Published online before print April 4, 2005
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* Inmunología, CIBICI (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Argentina; and
Centro de Excelencia en Productos y Procesos de la Provincia de Córdoba (CEPROCOR), Agencia Córdoba Ciencia S.E. and CONICET, Argentina
1 Correspondence: Inmunología, CIBICI (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Córdoba, Argentina. E-mail: scorrea{at}bioclin.fcq.unc.edu.ar
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, interleukin (IL)-12, IL-4, IL-10, and transforming growth factor-ß (TGF-ß), and the activation of T lymphocytes. We show here that the uptake of chitosan at inductive mucosal sites involves CD11b/c+ APC and that chitosan feeding increases the percentage of OX62+ dendritic cells, which up-regulate the major histocompatibility complex class II antigens without changing the expression of costimulatory CD80 or CD86 molecules. The polysaccharide elicits the release of IL-10 as well as the expression of IL-4 and TGF-ß in mucosa, and in spleen, the activation of CD3+ T cells occurs. Our results demonstrate that chitosan acts by enhancing the T helper cell type 2 (Th2)/Th3 microenvironment in the mucosa. A single dose of this polysaccharide exhibits local and systemic effects, and its activity could be relevant in the maintenance of the intestinal homeostasis.
Key Words: mucosa • immature dendritic cell • cytokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chitosan is a cationic biocompatible polysaccharide built by repeated units of N-acetyl-D-glucosamine and D-glucosamine, derived from the partial deacetylation of chitin, a natural polysaccharide extracted from the crustacean shells. Chitosan is also found in some microorganisms in yeasts and fungi [14 ]. It has been administered to humans by several routes without toxic effects [15 ]; it also has been used as dietary fiber [16 , 17 ] and represents a new generation system for antigen delivery [18 19 20 21 ]. At the mucosal level, this mucoadhesive polysaccharide interacts through positively charged amino groups with negatively charged sites on the cell surface. The interaction causes a redistribution of protein zonula occludens-1 and F-actin without affecting the viability of epithelial cells, although reversible perturbation of plasma membrane may occur [18 ]. As a consequence, changes in the permeability of epithelia take place, enhancing the uptake of peptides and proteins and increasing the contact with the immune system [18 , 19 ]. Given its ability to deliver drugs across the mucosal lining and its potential adjuvant activity, chitosan is considered an ideal candidate for mucosal immunization [22 , 23 ]. Coadministered with protein antigens, chitosan modifies the uptake and/or the distribution of the relevant antigen and enhances the release of regulatory cytokines associated to the antigen-specific stimulation early after feeding [24 ]. Antigens coadministered or encapsulated with the polysaccharide show improved mucosal and systemic humoral immune responses, although the mechanism is poorly understood [22 , 23 , 25 ]. Moreover, this polysaccharide is able to activate leukocytes in vitro and in vivo [24 , 26 ] with different effects depending on the metabolic status of the cell [27 ]. Its mucoadhesive properties and the adjuvant, activity reported previously encouraged us to study early events associated with the coadministration of chitosan with antigens frequently evaluated in oral tolerance protocols, such as type II collagen [24 ]. The effects observed in rats fed single or repeated doses of the polysaccharide prompted us to study the intrinsic ability of chitosan to promote a T helper cell type 2 (Th2)/Th3-biased environment at the mucosal level.
In this work, we characterized molecular events triggered after a single oral administration of chitosan. Sixteen hours after feeding, we studied in Peyer’s patches (PP), mesenteric lymph nodes (MLN), and spleen the uptake and distribution of the polysaccharide, the phenotype of recruited antigen-presenting cells (APC), the induction of cytokines tumor necrosis factor (TNF-), interleukin (IL)-12, IL-4, IL-10, and transforming growth factor-ß (TGF-ß), and the activation of T lymphocytes. We show here that at inductive mucosal sites, the uptake of chitosan involves CD11b/c+ APC. Early after chitosan feeding, the percentage of OX62+ dendritic cells (DC) increases, which up-regulates major histocompatibility complex (MHC) class II antigens without changing the expression of costimulatory CD80 or CD86 molecules. At mucosal tissues, the polysaccharide elicits the release of IL-10 as well as the expression of IL-4 and TGF-ß mRNA, and in the spleen, the activation of CD3+ T cells occurs. To the best of our knowledge, our work provides the first evidence in vivo of the activation of mucosal immune cells after feeding a low dose of this cationic polysaccharide in the absence of the protein antigen.
