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Four-week-old male C3H/HeN mice were bred with diets consisting of ovalbumin alone (OVA, control diet) or mixtures of OVA and cow's milk immunoglobulin G (IgG-added diets) as a protein source for 4 or 5 weeks, and both the cellular and humoral immune properties of the mice were investigated. The number of interleukin (IL)-12+CD11b+ cells in spleens and the formation of superoxide by peritoneal macrophages were higher in mice given the IgG-added diet than in those given the control diet. The number of natural killer cells in Peyer's patches or spleens and the cytotoxic activity of spleen cells toward an erythroleukemia cell line, K562, were also higher in mice given the IgG-added diet. In contrast, the numbers of interferon-gamma+CD4+ and IL-4+CD4+ cells in Peyer's patches or spleens and the levels of total or OVA-specific intestinal IgA and serum IgG were significantly lower in mice given the IgG-added diet than in those given the control diet. In addition, the number of cells expressing CD19 in spleens was significantly higher in mice given the IgG-added diet. These results indicate that oral ingestion of cow's milk IgG may stimulate some innate cellular immune systems, while suppressing humoral adaptive immune responses in the mouse.

Peyer's patches constitute both an inductive immune site and an enteropathogen invasion route. Peyer's patch mucosae from porcine jejunum were mounted in Ussing chambers, and either Salmonella choleraesuis vaccine strain SC-54 or non-pathogenic rodent and porcine Escherichia coli strains contacted the Peyer's patch mucosa for 90 min. Internalized bacteria were quantified by a gentamicin resistance assay. Monodansylcadaverine (300 microM, luminal addition), an inhibitor of clathrin-mediated endocytosis, significantly inhibited internalization of both E. coli strains relative to tissues untreated with the inhibitor; internalization of SC-54 was unaffected. The actin-disrupting agent cytochalasin D (10 microM, luminal addition), inhibited internalization of pig-adapted E. coli but not that of rodent-adapted E. coli or SC-54. Internalization of SC-54 and non-pathogenic E. coli in Peyer's patches appears to occur through different cellular routes.

Triptolide is a purified component from a traditional Chinese herb Tripterygium wilfordii Hook F. It has been shown to have anti-inflammatory and immunosuppressive activities by its inhibitory effect on T cells. But the effect of triptolide on Peyer's patch cells is unknown. Enteric mucosal immune system, including Peyer's patch, is regarded as one of the sites for inducing immunity tolerance, and this intolerance effect has been used to induce oral tolerance which can considerably reduce arthritis severity in several models of experimental polyarthritis and RA patients. In this study, we investigated the effect of triptolide on the Peyer's patch cells and peripheral lymphocytes in collagen induced arthritis (CIA) in rats. CIA in rat is a widely studied animal model of inflammatory polyarthritis with similarities to rheumatoid arthritis (RA). Our data show that triptolide could lower the arthritic scores and delay the onset of CIA. There are more Peyer's patches in triptolide treated rats than in control rats, while there is no difference in Peyer's patch numbers between CIA rats and triptolide treated rats. In the Peyer's patch, more CD4+ cells are observed in CIA rats, and the numbers of CD4+ cells in triptolide treated rats and control rats are similar. While more CD8+ cells are observed in triptolide treated rats, and the numbers of CD8+ cells in CIA rats and control rats are similar. In periphery, more CD4+ cells and less CD4+ cells in CIA rats and triptolide treated rats are respectively observed. Therefore, the regulation on Peyer's patch might explain some of the immunosuppressive activities of triptolide, and enteric immune response might be actively involved in CIA pathogenesis. It is suggested that the Peyer's patch is one of the primary targets of the immunosuppressive activity of triptolide.

A significant increase in lymphocyte apoptosis was detected by the TUNEL method in the thymus, spleen, and Peyer's patches (PP) following intraperitoneal (i.p.) administration of dimethyl sulfoxide (DMSO) (treatment, n = 47; control, n = 8). Interestingly, administration of low doses of DMSO caused apoptosis in only the PP, and suggested that i.p. administration of DMSO induced apoptosis for each lymphoid organ in a dose dependent manner. Moreover, in the early stage during the apoptotic change, a characteristic localization of lymphocytes undergoing apoptosis was observed. Briefly, early apoptosis occurred predominantly in the cortical mid-zone of the thymus, white pulp of the spleen, and germinal centers of PP. With increased time following administration, however, lymphocytes throughout lymphoid tissues, independent of characteristic localization during the early stage, seemed to undergo apoptosis, resulting in the severe loss of lymphocytes. In fact, the relative spleen weight significantly decreased at 24 h following DMSO administration (n = 7; P < 0.001 versus 8 control mice). Taken together, these results showed for the first time that the in vivo administration of DMSO to mice caused apoptosis in lymphoid organs, and also demonstrated that the apoptotic behavior varied between different lymphoid organs.

Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin, which exhibits particular properties: interacts with negatively charged sites on the cell surface; changes the permeability of intestinal epithelium, enhancing the uptake of peptides and proteins; and activates leukocytes. Antigens coadministered or encapsulated with the polysaccharide show improved mucosal and systemic humoral immune responses, although the mechanism is poorly understood. Herein, we characterized in Peyer's patches mesenteric lymph nodes and spleen molecular events triggered after oral administration of chitosan in the absence of protein antigen. Sixteen hours after feeding, we studied the uptake and distribution of the polysaccharide, the phenotype of recruited antigen-presenting cells (APC), the induction of cytokines such as tumor necrosis factor alpha, interleukin (IL)-12, IL-4, IL-10, and transforming growth factor-beta (TGF-beta), 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-beta 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.

The purpose of this study was to investigate the acute effect of ethanol on mucosa-associated lymphoid tissue at the level of Peyer's patches and the intestinal lamina propria in female rats and to determine whether this action of ethanol is modulated during the estrous cycle. Adult female rats showing proestrus or diestrus day 1 were treated intraperitoneally (ip) with ethanol (4 g/kg). Untreated and saline-injected rats were used as controls. The animals were sacrificed by decapitation 0.5 h after ethanol administration. Immunoglobulin A (IgA) immunoreactive cells were analyzed by indirect immunohistochemistry using mouse anti-rat IgA and a Dako LSAB+ kit. The number of IgA-immunoreactive cells in Peyer's patches was unaltered by ethanol treatment at both phases of the estrous cycle. However, stereological analysis revealed a significant increase in the number of IgA-immunoreactive cells (p < 0.01) in the intestinal lamina propria following acute ethanol administration at proestrus and on diestrus day 1. The results indicate that the intestinal lamina propria, the effector site of the mucosal immune system, can be affected by a single dose of ethanol at both phases of the estrous cycle.

