Intestinal Peyer's Patches Research Articles

Involvement of gut-associated lymphoid tissue of ruminants in the spread of transmissible spongiform encephalopathies

Scrapie is a transmissible spongiform encephalopathy (TSE) and its spread across the intestine of sheep is linked to the biology of intestinal Peyer's patches (PPs). Specialized epithelial cells, M cells, would appear to be the portal of entry for the scrapie agent, PrP Sc, while lymphoid nodules of PPs become major sites of accumulation of PrP Sc as the infection becomes established. Furthermore, evidence suggests that the enteric nervous system supplying the PPs is important for neuroinvasion. The gut-associated lymphoid tissue (GALT) of ruminants shows morphological and functional differences to the GALT of mice and humans. Recent investigations of aging scrapie-affected sheep revealed a substantial network of nerve fibres in the lymphoid nodules of PPs, contradicting the widely held notion that lymphoid nodules are poorly innervated. Advances in the understanding of the pathogenesis of scrapie may be achieved by a deeper appreciation of the development, morphology and function of GALT in small ruminants.

Identification and characterization of intestinal peyer's patch interferon-α producing (plasmacytoid) dendritic cells

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-α (IFN-α) producing cells in blood. In this study, C57BL/10 (B10;H2 b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 μg/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 CD8αα +. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of naı̈ve allogeneic (C3H; H2 k) 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-α. 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.

Apical membrane receptors on intestinal M cells: potential targets for vaccine delivery

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.

Endotoxin stimulates monocyte-endothelial cell interactions in mouse intestinal Peyer's patches and villus mucosa.

Although monocyte-endothelial cell interactions represent an initial step in controlling the recruitment of monocytes in inflamed tissues, their dynamic processes in microvessels of lymphoid (Peyer's patches) and non-lymphoid (villus) regions in gut-associated lymphoid tissue remain poorly understood. We monitored the migration of fluorescence-labelled monocytes derived from the spleen in intestinal microvessels with or without lipopolysaccharide (LPS) treatment and investigated the role of adhesion molecules, P-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In control mice, there were few interactions between infused monocytes and the endothelium of intestinal microvessels. The monocyte-endothelial interactions (both rolling and adhesion) were significantly increased in intestinal microvessels of LPS-treated mice compared with those in controls. Anti-P-selectin monoclonal antibody (MoAb) significantly suppressed the LPS-induced increase in monocyte rolling in postcapillary venules of Peyer's patches and submucosal venules. Anti-VCAM-1 MoAbs significantly suppressed the LPS-induced increase in monocyte adhesion to postcapillary venules (PCVs) of Peyer's patches, submucosal venules, and villus capillaries. In contrast, anti-ICAM-1 MoAb significantly suppressed the number of adherent monocytes in PCV of Peyer's patches but not in submucosal venules or villus capillaries. These observations demonstrated that LPS treatment resulted in a significant increase in recruitment of monocytes both in microvessels of lymphoid and non-lymphoid regions and that P-selectin, VCAM-1 and ICAM-1 appeared to play important roles in LPS-induced interactions.

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep.

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.

Dietary Nucleotides Accelerate Changes in Intestinal Lymphocyte Maturation in Weanling Mice