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Feeding and cell preparation
We used 85% deacetylated, low molecular weight chitosan (
80 kDa; Aldrich Chemical Co., Milwaukee, WI) prepared as described [24
, 28
] and fluorescein isothiocyanate (FITC)-chitosan (CarboMer, San Diego, CA), as we found previously that low molecular weight chitosan has a higher ability to stimulate APC than other chitosans [27
]. In the afternoon, we fed rats a final volume of 200 µl 0.1 M acetic acid (diluent group) or acetic acid containing 1 or 3 mg chitosan. Sixteen hours later, we removed PP, MLN, and spleens. We prepared single-cell suspensions by mechanical dispersion (PP and MLN) or spleen mononuclear cells using high-density gradient Ficoll-Paque according to standard procedures in RPMI medium supplemented with gentamicin, heparin, and 5% fetal calf serum (FCS) [24
]. Table 1
shows representative numbers of cells recovered from each tissue.
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Table 1. Absolute Number of Mononuclear and CD3+ Cells of Different Experimental Groups 16 h after Feeding
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20-cm sections (I–V) from each animal obtained from identical anatomical positions of the small intestine were obtained. We isolated two randomly selected PP per section, and we prepared cell suspensions as above. Cells were washed, fixed in 2% formaldehyde, and resuspended in phosphate-buffered saline (PBS); 50,000 events were analyzed using Cytoron Absolute (Ortho Diagnostic Systems, Raritan, NJ). In each experiment, we included PP cell suspensions from diluent-treated rats to control for intrinsic autofluorescence [29 ]. On the basis of forward- and side light-scatter, mononuclear cells were gated in, and dead cells were gated out. Positivity was defined as fluorescence that exceeded 98% of controls [30 , 31 ].
Flow cytometry
Analysis of phenotype and activation marker expression was performed as described [24
, 31
]. Briefly, 1 x 106 cells were incubated with FITC- or phycoerythrin (PE)-conjugated antibodies. Cells were stained for CD54 (1A29), CD71 (OX-26), CD3 (G4.18), CD45RA (OX-33), macrophage subset (HIS36), CD11b/c (OX-42), CD80 (3H5), and CD86 (24F; all from BD PharMingen, San Diego, CA) and MHC class II (OX-6; MCA46G) and OX62 (1029B; Serotec, Oxford, UK). All staining steps were performed at 4°C in PBS-EDTA-FCS. After incubation, cells were washed, fixed in 1% formaldehyde, and resuspended; 10,000 events were analyzed using Cytoron Absolute. Isotype controls (Sigma Chemical Co., St. Louis, MO) were run with each experiment and matched for fluorochrome. In each experiment, we included PP, MLN, and spleen cells from diluent-treated rats to control for intrinsic autofluorescence. On the basis of forward- and side light-scatter, mononuclear cells were gated in, and dead cells were gated out. Isotype-matched control histograms were included. Positivity for each probe was defined as fluorescence that exceeded 98% of controls [30
].
Cytokine assessment by enzyme-linked immunosorbent assay (ELISA) and flow cytometry
We determined IL-10 by ELISA using reagents and protocols obtained from BD PharMingen in supernatants of PP, MLN, or spleen cell suspensions, which were cultured with medium or restimulated with 10 µg/ml chitosan for 48 h at 37°C, 5% CO2. Levels of cytokines in cultures of cells from different groups without stimulation of any kind were below our level of detection. The in vitro restimulation was adapted from protocols used to assess cytokine production after oral administration of protein antigens [32
33
34
35
].
