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*
Departments of Microbiology and Oral Biology, The Immunobiology Vaccine Center, University of Alabama Medical Center, Birmingham, AL 35294;
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
DNAX Research Institute, Palo Alto, CA 94304; and
§
Departimento di Clinica Medica, Immunologia e Malattie Infetive, Universita Degli Studi di Bari, Bari, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-6, and IL-10 than did those from control mice, but only negligible
levels of IL-4 and IL-5. In summary, both intranasal IL-6 and IL-12
induced serum Abs that protected mice from systemic challenge with TT,
whereas only IL-12 induced mucosal S-IgA Ab responses. The significance
of IL-12-induced Th1-type responses for regulation of both mucosal and
systemic immunity is discussed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Th1-type) and IL-4 (Th2-type), which
mutually regulate one another (2). Furthermore, APCs favor
the development of Th2 or Th1 cells by secreting IL-6 and IL-12,
respectively (3, 4). A number of studies have demonstrated
the major role played by Th cell-derived cytokines in the development
of secretory IgA (S-IgA)3 Ab responses.
For example, cholera toxin (CT) promotes mucosal immune responses to
coadministered Ag only in the presence of IL-4 (5, 6, 7).
Furthermore, IL-4, IL-5, IL-6, and IL-10 are involved in the activation
and the terminal differentiation of IgA-secreting plasma cells
(8). Interestingly, mucosal S-IgA Ab responses are induced
by a vaccine regimen which promotes either Th1- or Th2-type cytokine
responses (7, 9, 10, 11). However, it is still unclear whether
the cytokine environment, specifically the APC-derived cytokines, could
provide necessary signals for the initiation of S-IgA Ab responses.
IL-6 is a pleiotropic cytokine that regulates both T and B cell
functions (12, 13). In T cells, IL-6 activates and
stimulates the cytotoxic activity of NK and CD8+ T cells
(14, 15), resulting in a reduction of metastases in
tumor-bearing mice (16, 17). IL-6 was also shown in a
recent study to support the development of CD4+ Th2-type
cells (4). This cytokine also induces B cells to
differentiate into plasma cells (12, 13, 18) and
selectively stimulate committed membrane IgA-positive Peyer’s patch B
cells to secrete IgA, in vitro (8). However,
contradictory results were reported on the role of IL-6 in mucosal IgA
Ab responses in vivo and both normal (19) and
impaired mucosal IgA Ab responses (20) have been reported
in the absence of IL-6. IL-12 is a potent inducer of IFN- production
by NK and T cells (21, 22, 23) and causes T cell precursors to
develop into functional Th1-type cells (22, 24). IL-12 was
recently shown to enhance Ig secretion by polyclonally activated mouse
spleen cells through both IFN--dependent and -independent mechanisms
(25). Our previous results have shown that CT-induced
S-IgA Ab responses were affected when IL-12 was administered by
parenteral but not by nasal or oral routes (7, 11),
suggesting that this regulatory cytokine affects the mucosal and
systemic compartments differently.
This study sought to determine whether APC-derived cytokines, which support either Th1 (IL-12)- or Th2 (IL-6)-type responses, could serve as adjuvant for enhancement of mucosal immune responses to coadministered protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from the Charles River Laboratories (Wilmington, DE) at 5–6 wk of age and were maintained in horizontal laminar flow cabinets in sterile cages in the Animal Facility of the University of Alabama Immunobiology Vaccine Center (Birmingham, AL). Sterile food and water were provided according to guidelines proposed by the committee for the Care of Laboratory Animal Resources, Commission of Life Sciences, National Research Council. The mice were pathogen free as determined by routine Ab-screening against common mouse pathogens and histological analysis of major organs and tissues. The mice were used at 8–12 wk of age in all experiments described here.