It is well established that glucocorticoids are key regulators of the immune system and act as immunosuppressive agents in high concentrations. In the pig, effects on the gut immune system and trafficking of lymphocytes between tissues and blood plasma were not investigated so far. Twelve pigs of 70 kg were fed 0.4 mg portions of dexamethasone (Dexa) twice daily for 9 days or remained untreated (controls) and were sacrificed for tissue collection at the end of Dexa treatment. Another six pigs with jugular vein catheters were left untreated for 7 days (control period) and then received Dexa for 9 days. Blood was drawn twice during the control period and at days 3, 6 and 9 of the Dexa period for characterization of peripheral blood leukocytes. Cells were obtained from thymus, mesenteric lymph nodes, jejunal mucosa and Peyer's patches. Lymphoid cells from gut tissue were isolated from two fractions: the EDTA-fraction, containing the intraepithelial lymphocytes (IEL), and the Collagenase-fraction, containing the lamina propria lymphocytes (LPL). In all samples, cell counts and phenotypic characterization of cells by flow cytometry (FCM) were performed. In thymus, Dexa led to a more than 90% reduction of the absolute cell number, which was mainly found in the CD4+CD8+ subpopulation. Dexa effects on lymphocytes from mesenteric lymph nodes were less severe (50%) and led mainly to a decrease (71%) of B-lymphocytes. The number of lymphocytes in the EDTA-fraction (IEL) of the jejunal mucosa decreased significantly by 56% in the Dexa-treated animals compared to the controls, whereas the number of lymphocytes in the Collagenase-fraction (LPL) decreased only moderately. In the Peyer's patches, a decreasing tendency in the number of lymphocytes in the EDTA-fraction was observed which, however, was not significant. In blood, monocytes and granulocytes were significantly increased in an order of 60%. The data show that supraphysiological amounts of Dexa remarkably reduce cell numbers in thymus and also in the intraepithelial compartment of the jejunal mucosa and ileal Peyer's patches. In blood, a notable homeostasis was observed for several leukocyte populations whereas both monocytes and granulocytes increased.

Chemical carcinogenesis studies are powerful tools to obtain information on potential mechanisms of chemical factors for malignancies. In this study Western blot analyses, using monoclonal antibodies specific for three different cytochrome P450 (CYP) isozymes (CYP1A1, CYP1A2 and CYP2B), were employed to examine the effect(s) of 3-methylcholanthrene and/or pristane (2,6,10,14-tetramethylpentadecane) on the basal and inducible levels of expression of CYP proteins within Copenhagen rat tissues. Pristane exposure led to tissue specific differences in the CYP isozymes expressed and elicited increased CYP protein expression over 3-methylcholanthrene induced levels in microsomes isolated from liver, Peyer's Patches, and thymus. Within the context of the chemical carcinogenesis model employed in this study, these observations correlated with the induction of B-cell malignancies by low doses of 3-methylcholanthrene and of thymic lymphomas by a high 3-methylcholanthrene dose. The data suggest that pristane treatment affects CYP isozyme expression. This pristane-mediated effect clearly could be a contributing factor in the chemical carcinogenesis of the previously observed lymphoid malignancies, and a possible basis for the tumor enhancing effects of pristane.

Circulating levels of vitamin A (retinol) and lactoferrin (Lf) are low in calves at birth. Bovine colostrum contains relatively high amounts of vitamin A and Lf, and both substances are intestinally absorbed by neonatal calves. There is evidence that these compounds interact with insulin-like growth factor binding proteins and thus influence the status and effects of insulin-like growth factor. The hypothesis was therefore tested that vitamin A and Lf influence epithelial growth, development, and absorptive capacity of the small and large intestine and modulate intestinal immune tissues (Peyer's patches; PP). Four groups of calves (n = 7 per group) were fed a milk-based formula with or without vitamin A and (or) Lf. Group F received formula (F) only; group F(A) was fed F supplemented with vitamin A; group F(L) was fed F supplemented with Lf, and group F(AL) received F plus vitamin A plus Lf. An additional group of calves (group C; n = 7) served as positive control and was fed colostrum (C) from pooled milk obtained on d 1, 2, and 3 of lactation. Amounts of nutritive components in formula and colostrum were similar. Blood samples were taken to measure vitamin A and Lf, and plasma xylose (added on d 4 to feeds) was measured postprandially for 8 h as a marker of intestinal absorptive capacity. Plasma vitamin A was low at birth and further decreased in groups F and F(L), but increased in groups F(A), F(AL), and C. Plasma Lf was low at birth and transiently increased up to 4 h after the first meal in group C. Xylose absorption was higher in group C than in other groups. Incorporation of 5-bromo-2'-deoxyuridine into DNA (as a measure of cell proliferation rate) was enhanced in intestinal crypts in groups F and F(L) at all intestinal sites. Ileum villus heights of groups F and F(L) were smaller than of groups F(A) and F(AL). Villus height to crypt depth ratios were smaller in F-fed groups (especially in groups F and F(L)) than in C-fed calves in the duodenum and jejunum. Incorporation of 5-bromo-2'-deoxyuridine into colon crypt cells of group F was greater than in groups F(L) and F(A). Sizes of follicles of PP in the ileum were greater in group F(A) than in group F. In the ileum, vitamin A and Lf tended to interact with PP size. In conclusion, feed supplementation of vitamin A and Lf influenced growth of the ileum and colon. Interactions were observed between vitamin A and Lf on epithelial cell maturation, villus growth, and size of follicles in PP of neonatal calves.

The structure of the Peyer's patches and mesenteric lymph nodes of rats at different periods after polychemotherapy was investigated by light microscopy. After the use of antitumor drugs the number of the blasts and mytoses in the lymphoid follicles with the mesenteric lymph node bright centres and in the Peyer's patch follicles lowered, that along with the decrease of the size of the mantle zone in the lymph node follicles and the decrease of the area of the bright centres in the follicles of the lympoid formations in the intestinal wall was evident of the proliferation inhibition and B cells differentiation in the lymphoid organs. After the polychemotherapy the size of the germinative centres of the Payer's patch follicles decreased while the size of the mantle zone remained unchanged. The size of the mantle zone in the follicles of the mesenteric lymph nodes decreased while the size of the germinative centres did not change. The different responses of the lymphoid organs could be associated with some remote location of the lymph nodes with respect to the antigen source (damaged epithelium and intestinal lumen contents).