We evaluated intracellular IL-10 and interferon-
(IFN-) content ex vivo in T lymphocytes of PP, MLN, and spleen cells prepared as described above and cultured for at least 6 h in RPMI–10 µg/ml brefeldin A. Then, cells were treated with FITC- or PE-labeled anti-CD3 antibodies, fixed in 4% formaldehyde for 15 min, permeabilized with PBS–1% FCS–0.1% saponine, and stained with PE-conjugated anti-IL-10 or FITC-conjugated anti-IFN- antibodies (BD PharMingen) as described [24
, 36
]. Isotype controls (Sigma Chemical Co.) were run with each experiment and matched for fluorochrome. After extensive washing with PBS–1% FCS–0.1% saponine, cells were resuspended in PBS, and 10,000 events per sample were analyzed.
Expression of mRNA content for TNF-, IL-4, IL-12, and TGF-ß
Evaluation of TNF-, IL-4, and IL-12, in PP and TGF-ß mRNA in PP, MLN, and mononuclear spleen cells was performed as described [24
, 37
]. Briefly, total RNA was extracted by the TRIzol® reagent method (Life Technologies, Gibco, Grand Island, NY). TNF-, IL-4, and IL-12 (4 µg) and TGF-ß (2 µg) of total RNA were incubated with 0.5 µg oligo(dT; Biodynamics, Buenos Aires, Argentina) for 5 min at 70°C and allowed to stand on ice for 5 min. The sample was incubated for 1 h at 42°C with 25 U RNase inhibitor (RNasin, Promega, Madison, WI), 1.25 mM deoxynucleoside triphosphate (Invitrogen, Life Technologies, Carlsbad, CA), and 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase (RT; Promega) in MMLV 5x reaction buffer (Promega) in a final volume of 25 µl. In a total volume of 25 µl polymerase chain reaction (PCR) buffer (Invitrogen, Life Technologies, Brazil), 1 µl (for ß-actin), 2.5 µl (for TNF-), or 5 µl (for TGF-ß, IL-12, and IL-4) cDNA was incubated with 1.25 U Taq DNA polymerase (Invitrogen), 1.5 mM (ß-actin, IL-12, TNF-, and IL-4) or 2 mM (TGF-ß) MgCl2 (Invitrogen), 1 mM deoxynucleotide triphosphate, and 0.2 µM (ß-actin, TNF-, and IL-12) or 1 µM (TGF-ß and IL-4) sense and antisense primers [24
]. Each sample was incubated in a thermal cycler (PTC-100 thermal cycler, M.J. Research, Watertown, MA) using one cycle at 94°C for 5 min; this was followed by 25 cycles for ß-actin, 30 cycles for IL-4, or 35 cycles for TGF-ß, TNF-, and IL-12; each cycle consisted of 1 min at 94°C, 1 min at 55°C (ß-actin, TNF-, and IL-12) or 58°C (TGF-ß and IL-4), and 1 min at 72°C, with a final extension at 72°C for 5 min. The linear range of amplification for each primer pair was established in independent preliminary studies. PCR products were analyzed by 2% agarose gel electrophoresis in the presence of 0.5 mg/ml ethidium bromide. Bands were analyzed with the Scion Image program and expressed as densitometric units. Results were depicted as the ratio of mRNA levels for each cytokine relative to ß-actin mRNA levels.
Statistical analysis
Data are shown as mean values ± SD. Statistical significance and differences among groups were determined by ANOVA and Student-Newman-Keuls tests. P< 0.05 values were considered statistically significant.
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To evaluate the uptake in different intestine sections, we fed rats a single dose of 1 or 3 mg FITC-chitosan:chitosan mixture. Sixteen hours later, the complete small intestine (up to the ileocaecal junction) was removed and divided in five sections (see Materials and Methods). Single-cell suspensions from two PP per section were prepared and analyzed by flow cytometry to determine the percentage and mean fluorescence intensity (MFI) of total FITC-positive cells or FITC-positive cells gated on R1, where on the basis of forward- and side light-scattering, APC are found (Fig. 1A ). We used PP cell suspensions from diluent-treated rats as negative controls. As shown in Figure 1 , the percentage of FITC-positive cells (Fig. 1B and 1D) and the MFI (Fig. 1C and 1E) for total cells (Fig. 1B and 1C) and cells from the R1 region (Fig. 1D and 1E) decreased down the gut, with the lowest values in the distal sections (V section). Compared with total cells, R1-gated cells showed higher percentages (P<0.05) and a lower MFI (P<0.05). The reduced MFI values in this subset could be a result of a more efficient processing of the polysaccharide by enzymes such as chitinases present in APC [39 ]. No differences were observed with 1 and 3 mg chitosan on the basis of per-section analysis. The correlation between fluorescence recovery (percentage or MFI) and gut segment showed a significant negative trend (Fig. 1F) . Together, these results demonstrate that the uptake of chitosan takes place mostly at the upper portion of the small gut and is mediated mainly by cells of the region, where on the basis of forward- and side light-scattering, APC are found.