ABSTRACTObjectiveNucleotides, the building blocks of nucleic acids, are normal components of the mammalian diet. These molecules have been implicated in biologic processes, such as the stimulation of the immunologic response. Nucleotides have also been considered as conditionally essential nutrients for infant formulas. The authors evaluated the influence of dietary nucleotides on the expression of several surface antigens by different intestinal lymphocyte populations in weanling mice.MethodsMice at weaning were fed a semipurified diet with or without 3 g/kg of each of the following nucleotides: adenosine monophosphate, cytosine monophosphate, guanosine monophosphate, and uridine monophosphate. Animals were killed at different times (0, 4, 7, 12, and 18 days) after weaning, and lymphocytes from intestinal Peyer's patches, epithelium, and lamina propria were isolated. The expression of different antigens (CD3, CD4, CD8α, CD8β, TCRαβ, TCRγδ, CD5, CD22 and CD45R) was analyzed by flow cytometry.ResultsThe expression of these antigens changed parallel to the maturation of the lymphocytes from Peyer's patches, epithelium, and lamina propria. However, developmental changes of expression for most of the antigens occurred sooner in the animals fed the diet supplemented with nucleotides. The expression of T and B antigens was different in the lymphocyte populations analyzed and also changed according to the diet within each population. In general, nucleotides promoted the expression of B‐ and T‐helper cell antigens.ConclusionsThe authors conclude that dietary nucleotides may affect the process of maturation and differentiation of intestinal lymphocytes, which usually takes place at weaning.

Dietary nucleotides accelerate changes in intestinal lymphocyte maturation in weanling mice.

Nucleotides, the building blocks of nucleic acids, are normal components of the mammalian diet. These molecules have been implicated in biologic processes, such as the stimulation of the immunologic response. Nucleotides have also been considered as conditionally essential nutrients for infant formulas. The authors evaluated the influence of dietary nucleotides on the expression of several surface antigens by different intestinal lymphocyte populations in weanling mice. Mice at weaning were fed a semipurified diet with or without 3 g/kg of each of the following nucleotides: adenosine monophosphate, cytosine monophosphate, guanosine monophosphate, and uridine monophosphate. Animals were killed at different times (0, 4, 7, 12, and 18 days) after weaning, and lymphocytes from intestinal Peyer's patches, epithelium, and lamina propria were isolated. The expression of different antigens (CD3, CD4, CD8alpha, CD8beta, TCRalphabeta, TCRgammadelta, CD5, CD22 and CD45R) was analyzed by flow cytometry. The expression of these antigens changed parallel to the maturation of the lymphocytes from Peyer's patches, epithelium, and lamina propria. However, developmental changes of expression for most of the antigens occurred sooner in the animals fed the diet supplemented with nucleotides. The expression of T and B antigens was different in the lymphocyte populations analyzed and also changed according to the diet within each population. In general, nucleotides promoted the expression of B- and T-helper cell antigens. The authors conclude that dietary nucleotides may affect the process of maturation and differentiation of intestinal lymphocytes, which usually takes place at weaning.

P-selectin and P-selectin glycoprotein ligand 1 are major determinants for Th1 cell recruitment to nonlymphoid effector sites in the intestinal lamina propria.

The recruitment of activated T cell subsets to sites of effector immune responses is mediated by homing receptors induced upon activation in secondary lymphoid tissue. Using an adoptive transfer model, the intestinal recruitment of CD4+ T cells activated with intraperitoneal antigen in complete Freund's adjuvant was examined. The data demonstrate that activated CD4+ T cells recruited to intestinal Peyer's patches (PP) and lamina propria (LP) up-regulate functional P-selectin glycoprotein ligand 1 (PSGL-1). Blockade of IL-12 inhibited functional PSGL-1 expression and reduced PP and LP CD4+ T cell recruitment by >40%. P-Selectin blockade reduced LP recruitment of activated cells by 56% without affecting PP recruitment. Studies of mice examined 3 d after adoptive transfer of differentiated T cell subsets revealed that Th1 but not Th2 cells were recruited to small intestine PP and LP. Mucosal addressin cell adhesion molecule blockade reduced Th1 recruitment to PP by 90% and to LP by >72%, whereas P-selectin blockade reduced Th1 recruitment to PP by 18% and Th1 recruitment to LP by 84%. These data suggest that IL-12–induced functional PSGL-1 expression is a major determinant for the recruitment of Th1 effector cells to noninflamed as well as inflamed intestine.

Decreased lymphocyte apoptosis by anti-tumor necrosis factor antibody in Peyer's patches after severe burn.

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.

Suppressive effects of Perilla frutescens on IgA nephropathy in HIGA mice.