Probiotics (PRO) are known to modulate immunity in animals and human subjects and to inhibit colon carcinogenesis in experimental models, but the effects of synbiotics (SYN) are not well understood. Therefore, the effects of PRO (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12), PRE (inulin-based enriched with oligofructose, 100 g/kg) and SYN (combination of PRO and PRE) on the immune system of rats were investigated in the azoxymethane (AOM)-induced colon cancer model. After 33 weeks, rats with and without AOM treatment were killed and immune cells were isolated from spleen, mesenterial lymph nodes (MLN) and Peyer's patches (PP). AOM treatment significantly reduced natural killer (NK) cell-like cytotoxicity in control rats and in PRO- and PRE-supplemented rats. SYN supplementation prevented the AOM-induced suppression of NK cell-like cytotoxicity in PP compared with control rats (P<0.01). SYN and PRE supplementation stimulated IL-10 production in PP in these rats (P<0.01) and in MLN of rats not treated with AOM (P<0.05). Interferon-gamma production in PP was decreased by PRO supplementation (PRO and SYN groups combined; P<0.05). Proliferative responsiveness of lymphocytes (PP) from AOM-treated rats was suppressed in SYN-supplemented rats (P<0.01). Overall, SYN supplementation in carcinogen-treated rats primarily modulated immune functions in the PP, coinciding with a reduced number of colon tumours. PRE and PRO provided in combination as SYN may contribute to the suppression of colon carcinogenesis by modulating the gut-associated lymphoid tissue.

Background: Bombesin, the amphibian analog of mammalian gastrin-releasing peptide, reverses total parenteral nutrition (TPN)-induced atrophy of gut-associated lymphoid tissue and increases intestinal and respiratory immunoglobulin A (IgA) levels. Structure-activity studies suggested that the biologically active portion of bombesin is a C-terminal heptapeptide (7AA). This study investigates the effect of 7AA on lymphocytes counts of the Peyer's patches (PP), the lamina propria (LP) and the intraepithelial layer (IE)., Methods: Forty-eight male mice were randomized to receive chow (n = 13), TPN only (n = 9), TPN + 15 microg 7AA 3 times per day (n = 13) or TPN + 150 microg 7AA 3 times per day (n = 13). After 5 days of feeding, PP, LP, and IE lymphocytes were determined. Intestinal IgA levels were measured with ELISA. Groups were compared with ANOVA., Results: All TPN-fed mice lost more weight than mice fed chow (p < .04). Lymphocyte counts in PP, LP, and IE were significantly lower in the TPN group than in the 3 other groups but did not differ between the groups fed chow, TPN + 15 microg 7AA 3 times per day, or TPN + 150 microg 7AA 3 times per day. Intestinal IgA levels were higher in chow-fed mice (148.4 +/- 16.9) than in mice fed TPN (98.4 +/- 14.0, p = .008), TPN + 15 microg 7AA 3 times per day (96.9 +/- 7.7, p = .003) or TPN + 150 microg 7AA 3 times per day (87.3 +/- 6.7, p = .001)., Conclusions: The C-terminal heptapeptide of bombesin prevented the TPN-induced decrease in intestinal lymphocyte populations but not the reduction in intestinal IgA levels.

In this study, we examined which cell population contributes to IL-5 production by Peyer's patch (PP) cells. Thy1.2(-) fraction of PP cells, but not those of splenocytes, secreted IL-5 in response to IL-2. We found that CD3epsilon(-)IL-2Ralpha(+) cells purified from the Thy1.2(-)B220(-) fraction of PP cells secreted IL-5 when stimulated with IL-2. CD3epsilon(-)IL-2Ralpha(+) cells were subdivided into CD4(+) and CD4(-) populations or c-kit(+) and c-kit(-) populations, and only the CD4(-) and c-kit(-) CD3epsilon(-)IL-2Ralpha(+) cells secreted IL-5 in response to IL-2. CD3epsilon(-)IL-2Ralpha(+) cells did not express NK cell-markers and exhibited a lymphoid morphology. We have therefore identified CD3epsilon(-)IL-2Ralpha(+) cells as a unique lymphoid population that are not classified into conventional IL-5-producing cell populations, such as T cells, mast cells and NK cells. Depletion of CD3epsilon(-)IL-2Ralpha(+) cells from PP resulted in reduced IL-5 production. Furthermore, IgA secretion by B cells was increased when PP B cells were cocultured with CD3epsilon(-)IL-2Ralpha(+) cells. Taken together, these results suggest that the novel subset of CD4(-)c-kit(-)CD3epsilon(-)IL-2Ralpha(+) PP cells are capable of secreting a high level of IL-5 in response to IL-2, contribute markedly to IL-5 production and help IgA secretion by B cells.

Background: Experimentally, total parenteral nutrition (TPN) diminishes gut-associated lymphoid tissue (GALT) cell numbers and function. Although glutamine supplementation is known to reverse TPN-induced changes in GALT, effects of another conditionally essential amino acid, L-arginine (ARG), on GALT remain unclear., Methods: Twenty-two male Institute of Cancer Research mice were randomized to standard TPN (0.3% arginine, STD-total parenteral nutrition) or 1% ARG-enriched TPN (ARG-total parenteral nutrition). After 5 days of feeding, lymphocytes were harvested from Peyer's patches (PP), the lamina propria, and intraepithelial (IE) spaces of the small intestine to determine cell yields. Lymphocyte phenotypes (alphabetaTCR, gammadeltaTCR, CD4, CD8, and B220 as a B cell marker) were determined using flow cytometry. IgA levels in washings of the small intestine, upper respiratory tract, and lungs were measured with ELISA., Results: ARG-total parenteral nutrition did not affect lymphocyte yields. The percentages of CD4+ cells in PP and IE, and alphabetaTCR+ cells in PP, were significantly higher in the ARG-total parenteral nutrition than in the STD-total parenteral nutrition mice, without marked differences in other phenotypes examined. There were no significant differences in intestinal and respiratory tract IgA levels between the 2 groups of mice., Conclusions: One percent ARG supplementation of TPN does not improve GALT cell number or mucosal IgA level but benefits to increase CD4+ cell percentages in GALT.

Apical membrane-located receptors on intestinal Peyer's patch M cells have been identified as pathogen binding sites which have potential as targets for the delivery of formulated subunit and recombinant vaccine antigens. Gene expression studies have correlated outputs from a series of models including human intestinal M-like cell cultures and human Peyer's patch tissue. Outputs comprise novel receptors with unknown function and also conserved binding sites for an ever-increasing list of pathogens. Screening for ligands to mimic pathways used by selected pathogens for invading the human intestine has the potential for increasing the efficiency of delivery of oral vaccine antigens to M cells and consequently to sub-epithelial sites for antigen processing. Synthesis of efficient and specific targeting ligands to human M cells may eventually lead to formulations based on the formats of targeted antigen-loaded nanoparticles or ligand-conjugated stable antigens.