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Figure 1. Analysis of chitosan uptake at different sections of the small gut. Rats were fed 1 mg (o) or 3 mg (•) FITC-chitosan:chitosan, and 16 h after polysaccharide administration, the gut was divided in five 20-cm sections (I–V), and cell suspensions were prepared out of two randomly selected PP per section. Cells were analyzed by flow cytometry, defining total and R1 cells on the basis of scatter (A). Data are shown as percentage and MFI of total positive cells (B and C) or positive cells gated on R1 (D and E). The correlation analysis between fluorescence (percentage or MFI) and gut segment is shown (F). A representative experiment of two similar ones is shown.
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7%), the highest percentage of CD11b/c+ FITC+ cells (33%) was observed in PP 16 h after feeding the polysaccharide. Approximately 15% of CD11b/c+ cells in MLN was FITC+, and in spleen, only 7% of CD11b/c+ cells were also FITC+. These results show that 16 h after feeding, chitosan-loaded cells are present in PP, MLN, and spleen. However, the polysaccharide is mainly distributed in the DC/macrophage subpopulation at the inductive sites of the mucosal immune system.
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Figure 2. Phenotype of chitosan-loaded cells in PP, MLN, and spleen. Rats were fed diluent or 1 mg FITC-chitosan:chitosan, and 16 h later, PP, MLN, and spleen cell suspensions were stained with PE-conjugated anti-CD3, anti-CD45RA, antimacrophage subset (mac), and anti-CD11b/c. For the analysis, PE-positive cells corresponding to the different subpopulations were gated on and evaluated for their levels of FITC fluorescence. (A) Data are the mean ± SD of the percentage of PE-positive cells (open bars) and FITC-positive cells (solid bars) present in each lymphoid tissue. The results are representative of two separate experiments using three animals per group. (B) Representative histograms of PP, MLN, and spleen FITC+ cells gated on PE-positive cells. The thick, bold line represents the staining of cells from diluent group with isotype control. The thin line with shading below represents the FITC fluorescence of gated PE+ cells.
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, IL-12, and IL-4 mRNA in PP 16 h after the administration of a single dose of chitosan. As shown in Figure 3A
, the polysaccharide did not modify the IL-12 mRNA expression or even decreased the TNF- mRNA expression at the 3 mg dose (P<0.05 vs. diluent). However, a significant increment in the IL-4 mRNA expression was observed with both doses of chitosan (P<0.05 vs. diluent).
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Figure 3. Cytokine production early after chitosan feading. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later PP, MLN, and the spleen were removed. (A and B) Total RNA was prepared out of cell suspensions and subjected to RT-PCR to determine mRNA expression for TNF-, IL-12, and IL-4 in PP, and TGF-ß and ß-actin in PP and MLN with specific primers as described in Materials and Methods. PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. Bands were analyzed with the Scion Image program and expressed as densitometric units. Results are depicted as the ratio of mRNA levels for each cytokine relative to ß-actin mRNA levels. Data are the mean ± SD of two experiments, each performed using total RNA obtained from two to four animals per group (*, P<0.05 vs. diluent). (C) PP, MLN, and spleen cell suspensions were restimulated with 10 µg/ml chitosan for 48 h to determine IL-10 in supernatants by ELISA. Shown is IL-10 concentration in picograms per milliliter and the SD of triplicate determinations. (*, P<0.05, vs. diluent and 1 mg dose; N.D., not detected).