Immunization and cytokine treatment
Mice were intranasally immunized on days 0, 7, and 14 with 15 µl of preparation (7.5 µl per nostril) consisting of 20 µg of tetanus toxoid (TT) (kindly provided by Dr. Patricia J. Freda Pietrobon, Connaught Laboratories, Swiftwater, PA) with or without cytokines. For recombinant cytokine treatment, mice were nasally administered with murine rIL-12 (generously provided by Dr. Stanley Wolf; Genetics Institute, Cambridge, MA) and/or human rIL-6 (Genzyme, Cambridge, MA) complexed with cationic liposomes (DOTAP, Boehringer Mannheim, Indianapolis, IN) as described previously (11). Earlier studies showed that optimal IL-12 effects required nasal administration of 1 µg/dose on days 0, 3, 7, 10, 14, and 17 (11) and established that murine rIL-6 or human rIL-6 induced comparable in vivo effects in C57BL/6 mice (data not shown). On days when mice were given intranasal cytokine-liposomes and TT, the protein TT was administered first followed 15 min later by cytokine-liposomes or liposomes only.
Cell isolation and purification
Spleen (SP) and cervical lymph nodes (CLN) were aseptically removed and single cell suspensions were obtained by gently teasing small fragments through sterile wire screen. The cells from lungs and lamina propria were obtained as described in previous studies (10, 26, 27) and were >95% viable as determined by trypan blue dye exclusion. Single cell preparations were resuspended in complete medium (RPMI 1640, Cellgro Mediatech, Washington, DC) containing 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin. Enriched CD4+ T cells were obtained from CLN and SP cell suspensions by panning on anti-L3T4 (GK 1.5) mAb-coated petri dishes as described elsewhere (28). This procedure resulted in CD4+ T cell-enriched cultures, which were >97% CD3+, CD4+ and contained <1% CD3+, CD8+ T cells. The CD3+, CD4+, CD8- T cell population was >98% viable as determined by trypan blue dye exclusion.
Analysis of Ab isotypes and IgG subclasses
Vaginal washes, fecal pellets, and blood samples for serum were
collected at weekly intervals (days 0, 7, 14, and 21) and saliva was
collected on day 21. Samples were processed as previously described and
stored at -70°C until assayed for TT-specific Abs (11, 27). Briefly, ELISA plates (Microtest III; Becton Dickinson,
Oxnard, CA) were coated with a 100-µl solution of TT (5 µg/ml; 1.25
Lf units/ml), and serial twofold dilutions of serum or fecal extracts,
saliva, or vaginal washes were added to individual wells. Titers of
IgM, IgG, or IgA Abs were determined by addition of a horseradish
peroxidase conjugated-goat anti-mouse -µ, -, or -
heavy
chain-specific antisera (Southern Biotechnology Associates, Birmingham,
AL). To determine serum IgG subclass titers, 100 µl of
biotin-conjugated, rat mAb anti-mouse -1 (G1 -7.3; 0.5
µg/ml), -2a (R19-15; 0.5 µg/ml), -2b (R12-3; 0.5 µg/ml), or
-3 (R40-82; 0.5 µg/ml) heavy chain-specific Abs were used
(PharMingen, San Diego, CA) as described previously (7, 11). Following incubation and washing, a 100-µl aliquot of
HRP-conjugated streptavidin (Life Technologies, Grand Island, NY) was
added and color developed with ABTS substrate (Sigma, St. Louis, MO).
The absorbance at 415 nm was determined after 15 min and the data
expressed as reciprocal endpoint titers of the last dilution exhibiting
an OD of >0.1 when compared with negative controls. Total IgE levels
and Ag-specific IgE Abs were determined by ELISA and a passive
cutaneous anaphylaxis assay, respectively, as previously described
(7, 11).
B cell ELISPOT for IgA Ab-forming cells (AFCs)
An enzyme-linked immunospot (ELISPOT) assay was used to
quantitate numbers of AFCs in the intestinal lamina propria, SP, CLN,
and lungs of mice intranasally immunized with TT in the presence or
absence of IL-6 and/or IL-12. TT-specific IgA, IgM, and IgG AFCs were
determined by ELISPOT assay as described previously (26).