An intestinal immune system modulating arabino-3,6-galactan (ALR-5IIa-1-1) has been found in rhizomes of Atractylodes lancea DC. [Planta Medica 1998, 64, 714-719; Carbohydr. Polyms. 2001, 46, 147-156], however other arabino-3,6-galactans from Larix and Acacia failed to express the modulating activity. Degradation of the galactosyl side chains in Araf-side chain-trimmed ALR-5IIa-1-1 (AF-ALR-5IIa-1-1) with an endo-beta-D-(1-->6)-galactanase remarkably decreased the activity of AF-ALR-5IIa-1-1. Structural analysis indicated that the major endo-beta-D-(1-->6)-galactanase-digestable side chains in ALR-5IIa-1-1 are composed of beta-D-(1-->6)-galactopyranosyl oligosaccharides having d.p. 1-8. Because degradation of the beta-D-(1-->3)-galactan backbone in AF-ALR-5IIa-1-1 also significantly reduced its activity, some of these galactosyl side chains attached to beta-D-(1-->3)-galactan backbone are suggested to be responsible for expression of the activity of ALR-5IIa-1-1.

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-alpha (IFN-alpha) producing cells in blood. In this study, C57BL/10 (B10;H2b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 microg/d; i.p.; 10 days) that expands DC selectively in vivo. Putative pDC (CD11c+B220+) were identified in the subepithelial dome and in interfollicular regions of intestinal Peyer's patches (PP) from both normal and Flt3L-treated animals. Freshly-isolated, immunobead-purified CD11c+ DC from PP were flow-sorted to obtain lineage- (CD11b-CD19-) CD11c+ B220+ DC (purity>96%). Flow cytometric analysis revealed that these sorted PPpDC were negative for surface markers associated with myeloid DC (CD11b) and expressed only low levels of the "lymphoid-related" DC marker CD8alphaalpha+. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of naïve allogeneic (C3H; H2k) T-cell proliferation. Cytospin preparations of sorted CD11c+B220+ cells revealed plasmacytoid morphology similar to that of human pDC. Immunocytochemistry and enzyme immunoassay revealed that, within 24-hour culture with Herpes simplex virus (10 p.f.u./cell), a subpopulation of stimulated (but not unstimulated) CD11c+B220+ DC produced and secreted IFN-alpha. This novel DC subset may play important roles in innate and adaptive immune responses of the gut and in the regulation of mucosal immune reactions.

In this study, we observed the in vivo acute immunotoxicity of a trimethyl arsenic compound, arsenobetaine (AsBe), which is present in large quantities in various marine animals that are daily ingested as seafood in many countries. The synthetic pure AsBe was orally administered to CDF(1) mice at a dose of 1.625 g/kg mouse weight once a day on days -6, -4, -2 and 0 (four times, total 6.5 g/kg mouse weight), and its effect on the immune organs and immune effector cells were assessed until day 8. Orally administered AsBe was temporally distributed to the immune organs, such as the spleen and thymus, but was not very toxic both quantitatively and qualitatively on these immune organs and immune effector cells, splenocytes, thymocytes, Peyer's patch lymphocytes and peritoneal macrophages. This finding suggests that the ingestion of AsBe contained in marine animals is relatively safe to the health of people who often consume marine animals in their daily diet.

Food protein intake interacts with the immune system. In earlier nutritional and immunological studies, nutrients, particularly milk whey proteins, were generally administered in soluble form and by gavage. However, orogastric intubation does not represent a natural way of ingesting nutrients such as lactoferrin (Lf). We examined how different modes of oral administration of Lf could affect the regulatory effect of this molecule on intestinal and systemic immune responses. Groups of 10 female BALB/c mice were administered Lf daily for 6 wk. To address the influence of the oral modes of administration, mice were given Lf either in solution, by gastric intubation or in the drinking water, or as a powder, by buccal deposition or in the diet. Mucosal and systemic immune responses, including specific immunoglobulin (Ig) secretion, cell proliferation, and cytokine production, were analyzed and compared with those of naïve mice given water under the same conditions or positive control mice that were administered Lf by i.m. injection. The addition of Lf to the drinking water had no visible effect on the immune status. Gastric intubation, single buccal doses, and continuous doses of Lf in the diet stimulated transient systemic and intestinal antibody responses against Lf. All of these oral modes of Lf exposure biased mucosal and systemic T-cell responses toward Thelper (Th)2-types and elevated IgA production by mucosal cells. However, the less natural gastric intubation also promoted Th1-type responses as evidenced by serum IgG(2a) antibodies and the secretion of Th1 cytokine by mucosal and systemic T cells in vitro. Thus, one should carefully consider the oral mode of administration for understanding regulation of immune responses by food proteins such as Lf.

The influence of the peroral administration of the macromolecular components of a herbal immunomodulator isolated from an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix on the function of Peyer's patches cells was investigated in mice. Peyer's patches cells isolated from mice which had received oral administration of the macromolecular fraction of the plant extract developed a significantly enhanced plaque-forming cell (PFC) response to sheep red blood cells after incubation with these cells in the presence of lipopolysaccharide or the extract fraction for 7 days in vitro. These results show that after oral administration of the herbal immunomodulator, the immunologically active macromolecules can contact the cells of the gut-associated lymphoid tissue and modulate the mucosal immune response.

Jejunal Peyer's patches (JPP) are innervated sites of immune induction and enteropathogen infection. We investigated the role of enteric nerves in modulating pathogen entry into porcine JPP. Presumptive norepinephrine (NE)-containing nerve fibers were localized in JPP domes and follicle-associated villi by secondary immunofluorescence histochemistry. NE or the neuronal conduction blocker saxitoxin increased intracellular internalization of pathogenic Salmonella choleraesuis and Escherichia coli O157:H7, but not nonpathogenic E. coli, into isolated JPP mucosa. NE action was prevented by the alpha-adrenergic antagonist phentolamine. Withdrawal of enteric neural activity or NE administration appears to modulate JPP interactions with pathogenic bacteria.