Perilla frutescens (perilla) is a herbal medicine used in Japanese traditional Kampo medicine. The present study was conducted to evaluate the anti-nephritic effects of perilla in HIGA mice that spontaneously develop high levels of serum immunoglobulin A (IgA) along with mesangial IgA deposition. A perilla decoction and its major active constituent, rosmarinic acid (RsA), were orally administrated to 10-week-old HIGA mice for 16 weeks. At study completion, we measured proteinuria and serum IgA levels and generated histological scores from kidney specimens. In addition, we measured concentrations of IgA in culture media of intestinal Peyer's patch cells and spleen cells obtained from the HIGA mice. Perilla suppressed proteinuria, proliferation of glomerular cells, serum levels of IgA, glomerular IgA and IgG depositions in HIGA mice. Cultured Peyer's patch cells and spleen cells from perilla-treated mice produced significantly less IgA than controls. Rosmarinic acid, by itself, suppressed serum IgA levels and glomerular IgA deposition in HIGA mice. Cultured spleen cells from RsA-treated mice produced less IgA than controls. The perilla decoction may suppress IgA nephropathy, in part, through modulation of the intestinal mucosal immune system. These effects were caused by RsA acting synergistically with other constituents.

PELA microspheres loaded H. pylori lysates and their mucosal immune response.

To prepare poly (D,L-lactide)-polyethylene glycol copolymer (PELA) microspheres loaded H.pylori lysates or Cystografin and observe their targeting in gastrointestinal mucous membrane or analyze the mucosal immune responses by oral administration. PELA microspheres loaded H.pylori lysates or Cystografin were prepared by double emulsion evaporation method. Their distribution in gastrointestinal mucous membrane was observed by CT. Balb/c mice orally immunized in mucosal immune responses, whose antibody production in salivary and gut washing and antibody secreting cells in Peyer's patches (PP) were estimated by ELISA and ELISPOT, respectively. The microspheres physical properties, such as particle size, protein level and morphology were investigated. All prepared microspheres were found to have a smooth surface morphology from 3.20-4.05 microm in diameter and high encapsulation efficiency from 74.9-82.2 %. No significant correlation in their physical properties was shown, depending on their molecular weight at the similar composition ratio. Immunization with all types of PELA-Hp microspheres elevated the saliva sIgA level at week 3 by approximately 3-4 times that with soluble antigen, which was greatly enhanced after boosting. At one week after last immunization with all types of PELA-Hp microspheres (week 8), the specific sIgA-ASCs, IgG-ASCs and sIgA in salivary rose obviously. In intestinal Peyer's patches, the specific sIgA-ASCs were 5.92-6.98X10(4)/ml cell and IgG-ASCs were 3.47-4.02X10(4)/ml cell, about 5-9 times higher than those with soluble antigen (P<0.01). ASCs in intestine were more than those in stomach and the majority of the ASCs were sIgA-ASCs. The sIgA in gut washing fluid was 1.62-1.85 OD, about 3-6 times tthat of those with soluble antigen. There were significant differences of the ASCs and sIgA in gut washing fluid as compared with those of PBS and MS-0 (P<0.05). There appeared to be good correlation between sIgA level in gut washing fluid and sIgA-ASCs in intestinal Peyer's patches. PELA microspheres may be used as vehicle to delivery antigen and adjuvant in designing oral vaccination.

Effects of burn with and without Escherichia coli infection in rats on intestinal vs. splenic T-cell responses.

