lacto-N-fucopentaose III; HSA, human serum albumin; HSA-LNFPIII, HSA conjugated to LNFPIII; LNnT, lacto-N-neotetraose; HSA-LNnT, LNnT conjugated to HSA; BSA-LNFPIII, LNFPIII conjugated to BSA; TMB, tetramethylbenzidine.
Received for publication November 13, 2000. Accepted for publication April 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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G. Raes, L. Brys, B. K. Dahal, J. Brandt, J. Grooten, F. Brombacher, G. Vanham, W. Noel, P. Bogaert, T. Boonefaes, et al. Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation J. Leukoc. Biol., March 1, 2005; 77(3): 321 - 327. [Abstract] [Full Text] [PDF]
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E. Van Liempt, A. Imberty, C. M. C. Bank, S. J. Van Vliet, Y. Van Kooyk, T. B. H. Geijtenbeek, and I. Van Die Molecular Basis of the Differences in Binding Properties of the Highly Related C-type Lectins DC-SIGN and L-SIGN to Lewis X Trisaccharide and Schistosoma mansoni Egg Antigens J. Biol. Chem., August 6, 2004; 279(32): 33161 - 33167. [Abstract] [Full Text] [PDF]
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 T. Watanabe, M. Okano, H. Hattori, T. Yoshino, N. Ohno, N. Ohta, Y. Sugata, Y. Orita, T. Takai, and K. Nishizaki Roles of Fc{gamma}RIIB in Nasal Eosinophilia and IgE Production in Murine Allergic Rhinitis Am. J. Respir. Crit. Care Med., January 1, 2004; 169(1): 105 - 112. [Abstract] [Full Text] [PDF]
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P. G. Thomas, M. R. Carter, O. Atochina, A. A. Da'Dara, D. Piskorska, E. McGuire, and D. A. Harn Maturation of Dendritic Cell 2 Phenotype by a Helminth Glycan Uses a Toll-Like Receptor 4-Dependent Mechanism J. Immunol., December 1, 2003; 171(11): 5837 - 5841. [Abstract] [Full Text] [PDF]
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L. Vervelde, N. Bakker, F. N.J. Kooyman, A. W.C.A. Cornelissen, C. M.C. Bank, A. K. Nyame, R. D. Cummings, and I. van Die Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen Glycobiology, November 1, 2003; 13(11): 795 - 804. [Abstract] [Full Text] [PDF]
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I. van Die, S. J. van Vliet, A. K. Nyame, R. D. Cummings, C. M.C. Bank, B. Appelmelk, T. B.H. Geijtenbeek, and Y. van Kooyk The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x Glycobiology, June 1, 2003; 13(6): 471 - 478. [Abstract] [Full Text] [PDF]
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G. Schramm, F. H. Falcone, A. Gronow, K. Haisch, U. Mamat, M. J. Doenhoff, G. Oliveira, J. Galle, C. A. Dahinden, and H. Haas Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs J. Biol. Chem., May 9, 2003; 278(20): 18384 - 18392. [Abstract] [Full Text] [PDF]
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A. van Remoortere, C. M.C. Bank, A. K. Nyame, R. D. Cummings, A. M. Deelder, and I. van Die Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R Glycobiology, March 1, 2003; 13(3): 217 - 225. [Abstract] [Full Text] [PDF]
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Z. Liu, Q. Liu, J. Pesce, J. Whitmire, M. J. Ekkens, A. Foster, J. VanNoy, A. H. Sharpe, J. F. Urban Jr., and W. C. Gause Nippostrongylus brasiliensis Can Induce B7-Independent Antigen-Specific Development of IL-4-Producing T Cells from Naive CD4 T Cells In Vivo J. Immunol., December 15, 2002; 169(12): 6959 - 6968. [Abstract] [Full Text] [PDF]
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C. Faveeuw, V. Angeli, J. Fontaine, C. Maliszewski, A. Capron, L. Van Kaer, M. Moser, M. Capron, and F. Trottein Antigen Presentation by CD1d Contributes to the Amplification of Th2 Responses to Schistosoma mansoni Glycoconjugates in Mice J. Immunol., July 15, 2002; 169(2): 906 - 912. [Abstract] [Full Text] [PDF]
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L. I. Terrazas, K. L. Walsh, D. Piskorska, E. McGuire, and D. A. Harn Jr. The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections J. Immunol., November 1, 2001; 167(9): 5294 - 5303. [Abstract] [Full Text] [PDF]
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O. Atochina, T. Daly-Engel, D. Piskorska, E. McGuire, and D. A. Harn A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-{gamma} and Nitric Oxide-Dependent Mechanism J. Immunol., October 15, 2001; 167(8): 4293 - 4302. [Abstract] [Full Text] [PDF]
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),
boost (
), and challenge (
) as described in Materials and
Methods. Ab of IgG (A and E),
IgM, IgA, and IgG subclasses (B) specific for HSA were
measured by indirect ELISA and estimated by endpoint titer. IgE titers
specific for HSA (C and F) and the volume
of serum total IgE (D) were determined by sandwich
ELISA. Levels of HSA-specific IgE were presented as the absorbance at
450 nm from duplicate wells of 1/4 serum dilution. Results show the
mean ± SEM of four individual serum per group. Data are
representative of three (A–D) and two
(E and F) separate experiments.