Severe burn results in immunosuppression, with increased lymphocyte apoptosis in both the central and peripheral immune system. As atrophy of the small intestine has been described in mouse models and intestinal lymphocytes have been implicated in the burn inflammatory response, we examined the effects of burn and tumor necrosis factor (TNF)-alpha on lymphocytes in intestinal Peyer's patches. Anesthetized C57BL6 mice received a 30% full-thickness scald burn on the upper back. Sham-burned animals served as controls. Anti-TNF or control immunoglobulin (Ig) G antibody (200 microg) was given immediately after the burn. The animals were initially resuscitated with 2 mL of normal saline, and were then sacrificed 12 h postburn. Terminal deoxyuridine nick-end labeling (TUNEL) and proliferative cell nuclear antigen (PCNA) staining was performed. Apoptosis was quantified as apoptotic lymphocytes/high-powered field (hpf). Results, expressed as mean +/- SEM, were compared using analysis of variance (ANOVA) and the Student-Newman-Keuls test. All mice survived the burn. An initial time-course experiment demonstrated maximal Peyer's patch apoptosis 12 h after the burn. Sham mice had 25 +/- 7 TUNEL-stained cells/hpf in Peyer's patches, whereas burned mice had 93 +/- 18 cells/hpf (P < 0.05). In contrast, burned mice receiving anti-TNF antibody had 28 +/- 8 TUNEL-stained cells/hpf (P < 0.05 vs. burn), whereas sham mice receiving anti-TNF antibody had 20 +/- 4 cells/hpf. There were no significant differences in PCNA staining between the groups. Scald burn results in lymphocyte apoptosis in Peyer's patches. This apoptosis can be abrogated by the addition of anti-TNF antibody. Apoptotic changes may lead to the failure of the intestinal immunological barrier and increased risk of sepsis.

Objective: The purpose of this study was to determine whether fructooligosaccharides (FOS) exert an immunomodulating effect on Peyer's patches (PP), the main inductive site of the intestinal immune system. We investigated the effects of FOS in healthy and endotoxemic animals., Methods: Six-week-old female Balb/c mice were fed a control diet or a diet supplemented with 10% FOS over a period of 16 d. To induce endotoxemia, mice were challenged intraperitoneally with lipopolysaccharide (LPS) on day 15. PP were excised from mice, and lymphocyte subpopulations (B lymphocytes, T lymphocytes, CD4(+) cells, and CD8(+) cells) were determined by flow cytometry., Results: The FOS-enriched diet increased the total cell yield in healthy and endotoxemic mice (P < 0.001). Similarly, B lymphocytes were increased in both groups (P < 0.001). In contrast, T lymphocytes were unaltered in healthy mice but increased in LPS-challenged mice after FOS enrichment (P < 0.001). In endotoxemic mice but not in control animals, the increase of CD4(+) cells (P < 0.001) was more pronounced than that of CD8(+) cells (P < 0.001), thus increasing the CD4:CD8 ratio (P < 0.01)., Conclusion: FOS showed an immunostimulating effect on PP lymphocytes under healthy and endotoxemic conditions. Thus it can be concluded that FOS administration affects not only the large intestine but also the main inductive part of the mucosal immune system in the small intestine.

Dietary exposure to the common foodborne mycotoxin deoxynivalenol (DON) selectively upregulates serum immunoglobulin A (IgA) in the mouse, most of which is polymeric, thus suggesting that the mucosal immune system is a primary target. When ingested, DON has no adjuvant or antigen properties but, rather, induces polyclonal IgA synthesis and serum elevation in an isotype-specific fashion. Resultant hyperelevated IgA is polyspecific, autoreactive and is likely to be involved in immune complex formation as well as kidney mesangial deposition. These latter effects mimic IgA nephropathy, the most common human glomerulonephritis. At the cellular level, DON upregulates production of T helper cytokines and enhances T cell help for IgA secretion. Analogous effects are observed in the macrophage with IL-6 being of particular importance based on ex vivo reconstitution and antibody ablation studies as well as experiments with IL-6 deficient mice. Upregulation of cytokines by DON involves both increased transcriptional activation and mRNA stability which are mediated by activation of mitogen-activated protein kinases. Interestingly, dietary omega-3 fatty acids can downregulate these processes and ameliorate DON-induced IgA nephropathy. From the perspective of gut mucosal immunotoxicology, these studies demonstrate that the capacity of a chemical to affect mucosal immune response can have systemic repercussions and, further, that these effects can be modulated by an appropriate nutritional intervention.

Tryptanthrin, a biologically active compound found in the medicinal plant Polygonum tinctorium, reportedly has several biological activities. We investigated the effects of tryptanthrin on cytokine production by lymphocytes in response to staphylococcal enterotoxin B (SEB), which causes a variety of disorders in humans based on its induction of large amounts of immunostimulatory cytokines. Tryptanthrin dose-dependently inhibited interferon-gamma (IFN-gamma) and interleukin-2 production by mouse spleen cells and Peyer's patch (PP) lymphocytes in vitro. The efficacy of tryptanthrin was further studied in a mouse model in vivo. Tryptanthrin was administered orally 2 h after an oral challenge with SEB. Nineteen hours after SEB administration, PP lymphocytes were prepared, and IFN-gamma production by PP lymphocytes was examined. The production of IFN-gamma increased after SEB administration, and the elevated IFN-gamma production was significantly inhibited by tryptanthrin treatment. These results suggest that tryptanthrin may be effective in the treatment of disorders of the intestines, such as food poisoning, that are associated with activated lymphocytes.

Background: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion molecule that directs naive T and B cells into Peyer's patches for sensitization and distribution to intestinal and extraintestinal sites. With no enteral stimulation, its expression drops rapidly in association with reduced Peyer's patch cell populations and increases rapidly with reinstitution of enteral feeding. Because both glutamine (GLN) and bombesin (BBS) preserve mucosal immunity, this study examined whether they preserve MAdCAM-1 expression., Methods: In 2 separate experiments, animals were randomized to IV cannulation with chow, total parenteral nutrition (TPN), and (experiment 1) 15 microg/kg BBS 3 times per day or (experiment 2) an isocaloric, isonitrogenous 2% GLN-supplemented solution. After 5 days of feeding, MAdCAM-1 expression in Peyer's patches, spleen, and intestine was measured using a dual radiolabeled monoclonal antibody technique., Results: MAdCAM-1 expression was not significantly improved from TPN levels either with BBS or GLN supplementation. Levels of MAdCAM-1 expression remained unchanged in non-Peyer's patch sites., Conclusions: Although MAdCAM-1 is considered the gateway molecule for cell entry into mucosal immunity, this does not seem to be the mechanism for mucosal immunity preservation in nonenterally fed mice receiving bombesin or glutamine.