Briefly, 96-well nitrocellulose-based plates were coated with a
100-µl solution of TT (5 µg/ml) diluted in PBS, while control wells
received only PBS. Following washing, the wells were blocked with 1%
BSA in PBS. Fivefold serial dilutions of cell suspensions (starting at
1 x 106 cells) were added to the wells in duplicate
and incubated for 6 h. AFCs were detected with peroxidase-labeled
anti-mouse -µ, -, or - chain-specific Ab (Southern
Biotechnology Associates), visualized by adding the chromogenic
substrate 3-amino-9-ethylcarbazole (Moss, Pasadena, MD), and counted
with the aid of a dissecting microscope (SZH Zoom Stereo Microscope
System, Olympus, Lake Success, NY).
Ag-specific CD4+ T cell responses
For the stimulation of Ag-specific CD4+ T cells in vitro, TT was adsorbed onto latex microspheres as described previously (7, 27), and CD4+ T cells were restimulated in vitro according to previously described methods (7, 28). CD4+ T cells (2 x 106 cells/ml) were cultured with rIL-2 (10 U/ml, PharMingen) and T cell-depleted, irradiated SP feeder cells from naive mice in flat-bottom 96-well (200 µl/well) or 24-well (1 ml/well) tissue culture plates (Corning Glass Works, Corning, NY) for proliferation and cytokine synthesis, respectively. To measure Ag-specific T cell proliferation, 0.5 µCi of tritiated [3H]thymidine (Amersham, Arlington Heights, IL) was added after 5 days of culture and 16 h prior to harvest. Optimal proliferative responses and cytokine levels in culture supernatants were obtained with approximately 10 TT-coated particles/cell.
Cytokine measurements
For assessment of cytokine production in Ag-specific
CD4+ T cell cultures, supernatants were harvested following
5 days in culture. Controls consisted of cultured cells only or of
cells incubated with unadsorbed beads or with OVA-coated beads. All T
cell cultures were maintained at 37°C in a 5% CO2
incubator in moist air. Cytokine levels in culture supernatants and
serum were determined by a ELISA using appropriate combinations of mAbs
as described in our previous studies (11). Falcon
Microtest III plates (Becton Dickinson) were coated with 100 µl of
anti-cytokine mAb diluted in 0.1 M bicarbonate buffer (pH 8.2) and
incubated overnight at 4°C. The wells were blocked with PBS
containing 1% BSA at room temperature for 1 h. Serial twofold
dilutions of supernatants or serum were added to duplicate wells and
incubated overnight at 4°C. The wells were then washed with PBS-Tween
(PBS-T) and incubated with the appropriate biotinylated
anti-cytokine mAb diluted in PBS-T with 1% BSA for 2 h.
Following three rinses, wells were incubated with peroxidase-labeled
anti-biotin mAb (0.5 µg/ml; Vector Laboratories, Burlingame, CA)
for 1 h and developed with ABTS substrate (Sigma). Standard curves
were generated using murine IFN-, rIL-5, rIL-6, and rIL-10
(Genzyme); rIL-2 (PharMingen); and rIL-4 (Endogen, Boston, MA). The
ELISA used in this study could detect 15 pg/ml of IFN-; 5 pg/ml of
IL-2, IL-4, or IL-5; 100 pg/ml of IL-6; and 200 pg/ml of IL-10.
Tetanus toxin challenge
Tetanus toxin was obtained from Drs. Jean Halpern and William Habig, Division of Bacterial Products, Food and Drug Administration, Bethesda, MD. The toxin was diluted in gelatin-saline and appropriate doses were injected s.c. into normal or immunized mice. The mice were monitored daily for paralysis and death.
Statistics
The results are expressed as the mean ± 1 SD. The Ab and
cytokine data were analyzed by the nonparametric Mann-Whitney
U test using the Statview software (Abacus, Berkeley, CA).