To evaluate the effect of burn injury with and without an Escherichia coliseptic complication on T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses in intestinal Peyer's patch and splenic T cells. Prospective, randomized, sham-controlled animal study. University medical center research laboratory. Adult male Sprague-Dawley rats. Rats were subjected to a 30% total body surface area, full skin thickness burn. Infection in rats was induced via intraperitoneal inoculation of E. coli, 10(9) colony forming units/kg, with or without a prior burn. Rat Peyer's patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol. T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of (3)H-thymidine into T-cell cultures. Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay. Intracellular T-cell Ca2(+ )concentration, [Ca(2+)](i), was measured by the use of Ca(2+)-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca(2+)](i) was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells. T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer's patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E. coli inoculation at 24 hrs after the initial burn. There seemed to be no demonstrable additive effects of E. coli infection on the effects produced by burn injury alone. The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca(2+) signaling. E. coli infection alone suppressed T-cell proliferation in Peyer's patch but not in splenic T cells at 2 days postbacterial inoculation; E. coli infection had no effect on Peyer's patch or splenic T cells at 1 day postinjury. On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer's patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer's patch but not in the spleen. An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation. An earlier onset of T-cell suppression in Peyer's patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury. Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression.

Prevention of dexamethasone-induced lymphocytic apoptosis in the intestine and in Peyer patches by enteral nutrition.

Apoptosis is a programmed cell death, genetically controlled, that can be activated by physiological and pathophysiological events and by the administration of several drugs, including the exogenous administration of corticosteroids. The aim of this study was to develop a rat model of intestinal lymphocytic apoptosis induced by dexamethasone to determine if apoptosis could be prevented by enteral or parenteral nutrition. Male Sprague-Dawley rats were used. On day 0, the right internal jugular vein was catheterized for parenteral nutrition, and a silicone tube was inserted into the duodenum for enteral feeding. Animals (n = 6/group) were randomly assigned to one of the following 3 feeding regimens: an immune-enhancing enteral diet; its placebo (the same formula without immunonutrients); and isocaloric isonitrogenous parenteral nutrition. On the seventh day, 200 microg of dexamethasone or vehicle were administered by i.v. bolus, and the diets were continued for 1 more day. Intestinal Peyer patches and thymic lymphocytic apoptosis were determined both by flow-cytometry and immunohistochemistry. A single dexamethasone dose (200 microg/rat) administered to surgically treated rats fasted for 18 hours, 24 hours later, caused massive intestinal and Peyer patches lymphocytic apoptosis (96 +/- 2% and 85 +/- 5%, respectively; p < .0001 versus vehicle in both kinds of tissue). Lymphocytic apoptosis was reduced to almost indetectable levels in intestinal and lamina propia lymphocytes (from 96 +/- 2% to <0.6%; p < .0001). Peyer patches lymphocytic apoptosis was reduced as well (from 85 +/- 5% to 15 +/- 7.1%; p < .001) in animals prefed the 2 enteral nutrition formulas. Those prefed parenteral nutrition only showed a partial decrease of lymphocytic apoptosis in the intestine (from 96 +/- 2% to 53 +/- 23%; p < .001). Nutrition had no effect on the dexamethasone-induced thymus involution. Enteral nutrition prevents intestinal intraepithelial and lamina propia lymphocytic apoptosis due to dexamethasone. These findings support the use of early enteral nutrition in critically ill patients treated with corticosteroids.

Peyer's patch eosinophils: identification, characterization, and regulation by mucosal allergen exposure, interleukin-5, and eotaxin

AbstractThe gastrointestinal immune system is traditionally thought to be composed of lymphocytes located within Peyer's patches and the lamina propria. We have recently reported that eosinophils also reside in the gastrointestinal tract during healthy states, in particular, within the lamina propria, and that these cells substantially increase after oral allergen exposure. We now demonstrate the presence of eosinophils in Peyer's patches and characterize the signals that regulate the accumulation of eosinophils in Peyer's patches. In contrast to the lamina propria, intestinal Peyer's patches have very low levels of eosinophils under healthy states. However, elevated levels of interleukin-5 (IL-5), generated by transgenic or pharmacologic approaches, result in a dramatic increase in eosinophil levels in Peyer's patches. Most eosinophils are located in the outer cortex and interfollicular regions of the Peyer's patches. To dissect the mechanism of eosinophil trafficking to Peyer's patches, the role of eotaxin was examined. Mice transgenic for IL-5 and genetically deficient in eotaxin were found to have reduced levels of eosinophils in Peyer's patches compared with IL-5-transgenic mice. To prove that eosinophils also traffic to Peyer's patches in wild-type mice, allergic hypersensitivity was induced and Peyer's patches were examined. Exposure to mucosal allergen promoted marked accumulation of eosinophils in Peyer's patches and this process was attenuated in eotaxin-deficient mice. In summary, these data demonstrate that elevated levels of IL-5 and mucosal allergen exposure promote eotaxin-dependent eosinophil trafficking to Peyer's patches. These studies suggest that eosinophils may cooperate with lymphocytes in the development of mucosal immune responses in the gastrointestinal tract.