The crypt and villus epithelium associated with Peyer's patches (PPs) is largely spared from methotrexate (MTX)-induced damage, compared with the non-patch (NP) epithelium. To assess the mechanism(s) preventing damage to the PP epithelium after MTX treatment, epithelial proliferation, apoptosis, and cell functions were studied in a rat-MTX model. Small intestinal segments containing PPs were excised after MTX treatment. Epithelial proliferation and apoptosis were assessed by detection of incorporated BrdU and cleaved caspase-3, respectively. Epithelial functions were determined by the expression of cell type-specific gene products at mRNA and protein level. Before and after MTX treatment, the number of BrdU-positive cells was higher in PP crypts than in NP crypts. BrdU incorporation was diminished in NP crypts, while in PP crypts incorporation was hardly affected. In PP and NP crypts, similar and increased levels of cleaved caspase-3-positive cells were observed after MTX. The enterocyte markers, sucrase-isomaltase, sodium-glucose co-transporter 1, glucose transporters 2 and 5, and intestinal and liver fatty acid binding protein, were down-regulated after MTX in NP epithelium but not in PP epithelium. In contrast, expression of the goblet cell markers, Muc2 and trefoil factor 3, and the Paneth cell marker, lysozyme, was maintained after MTX in both PP and NP epithelium. In conclusion, as MTX-induced apoptosis was similar in PP and NP crypts, the protection of the PP epithelium seems to be based on differences in the regulation of epithelial proliferation. Enterocyte function in the PP epithelium was unaffected by MTX treatment. Goblet and Paneth cell function was maintained in both NP and PP epithelium., (Copyright 2002 John Wiley & Sons, Ltd.)

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.

Tacrolimus (FK) and cyclosporine (Cs) are potent immunosuppressants that effectively prevent the rejection of transplanted organs including liver and small intestine. Our study examined the effects of these immunosuppressants on Peyer's patches, which play an important role in mucosal immune response through uptake and transport of enteric microorganisms and macromolecules in the gut-associated lymphoid tissues. After administration of FK and Cs, we assessed changes in lymphoid follicle structure and quantified the uptake and transport of particles in the follicle associated epithelium (FAE) including M cells, using fluorescent latex microspheres in rabbit Peyer's patches. Rabbits, five in each group, received oral administration of FK (3.2 mg/kg), Cs (10 mg/kg), or phosphate-buffered saline daily for 7 days. After 2 days of withdrawal, rabbits were anesthetized, and received injections with 2 ml of the suspension of 0.5-microm fluorescent microspheres (1010/ml) into ligated intestinal segments containing Peyer's patches. After 2 hr of gentle agitation, segments were removed, rinsed, fixed with periodate-lysine-2% paraformaldehyde, frozen, and sections were stained with fluorescent phalloidin to label brush border actin filaments. The size of the lymphoid follicles in each group was measured under a light microscope. The number of microspheres in follicles was assessed in graphically defined areas of follicles from each group. In addition, immunohistochemical analysis of CD43 and MHC-II positive cells in FAE of lymphoid follicles of each group was performed. FK and Cs significantly reduced the height of lymphoid domes and the height and width of follicles, as compared to those of controls. In both FK and Cs groups, the numbers of microspheres that adhered, were taken up and were transported into lymphoid follicles were smaller than in controls, indicating that their movement rates into deep layers were markedly reduced. Furthermore, FK and Cs reduced the mean numbers of CD43 and MHC-II positive cells in FAE per unit area (mm2) as compared with controls. These findings suggest that FK and Cs may produce immunosuppressive effects, at least in part, through reduction of the uptake and transport of particles into Peyer's patches, and by reduction of the number of immunoreactive cells in FAE of Peyer's patches.

ICR:CD-1 male mice were orally administered with Nivalenol(NIV) at the dose levels of 5, 10 and 15 mg/kg body weight, and examined at 12, 24 and 48 hours after inoculation (HAI), respectively, to elucidate the process of development of apoptosis in the thymus, spleen and Peyer's patch. There were no signs of clinical disorders and no changes in body and organ weights until 48 HAI except for that the thymus weight significantly decreased at 48 HAI. Immunohistochemically, the number of apoptotic lymphocytes evaluated by in situ detection for fragmented DNA showed a dose-dependent increase at 12 HAI in both the thymus and the Peyer's patch, while it became to increase at 24 HAI in the spleen. Dead lymphocytes in the thymus, spleen and Peyer's patch showed ultrastructural characteristics of apoptosis. Moreover, the DNA ladder was first detected by agarose gel electrophoresis at 12 HAI in the thymus of 15 mg/kg-group. The results clearly indicate that NIV is able to induce apoptosis in the lymphoid tissues of mice.

The effects of oral administration of milk and soy milk kefirs on tumor growth in tumor-bearing mice and the mucosal immunoglobulin A response in mice were studied. Oral administration of milk and soy milk kefirs to mice inoculated with sarcoma 180 tumor cells resulted in 64.8% and 70.9% inhibition of tumor growth, respectively, compared with controls. In addition, oral administration of the two kefir types induced apoptotic tumor cell lysis. Total immunoglobulin A levels for tissue extracts from the wall of the small intestine were also significantly higher for mice fed a milk kefir or a soy milk kefir regimen for 30 days. These results suggest that milk and soy milk kefirs may be considered among the more promising food components in terms of cancer prevention and enhancement of mucosal resistance to gastrointestinal infection.

In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer's patches (PPs), and mesenteric lymph nodes. Mice were implanted s.c. with a morphine slow release pellet. Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later. Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days. Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo. Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants. Morphine also inhibited CT-specific IgA and IgG levels in serum. By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo. The effect of morphine on TGF-beta, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT. Morphine significantly decreased TGF-beta mRNA compared with that in the placebo group, and naltrexone blocked this effect. These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-beta, a putative IgA switch factor, in the gastrointestinal tract.

Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-beta receptor-Ig fusion protein (LTbetaR-Ig) in utero, a treatment that blocked the formation of both Peyer's and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTbetaR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-gamma gene knockout (IFN-gamma(-/-)) mice. Four weekly treatments with LTbetaR-Ig resulted in deletion of Peyer's and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-gamma(-/-) mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTbetaR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-gamma. Thus, LTbetaR-Ig may offer a possible treatment for the Th2-dominant form of colitis.

Development of apoptosis and changes in lymphocyte subsets were examined mainly by flow cytometer in thymus, mesenteric lymph nodes and Peyer's patches of mice up to 24 hours after oral inoculation with T-2 toxin (10 mg/kg). T-2 toxin attacked Peyer's patches first, then mesenteric lymph nodes, and finally thymus in relation to the course of enteric absorption of orally inoculated T-2 toxin. The degree of lymphocyte apoptosis was prominent in the thymus, moderate in the Peyer's patches, and somewhat mild in the mesenteric lymph nodes, suggesting the difference in lymphocyte population susceptible to T-2 toxin. As to the changes in lymphocyte subsets, CD4+ CD8+ T cells were most sensitive to T-2 toxin, and CD4+ CD8- T cells were more severely depressed than CD4- CD8+ T cells in the thymus. In the mesenteric lymph nodes, CD3+ cells was more clearly affected than CD19+ cells, and the numbers of CD4+ and CD8+ cells were similarly decreased. In the Peyer's patches, the numbers of CD3+, CD 19+, CD4+ and CD8+ cells were unexceptionally decreased. In addition, among IgM+, IgG+ and IgA+ B cells, the number of IA+ B cells which are more important in the mucosal immunity was most severely affected.