Results were considered statistically significant if p
values were <0.05. For statistical analysis of cytokine levels, those
below the detection limit were recorded as one-half the detection limit
(e.g., IFN- = 7.5 pg/ml). Mouse survival data were analyzed by a
Fisher exact test and differences were considered significant for
p values of <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In order to calibrate the impact of varying dosages and
frequencies of IL-6 on the immune response to TT, mice received TT
alone or in combination with a wide range of IL-6 doses (1–1000
ng/dose). Mice immunized with doses of 100 ng or less showed slightly
higher TT-specific serum IgG Abs than did control mice nasally
immunized with TT only (Fig. 1
). A
significant increase in TT-specific serum IgG Abs was achieved after
administration of three doses containing 1000 ng of IL-6 (Fig. 1). When
the frequency of IL-6 administration was increased to six doses (i.e.,
on days 0, 3, 7, 10, 14, and 17), each containing 1000 ng, the
TT-specific IgG Ab levels were enhanced and did not further increase
when doses were increased up to 3000 ng. Therefore, six doses of 1000
ng were used for IL-6 treatment throughout the study.
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). In contrast, IgG Ab titers were
significantly increased in all mice receiving nasal cytokines, although
the administration of IL-12 alone or with IL-6 resulted in higher
TT-specific Ab responses than administration of TT with IL-6 (Fig. 2).
Interestingly, nasal IL-12 also resulted in significant titers of
TT-specific IgA Abs, which were not further increased by
coadministration of IL-6. In contrast, nasal administration of IL-6 and
TT did not induce significant titers of TT-specific serum IgA Abs (Fig. 2). Administration of liposomes without cytokine after nasal
immunization with TT did not enhance anti-TT Ab responses
(Fig. 2).
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). When
sera from mice intranasally immunized with TT and IL-12 were analyzed,
a significant increase in all IgG subclasses was noted. Specifically,
comparable titers of IgG1, IgG2a, and IgG2b Abs with low but
significant IgG3 Ab responses were noted (Fig. 3). Codelivery of IL-6
and IL-12 resulted in an identical TT-specific IgG subclass
distribution and titers comparable with those measured following
delivery of IL-12 alone. None of the nasal cytokine treatments induced
detectable levels of TT-specific IgE (data not shown).
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Since intranasal administration of cytokines resulted in increased
titers of TT-specific serum Abs, we next investigated whether
intranasal administration of IL-6 or IL-12 supported S-IgA Ab
responses. Nasal administration of TT and IL-6 resulted in modest IgA
Ab titers in fecal extracts and negligible IgA Ab titers in saliva and
vaginal washes (Fig. 4), while nasal
administration of TT and IL-12 resulted in high TT-specific IgA Ab
titers in all mucosal secretions that were not further increased by
coadministration of IL-6 (Fig. 4). The source of mucosal TT-specific
IgA Abs measured in external secretions was confirmed by enumeration of
IgA AFCs in both mucosal immune sites such as the lamina propria and
lungs and in systemic immune sites such as the SP and CLNs. As shown in
Table I, intranasal administration of
IL-6 alone slightly increased the frequency of IgA AFC in the
intestinal lamina propria. On the other hand, intranasal delivery of
IL-12 or IL-12 plus IL-6 resulted in high frequencies of TT-specific
IgA AFC in all mucosal compartments analyzed.
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We studied the array of Th1-type and Th2-type cytokines secreted
by TT-specific CD4+ T cells following in vitro
restimulation. CD4+ T cells from CLN and SP of mice nasally
immunized with TT failed to respond to TT (data not shown) and produced
low or negligible levels of cytokines (Fig. 5). When SP or CLN CD4+ T
cells from mice receiving nasal TT and IL-6 were restimulated in
vitro, high proliferative responses were seen, accompanied by high
levels of IFN-, IL-6, and IL-10 synthesis (Fig. 5). No significant
differences in profiles of TT-specific CD4+ T cell
cytokines were observed between mice that received intranasal IL-12 or
IL-12 plus IL-6. In fact, these treatments resulted in large increases
in IFN-, IL-6, and IL-10 secretion by TT-specific CD4+ T
cells (Fig. 5). Furthermore, TT-specific CD4+ T cells from
mice that received IL-12 or IL-12 plus IL-6 produced higher levels of
IFN- and IL-6 than did T cells from mice treated with IL-6 only
(Fig. 5).