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Assessment of T cell phenotype and cytokine production
It is well accepted that immediately after oral administration of protein antigens, peripheral T cells become activated in vivo [34
]. We evaluated the expression of CD71 (early activation marker) and CD54 (activation marker) molecules in CD3+ cells after feeding a single 1-mg dose of chitosan (Fig. 4A
). The percentage of CD3+ CD71+ cells remained unchanged in PP, MLN, and spleen, although a significant increment of CD3+ CD54+ cells (P<0.05 vs. diluent) was observed 16 h after feeding the polysaccharide.
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Figure 4. Phenotypic and functional changes of CD3+-positive cells after chitosan administration. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later, PP, MLN, and spleen cells (1x106) were stained with FITC-labeled anti-CD3 antibodies and PE-labeled anti-CD71 or anti-CD54 antibodies (A), PE-conjugated anti-IL-10 (B), or PE-conjugated anti-IFN- (C). For intracellular cytokines, after CD3 staining, cells were fixed in PBS–4% formaldehyde, permeabilized, and treated with the appropriate monoclonal antibody (mAb); 10,000 events per sample were analyzed. (B) Shown are representative histograms for intracellular IL-10 levels, and the percentage of CD3+ IL-10+ cells of each group is indicated. The thick, bold line represents the staining of cells with isotype control. The thin line with shading below represents the intracellular staining of CD3+ cells with anti-IL-10 antibody. A representative experiment out of three is shown. (C) Data are the mean ± SD of the percentage of intracellular, IFN--positive cells of three animals per group. The results are representative of two separate experiments (*, P<0.05, vs. diluent).
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. Representative histograms show an increment of 41% in the percentage of CD3+ IL-10+ cells in PP of 3 mg chitosan-fed rats compared with the diluent group (Fig. 4B)
. Similar results were obtained with 1 mg chitosan (data not shown). A slight but significant (P<0.05 vs. diluent) increase in the percentage of CD3+ IFN-+ cells was observed in PP at both doses (Fig. 4C)
.
Assessment of costimulatory molecules in OX62+ cells in mucosal tissues
APC from inductive mucosal sites include immature DC, which show high expression of MHC class II antigens, display low stimulatory activity, and secrete TGF-ß and IL-10 [40
41
42
]. In rat, a subpopulation of DC expresses the 
2 integrin recognized by the OX62 mAb [43
, 44
]. In our experimental condition, FITC-chitosan+ cells are mainly located in R1 (Fig. 1)
and CD11b/c+ cell macrophages and DC uptake chitosan (Fig. 2)
. In consequence, we examined PP and MLN cell suspensions gated in a similar region (R1) for OX62, MHC class II antigens, and costimulatory CD80 and CD86 molecule expression. After the administration of the 3 mg dose, the percentage of OX62+ cells increased in PP (P<0.05 vs. diluent) without changes or even diminution in MLN (Fig. 5
). OX62+-gated cells showed a significant increment on the expression of MHC class II antigens after 3-mg chitosan administration (P<0.05 vs. diluent), with no changes in the levels of CD80 and CD86 molecules.
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Figure 5. Phenotype of DC of PP and MLN after chitosan feeding. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later, PP and MLN (1x106) were stained with FITC-labeled anti-OX62 and PE-labeled anti-MHC II anti-CD80 or anti-CD86 antibodies. The upper-left graph is a representative granularity versus size-density plot, where the R1 region was defined. Results are expressed as percentage of OX62+ cells in R1 in cell suspensions of PP and MLN or MFI of OX62+-gated cells for MHC class II, CD80, and CD86 markers. Average data of three rats per group are depicted (*, P<0.05, vs. diluent). RT-SC, Right-scatter; FW-SC, forward-scatter.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unique features of immune responses in the gut are related to the microenvironment and to the presence of specialized APC populations. Freshly isolated PP DC seem phenotypically and functionally immature, express comparably low levels of costimulatory CD80 and CD86 molecules, and release IL-10 and TGF-ß after stimulation [46
, 47
]. Mucosal resident DC have a propensity to induce responses rich in IL-10 and TGF-ß cytokines, although Th1 responses can be induced following intestinal infections. We demonstrate here that a single dose of chitosan, in the absence of protein antigen, triggers at the local microenvironment the release of IL-10 as well as the expression of IL-4 and TGF-ß mRNA. Cytokines such as IL-10, TGF-ß, and IL-4 are known to suppress the function of APC. For instance, IL-10 selectively inhibits the expression of B7 on murine macrophages without effect on the up-regulation of MHC class II expression [48
]. In agreement, in our experimental condition, the minor release of IFN- in PP and the up-regulation of MHC class II molecules seem to be counterbalanced by the huge production of IL-10 triggered by the polysaccharide.