The changes in T cell receptor (TCR) Vbeta expression, use, and clonality in mice orally challenged with Escherichia coli heat-labile enterotoxin (LT) were assessed. Use of the TCR Vbeta family and clonality were significantly changed at the single-cell level. In Peyer's patches of treated mice, use of TCR Vbeta6, Vbeta8, and Vbeta14 increased in CD4(+)CD44(+) T cells, compared with use in nontreated mice. On the other hand, use of TCR Vbeta1 and Vbeta8 was enhanced in splenic CD4(+)CD44(+) T cells. Intraepithelial lymphocytes isolated from LT-challenged mice showed expanded clonality (e.g., Vbeta1, Vbeta2, Vbeta9, and Vbeta18) and altered TCR Vbeta use (e.g., Vbeta15, Vbeta16, and Vbeta17). These findings reveal that oral administration of LT has distinct effects on mucosal versus systemic alphabeta T cells for induction of CD4(+) T cells with selected Vbeta use. This most likely reflects the function of LT as a mucosal modulator.

Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut-associated lymphoid tissue (GALT), which includes Peyer's patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) alpha-/- mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTbeta-/- mice and LTalpha/LTbeta+/- animals do not have PP but could be orally tolerized, as measured by IFN-gamma production and delayed-type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTbeta-receptor-IgG-fusion-protein (LTbetaRigG)-treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high- or low-dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.

The susceptibility to oral tolerance in post-neonatal infant mice and the effect of a herbal drug, Hochu-ekki-to (HOT), on the susceptibility were investigated. To induce oral tolerance induction, infant and adult mice at 4 and 8 weeks of age, respectively, were orally administered a single high dose of OVA before an intraperitoneal immunization with OVA adsorbed on aluminum hydroxide. HOT (1000 mg/kg) was administered orally for 7 days before the induction. HOT significantly decreased the serum levels of OVA-specific IgE and IgG1 and the antigen-specific proliferation of spleen cells in infant mice, both of which were greatly enhanced compared to in adult mice. HOT increased the number of both CD4+ T cells and antigen-presenting cells expressing MHC class II as well as costimulatory molecules (CD40, CD80 and/or CD86) in the Peyer's patch (PP) of infant mice, which had fewer cells than adult mice. In the PP, moreover, HOT augmented the IL-12p40 mRNA expression and spontaneous or CD40-stimulated IL-12 production, and increased the number of CD4+ cells expressing CD40 ligand, which is up regulated by IL-12. These results suggest that HOT increases the number and improves the function of PP cells that are fully susceptible to the induction of oral tolerance.

In this study, we investigated whether restraint stress induces either apoptotic cell death or lymphocyte migration in Peyer's patches. Exposure to stress induced a striking decrease in the number of CD3+CD4+, CD3+CD8+ and B220+ cells. Such decreases were accompanied by the enhanced induction of apoptosis in Peyer's patches. The apoptosis of Peyer's patch cells was completely reversed by pretreatment with either high or low doses of RU-486, a glucocorticoid receptor antagonist. In contrast, the stress-induced lymphopenia was little affected by administration of low doses of RU-486, although such lymphopenia was perfectly inhibited by treatment with high doses of RU-486. Taken together, these results suggest that the stress-induced lymphopenia in Peyer's patches is partly due to apoptotic cell death, although other systems such as lymphocyte migration, may contribute to such a reduction in the number of Peyer's patch cells., (Copyright 2002 S. Karger AG, Basel)

Such allergic diseases as rhinitis and asthma are IgE-mediated type I reactions and are controlled primarily by Th2 cells. One of the major dust mites, Dermatophagoides pteronyssinus (Dp), is considered to cause allergic reactions. Oral tolerance, largely used to modulate immune response, opens the possibility of modulating Th2 allergic responses. We observed downmodulation of total and specific IgE antibody levels as well as the number of specific IgE-secreting cells with Dp feeding in previously sensitized mice. Analysis of the cytokine profile in mucosal lymphoid tissues in the protocol revealed altered patterns of interferon-gamma (IFN-gamma), interleukin-5 (IL-5), and transforming growth factor-beta (TGF-beta) secretion in Dp-fed animals. The results suggest that both the Th and B cell populations are modulated in mice made tolerant by oral Dp feeding. Understanding the mechanisms at the mucosal level that underlie oral tolerance can improve its use in allergy immunotherapy.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.

Background: The purpose of this study was to examine the effects of a steam burn injury on apoptosis in gut-associated lymphoid tissue and to determine whether endogenous glucocorticoid and Fas ligand signaling were involved in this process., Methods: Histologic analysis, in situ deoxynucleotidyl transferase dUTP nick-end labeling staining and annexin V and 7-amino-actinomycin-D flow cytometry of lymphocyte populations were evaluated in intraepithelial lymphocytes and Peyer's patch. Additional mice were pretreated with a glucocorticoid receptor antagonist (mifepristone) before the steam burn. Similarly, C3H/HeJ-FasL(gld) mice lacking functional Fas ligand were also studied., Results: Apoptosis was significantly increased in intraepithelial lymphocytes and Peyer's patch after the burn injury. Mifepristone pretreatment significantly reduced apoptosis in both T- and B-cell populations in intraepithelial lymphocytes after the burn injury. In contrast, the increased apoptosis seen in B-cells from Peyer's patch was not seen in C3H/HeJFasL(gld) mice, whereas the increased apoptosis in CD8+ T-cells was unaffected., Conclusion: Both corticosteroids and FasL contribute to the apoptosis in gut-associated lymphoid tissues early after burn injury.