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It was important to determine whether the immune responses induced
by mucosal cytokine treatment resulted in protective immunity.
Therefore, we tested mice that received nasal IL-6 and/or IL-12
treatment with TT to determine sensitivity to a systemic challenge with
tetanus toxin. Naive mice and those nasally immunized with either TT
alone or with cytokine alone did not survive the challenge. In
contrast, mice receiving TT plus IL-6 or TT plus IL-12 or TT plus both
IL-12 and IL-6 were completely protected, demonstrating that protective
immunity is induced by these mucosal cytokine treatments (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early studies using in vitro systems suggested a role of IL-6 as a key factor for B cell differentiation into plasma cells (12, 13, 18). However, the role of IL-6 in Ag-specific responses remain controversial. It has been suggested that IL-6 is not required for Ag-specific Ab responses (28) and both normal and impaired Ab responses have been reported in mice lacking IL-6 (19, 20). Here we show that nasally administered IL-6 can act as adjuvant for systemic immunity to coadministered vaccine protein. Our results are consistent with observations by others that parenteral administration of IL-6 enhances Ag-specific Ab responses in mice (29). Interestingly, although IL-6 was reported to selectively stimulate mIgA+ B cells residing in the Peyer’s patches to secrete IgA in vitro (8), no significant Ag-specific S-IgA Ab responses were induced by nasal IL-6 treatment. This observation is in contrast with the study by others in which oral treatment with IL-6 before oral infection Campylobacter jejuni induced Campylobacter-specific serum and mucosal S-IgA Ab responses (30). It is unlikely that induction of S-IgA Abs was due to the oral route of IL-6 delivery in these studies. In this regard, bacterial LPS is known to be a trigger for a large array of effects on B lymphocytes and APCs (31), and thus, this molecule could have exerted a costimulatory effect for the induction of Campylobacter-specific S-IgA Ab responses.
In contrast to IL-6, which is also produced by B and T cells, the
production of IL-12 is mainly restricted to APCs (i.e., macrophages and
dendritic cells) (23). IL-12 is primarily involved in the
induction of IFN- and Th1-type responses. However, IL-12 was also
reported to stimulate B cells growth (32) and Ig secretion
in vitro (25). We have previously reported that
IL-12 administered by mucosal routes (i.e., either oral or nasal) could
redirect Th2-type immune responses induced by the mucosal adjuvant CT
toward a Th1 type (11, 40). In the present study, nasal
treatment with IL-12 increased serum Ab responses to co-administered TT
vaccine, clearly showing an adjuvant effect by this cytokine when
administered by a mucosal route.
A striking observation in our previous studies was the fact that CT-induced S-IgA Ab responses were not affected by mucosal administration of IL-12 (11, 40). Interestingly, we report here that nasal treatement with IL-12 could also trigger S-IgA Ab responses. Our observation supports a recent report by others that IL-12 displays mucosal adjuvant activity when delivered by the nasal route (33). Even though IL-6 is a stimulatory factor for B cell differentiation (13, 18), the absence of an enhancing effect when IL-6 was coadministered with IL-12 suggests that IL-12 does not require additional signals for its mucosal adjuvanticity.