We show here that chitosan has biological activity at mucosal and systemic levels. The uptake and distribution of the polysaccharide as well as the activation of spleen T cells that we found are similar to previous results with peptides, proteins, or food antigens [32 , 34 , 49 ]. The transient T cell activation induced by food antigens has been related to the maintenance of the peripheral T cell population and to the shape of the T cell repertoire [32 ]. Considering that chitosan is a weak base that precipitates at neutral pH or higher [18 ], it is tempting to speculate that free chitosan is not reaching systemic lymphoid tissues as described for soluble antigens. In agreement, soluble antigens crossing the epithelial-cell barrier through the epithelium converge in the liver, and those transported by M cells or DC that reach lymphatics converge in the spleen [4 ]. Substances that can adhere to the epithelium or M cells are transported in much larger amounts. It is interesting that the route by which antigens cross the intestinal epithelial-cell barrier is likely to dictate the type of response that is generated [45 ].
Chitosan could exhibit natural properties to modulate the mucosal immune response: As a dietary fiber, it can influence local immune function by changing the intestinal flora and mucosal microenvironment [16
, 17
]. As a delivery agent, chitosan can decrease the clearance rate and stimulate the uptake of antigens by M cells [21
22
23
]. As a component of fungal cell walls and parasite sheaths, chitosan could provide "danger signals" acting as an adjuvant [50
, 51
], possibly through the activation of components of the innate immune system such as macrophages [26
, 27
]. Immunizations with several immunogens and chitosan administered at the mucosal interface increase antibody responses [15
, 20
21
22
23
], particularly the immunoglobulin A production, a TGF-ß- and IL-10-driven process [2
, 4
, 47
]. When fed with a single low dose of protein, chitosan enhances the antigen-specific release of IL-10 and TGF-ß [24
]. Besides, the IL-10, IL-4, and TGF-ß mRNA expression in PP is noticeably higher in rats fed multiple doses of chitosan before the antigen administration [24
]. In the same way, the intranasal administration of the immunodominant epitope of the Dermatophagoides pteronyssinus allergen adsorbed to chitosan suppresses airway inflammation and triggers the IL-10 production by antigen-specific T cells [52
]. In that experimental condition, an increment of IFN- is observed and is associated to the induction of a tolerogenic state [52
].
The strong stimulatory activity of chitinous derivatives has been mainly attributed to the N-acetyl-D-glucosamine residues [22
, 27
], and the effect could be mediated by a macrophage lectin receptor, as with mannose specificity [53
]. The activity of the polysaccharide seems to depend on the activation status of the cell: Chitosan inhibits the production of inflammatory mediators by proteose-peptone-elicited peritoneal macrophages [27
] or LPS/IFN--activated RAW 264.7 cells [54
, 55
], but it stimulates the release of proinflammatory mediators by resident peritoneal macrophages [27
] or unstimulated RAW 264.7 cells [54
, 55
]. The apparent conflicting results obtained in vitro and in vivo are not necessarily opposite effects but could reflect differences in experimental conditions or parameters evaluated. It is important to point out here that previous reports using chitosan as an adjuvant, as well as this study where the intrinsic activity of chitosan was evaluated, show that in vivo, this polysaccharide consistently enhances the natural phenomena that occur at the mucosal level. Together, our results demonstrate that orally administered chitosan, in the absence of protein antigen, enhances a naturally Th2/Th3-biased microenvironment at the mucosal level by stimulating the production of regulatory cytokines. Its activity could be relevant in the maintenance of the intestinal homeostasis. Our results open the possibility that in a near future, this or other polysaccharides could be used to modulate the immune response to orally administered antigens.
Received September 21, 2004; revised February 24, 2005; accepted March 4, 2005.
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+, and double-negative Peyer’s patch dendritic cells J. Immunol. 166,4884-4890This article has been cited by other articles:
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