Background: Total parenteral nutrition (TPN) leads to atrophy of the gut-associated lymphoid tissue (GALT) and a significant decrease in intestinal immunoglobulin A (IgA) levels, a major constituent of mucosal immunity. Bombesin (BBS) prevents TPN-induced GALT atrophy and maintains intestinal IgA levels. BBS, a neuropeptide analogous to gastrin-releasing peptide in humans, stimulates the release of other gut neuropeptides including cholecystokinin (CCK), gastrin, and neurotensin (NT). This study investigates the ability of CCK, gastrin, or NT to individually prevent TPN-induced GALT atrophy and preserve respiratory immunity., Methods: Experiment 1: Male mice were randomly assigned to receive chow, TPN, TPN plus CCK, TPN plus gastrin, or TPN plus NT. After 5 days of feeding, Peyer's patches (PP) from the proximal and distal small bowel were harvested and analyzed for cell yields. PP cells were also analyzed for GALT cell type. Small bowel IgA levels were measured by enzyme-linked immunosorbent assay (ELISA). Experiment 2: Mice were randomly assigned to receive either liposomes containing Pseudomonas antigen or liposomes without antigen. After 10 days, mice were randomly assigned to the same five treatment groups, fed for 5 days, and then given intratracheal Pseudomonas. Mortality was assessed after 48 hours., Results: Experiment 1: GALT cell reductions due to IV-TPN were greater in the distal than proximal small bowel. All three neuropeptides prevented most TPN-induced GALT atrophy due mainly to the maintenance of the B-cell and T-cell populations in the PP of the distal bowel. Intestinal IgA levels were significantly higher in the animals treated with neuropeptides than animals treated with TPN only; however, these IgA levels were not maintained at levels observed in chow-fed animals. Experiment 2: Immunization resulted in significantly lower mortality in animals fed chow, TPN plus CCK, and TPN plus gastrin. TPN alone and TPN plus NT resulted in loss of immunity and mortality rate at comparable levels to unimmunized animals., Conclusions: Supplementation of IV-TPN with CCK, gastrin, and NT prevents GALT atrophy, primarily in the distal bowel. Intestinal IgA levels improve but not to normal levels. CCK and gastrin reversed IV-TPN-induced effects on antibacterial pneumonia in immunized animals while NT did not.

Background and Aims: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice., Methods: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA., Results: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups., Conclusion: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content., (Copyright 2000 Harcourt Publishers Ltd.)

Background: M cells play an important role in the intestinal immune system as they have a high capacity for transcytosis of a wide range of microorganisms and macromolecules. However, little is known about the role of M cells during intestinal inflammation., Aim: We studied M cell development during indomethacin-induced intestinal inflammation in rats., Methods: Ileitis in rats was induced by two subcutaneous injections with indomethacin (7.5 mg/kg) given 24 h apart. Rats were sacrificed after 14 days and tissue was analysed by fluorescence microscopy and electron microscopy. M cells could be visualized by using the FITC-labelled mAb anti-cytokeratin (CK)-8 (clone 4.1.18), which was recently identified as specific M cell marker in rats. The number of cytokeratin-8 positive M cells was related to the surface of the follicle associated epithelium. For morphological studies, we used both transmission electron microscopy (T.E.M.) and scanning electron microscopy (S.E.M.)., Results: In non-inflamed ileum M cells were scarce. Only 4% of the follicle associated epithelium were M cells, whereas an increase of M cells up to 11% was found in inflamed follicle associated epithelium (P < 0.001). The rate of M cell induction depended on the macroscopic degree of inflammation. T.E.M./S.E.M. studies showed that in inflamed tissue most M cells underwent apoptosis with typical morphological signs. In contrast to apoptotic M cells, the neighbouring enterocytes usually appeared intact. The number of mononuclear cells below the follicle associated epithelium was significantly increased. S.E.M. studies revealed that during induced ileitis mononuclear cells migrated from the lamina propria into the gut lumen by passing through apoptotic M cells., Conclusions: During indomethacin-induced ileitis in rats the increase in M cell number in association with apoptosis of M cells may alter the intestinal barrier function. These observations may play a pivotal role in the pathogenesis of chronic intestinal inflammation, e.g. in inflammatory bowel disease.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Objective: To study the ability of bombesin (BBS) to recover gut-associated lymphoid tissue (GALT) and preserve immunity in a lethal model of Pseudomonas aeruginosa (Ps) pneumonia in mice receiving total parenteral nutrition (TPN)., Summary Background Data: TPN causes depression of mucosal immunity compared with enterally fed animals, which may explain the increased incidence of pneumonia in parenterally fed trauma patients. BBS prevents this TPN-induced GALT atrophy, depressed gastrointestinal and respiratory tract IgA levels, and impaired antiviral IgA-mediated mucosal immunity. The authors examined whether some supplement could be added to TPN to avoid this GALT atrophy and lower the incidence of infectious complications in the parenterally fed animal., Methods: Male mice were randomized to chow or intravenous (IV) TPN. After 5 days of IV TPN, mice received 0, 1, 2, or 3 days of BBS IV three times a day and then were killed to harvest Peyer's patch, intraepithelium, and lamina propria for cell yields. Gastrointestinal and respiratory tract IgA levels were analyzed by enzyme-linked immunosorbent assay. Next, mice underwent intranasal inoculation with liposomes alone (nonimmune) or liposome-containing Ps polysaccharide. Ps immune mice were catheterized and randomized to chow, IV TPN, or IV TPN + BBS. The liposome group received chow but no IV catheter. These mice were given an LD90 dose of intratracheal Ps, and death rates were recorded., Results: GALT and gastrointestinal and respiratory tract IgA levels improved to those in chow-fed mice after 3 days of BBS. Immunization reduced the death rate from 92% in chow-fed liposome-only animals to 20% in immunized animals. TPN-fed animals lost their mucosal immunity, with a death rate of 86% compared with 21% in the TPN + BBS group., Conclusion: The results demonstrate that BBS reverses TPN-induced changes in GALT and preserves mucosal immunity. Ps immunization reduces the death rate in a gram-negative pneumonia model and maintains gastrointestinal and respiratory immunity in Ps immune mice receiving IV TPN.

Hemorrhagic shock (HS) can cause whole body ischemia including the gastrointestinal tract. We investigated whether cells from small intestine Peyer's patches (PP) were capable of producing superoxide radical when animals underwent HS or HS followed by resuscitation (HS/RS). HS was initiated by removing 60% of the blood volume of surgically prepared guinea pigs. PP lymphoid cells were purified and stimulated with phorbol 12-myristate 13-acetate in the presence of spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Electron paramagnetic resonance spectra of PP lymphoid cells from sham-treated control, HS, and HS/RS animals produced DEPMPO radical adducts characterized as the adducts of superoxide (DEPMPO/*OOH) and hydroxyl (DEPMPO/*OH) radicals. The formation of both radical adducts was totally inhibited by superoxide dismutase or a nicotinamide adenine dinucleotide phosphate (reduced form) oxidase inhibitor, diphenyleneiodonium chloride. HS/RS increased radical adduct formation, expressed as a percentage control, by 160% and 225% for DEPMPO/*OOH, and DEPMPO/*OH, respectively. When animals were allowed to recover for 24 h post-HS/RS treatment, PP cells decreased the superoxide generation to the same level as controls. Thus, RS following HS may prime PP lymphoid cells for increased nicotinamide adenine dinucleotide phosphate (reduced form) oxidase-dependent superoxide generation, and this process may have cytotoxic and/or immunomodulatory effects on the host.