Since the adjuvant effects of IL-6 and IL-12 are likely mediated by Th cell-derived cytokines (4, 22), we further characterized serum IgG subclasses and IgE Ab responses. Indeed, Th cytokines control the pattern of IgG subclass Ab responses (34), and IgE Abs were shown to correlate with IL-4 production (11, 35). The adjuvant effect of IL-6 resulted in Ag-specific serum IgG1 and IgG2b Abs followed by IgG2a Abs, while nasal IL-12 induced comparable levels of IgG1, IgG2a, and IgG2b as well as high IgG3 Ab responses. Thus, IL-6 induced a pattern of IgG subclasses corresponding to Th2-type responses (34). Although IL-12 is known to be a potent Th1 cell inducer (3), its adjuvant effect resulted in IgG subclass responses that correspond to a mixed Th1- and Th2-type response. Despite this difference in the profiles of IgG subclasses, no IgE Ab response was induced by either IL-6 or IL-12 as nasal adjuvant.
In order to clearly establish that Th cell-derived cytokine responses
are involved in the adjuvanticity of IL-6 and IL-12, we analyzed
cytokine secretion patterns by TT-specific CD4+ T cells
from mice that received nasal IL-6 and/or IL-12 as adjuvant. Our
results show that the adjuvant effect of both IL-6 and IL-12 involved
Th1- and Th2-type cytokines. In fact, increased IFN- secretion was
measured in culture supernatants of TT-specific CD4+ T
cells from mice treated with either IL-6 and/or IL-12. This observation
is consistent with the ability of IL-12 to induce Th1-type cells
(23) and the reported impaired Th1-type development seen
in IL-6-deficient mice (36). Furthermore, mice that
received IL-12 treatment secreted larger amount of IFN- than those
treated with IL-6, confirming the greater ability of IL-12 to promote
Th1-type responses. Similar patterns of Th2-type cytokines were
observed after IL-6 and IL-12 treatment with increased IL-6 and IL-10
secretion with no significant levels of IL-4 and IL-5. Interestingly,
the IL-12 treatment resulted in significantly higher IL-6 secretion in
culture supernatants than the IL-6 treatment itself, suggesting that
IL-12 was a better inducer of Th2-type responses than was IL-6.
Furthermore, coadministration of IL-6 enhanced the IL-12-induced IL-6
and IL-10 secretion by TT-specific CD4+ T cells,
demonstrating an absence of interference between these two cytokines
even though a subunit of IL-12 shares homologies with the IL-6R
(23). It was reported that IL-12 promotes differentiation
of Th2-type cells in the absence of IFN- (37) and
enhances rather than suppresses ongoing Th2-type responses (38, 39). On the other hand, we have shown that serum elevated serum
IFN- occurs 12 h following nasal IL-12 administration (40).
Thus, it is possible that Th2 cells were the first stimulated following
nasal administration of IL-12 and TT and that Th1 cells were later
induced through IFN- production. In this regard, we have reported
that administration of IL-12 from a site distant from that where the
immune responses is initiated could both enhance CT-induced Th2-type
response and promote Th1-type responses (40).
It is important to note that the ability of IL-12 (but not IL-6) to
display a mucosal adjuvant activity correlates with a greater ability
of IL-12 to induce IL-6 and IFN- secretion by Ag-specific
CD4+ T cells. These results are consistent with the ability
of Salmonella vectors to induce mucosal S-IgA Ab responses
with Th responses characterized by IFN-, IL-6, and IL-10 secretion
(9). Thus, strong Th1 (i.e., IFN-) responses together
with IL-6 might provide necessary signals for S-IgA Ab responses. An
alternative explanation for the mucosal adjuvanticity of IL-12 is a
specific effect of IL-12 on B cells. Indeed, while the role of IL-6 is
restricted to stimulation of mIgA+ cells to become
IgA-producing plasma cells (8), IL-12 may influence both
the switch of B cells to mIgA+ cells and their terminal
differentiation into plasma cells.
We have shown that nasal administration of IL-6 and/or IL-12 resulted in systemic immunity to nasally-delivered vaccine and protected animals against a systemic challenge. Furthermore, only IL-12 could induce S-IgA Ab responses although a similar pattern of Ag-specific Th1- and Th2-type responses were induced by IL-6 and IL-12. Our results have important implications for the design of cytokine-based mucosal vaccines and unmask an important role for IL-12 in the development of S-IgA responses.
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Acknowledgments |
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Footnotes |
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2 P.N.B. and M.M. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Jerry R. McGhee, Immunobiology Vaccine Center, University of Alabama, 761 BBRB 845 19th Street South, Birmingham, AL 35294-2170. E-mail address:
4 Abbreviations used in this paper: AFC, antibody-forming cells; CLN, cervical lymph nodes; CT, cholera toxin; ELISPOT, enzyme-linked immunospot; PP, Peyer’s patches; S-IgA, secretory IgA; SP, spleen; TT, tetanus toxoid.
Received for publication June 25, 1998. Accepted for publication September 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cell stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869. production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.-dependent and -independent mechanisms. Eur. J. Immunol. 27:1958.[Medline]
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L. A. Dunn, J. A. Upcroft, E. V. Fowler, B. S. Matthews, and P. Upcroft Orally Administered Giardia duodenalis Extracts Enhance an Antigen-Specific Antibody Response Infect. Immun., October 1, 2001; 69(10): 6503 - 6510. [Abstract] [Full Text] [PDF]
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P. N. Boyaka, M. Marinaro, R. J. Jackson, F. W. van Ginkel, E. Cormet-Boyaka, K. L. Kirk, C. R. Kensil, and J. R. McGhee Oral QS-21 Requires Early IL-4 Help for Induction of Mucosal and Systemic Immunity J. Immunol., February 15, 2001; 166(4): 2283 - 2290. [Abstract] [Full Text] [PDF]
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 J. D. Rempel, M. Wang, and K. T. HayGlass Failure of rIL-12 administration to inhibit established IgE responses in vivo is associated with enhanced IL-4 synthesis by non-B/non-T cells Int. Immunol., July 1, 2000; 12(7): 1025 - 1034. [Abstract] [Full Text] [PDF]
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A. McI. Mowat, M. Steel, A. J. Leishman, and P. Garside Normal Induction of Oral Tolerance in the Absence of a Functional IL-12-Dependent IFN-{gamma} Signaling Pathway J. Immunol., November 1, 1999; 163(9): 4728 - 4736. [Abstract] [Full Text] [PDF]
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J. Skok, J. Poudrier, and D. Gray Dendritic Cell-Derived IL-12 Promotes B Cell Induction of Th2 Differentiation: A Feedback Regulation of Th1 Development J. Immunol., October 15, 1999; 163(8): 4284 - 4291. [Abstract] [Full Text] [PDF]
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B. Jiang, M. Jordana, Z. Xing, F. Smaill, D. P. Snider, R. Borojevic, D. Steele-Norwood, R. H. Hunt, and K. Croitoru Replication-Defective Adenovirus Infection Reduces Helicobacter felis Colonization in the Mouse in a Gamma Interferon- and Interleukin-12-Dependent Manner Infect. Immun., September 1, 1999; 67(9): 4539 - 4544. [Abstract] [Full Text] [PDF]
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); a second group received nasal
liposomes only (
); a third group received IL-6-liposomes (
1000
ng/dose on days 0, 3, 7, 10, 14, and 17); a fourth group received nasal
IL-12-liposomes (
1000 ng/dose on days 0, 3, 7, 10, 14, and 17); and
the last group received both IL-12 and IL-6 in liposomes (
1000
ng/dose of IL-6 plus 1000 ng/dose of IL-12 on days 0, 3, 7, 10, 14, and
17). Serum Ab titers were measured by ELISA on day 21. Results are
expressed as the mean ±SD of four separate experiments
(*, p < 0.05).
, 1000 ng/dose on days 0, 3, 7,
10, 14, and 17); and the last group received both IL-12 and IL-6 in
liposomes (