Peyer's patch eosinophils: identification, characterization, and regulation by mucosal allergen exposure, interleukin-5, and eotaxin

The gastrointestinal immune system is traditionally thought to be composed of lymphocytes located within Peyer's patches and the lamina propria. We have recently reported that eosinophils also reside in the gastrointestinal tract during healthy states, in particular, within the lamina propria, and that these cells substantially increase after oral allergen exposure. We now demonstrate the presence of eosinophils in Peyer's patches and characterize the signals that regulate the accumulation of eosinophils in Peyer's patches. In contrast to the lamina propria, intestinal Peyer's patches have very low levels of eosinophils under healthy states. However, elevated levels of interleukin-5 (IL-5), generated by transgenic or pharmacologic approaches, result in a dramatic increase in eosinophil levels in Peyer's patches. Most eosinophils are located in the outer cortex and interfollicular regions of the Peyer's patches. To dissect the mechanism of eosinophil trafficking to Peyer's patches, the role of eotaxin was examined. Mice transgenic for IL-5 and genetically deficient in eotaxin were found to have reduced levels of eosinophils in Peyer's patches compared with IL-5-transgenic mice. To prove that eosinophils also traffic to Peyer's patches in wild-type mice, allergic hypersensitivity was induced and Peyer's patches were examined. Exposure to mucosal allergen promoted marked accumulation of eosinophils in Peyer's patches and this process was attenuated in eotaxin-deficient mice. In summary, these data demonstrate that elevated levels of IL-5 and mucosal allergen exposure promote eotaxin-dependent eosinophil trafficking to Peyer's patches. These studies suggest that eosinophils may cooperate with lymphocytes in the development of mucosal immune responses in the gastrointestinal tract.

B-cell depletion fails to abrogate the induction of oral tolerance of specific Th1 immune responses in mice.

Antigen presentation by resting B cells has been shown to induce peripheral tolerance to intravenous (i.v.) administered soluble protein antigens. We further examined the role of resting B cells in the induction of oral tolerance. Mice were treated continuously from birth with rabbit antimouse IgM serum for 5 weeks. Immunohistological studies revealed that anti-IgM treatment depleted B cell-aggregated follicles in intestinal Peyer's patches. At 4-weeks-old, B cell-depleted mice were fed 25 mg ovalbumin or given 10% chicken egg white to drink for 5 days. Anti-IgM treatment was stopped 2 days after the last feed. Ten weeks later, the mice were immunized with 100 microg ovalbumin emulsified with complete Frund's adjuvant. Their T helper 1 (Th1) cell-regulated systemic delayed-type hypersensitivity, IgG2a antibody responses and spleen cell production of interferon-r and interleukin-2 were suppressed by prior ovalbumin or egg white feeding during anti-IgM treatment. Active suppression of Th1 immune responses was also demonstrated following adoptive transfer of egg white-fed donor spleen cells collected during anti-IgM treatment to naïve recipients. Although enormous small resting B cells are aggregated in the mantle zones of follicles of intestinal Peyer's patches, they are not the antigen-presenting cells seen in the induction of oral tolerance.

Organogenic role of B lymphocytes in mucosal immunity.

Follicle-associated epithelium (FAE) in the intestinal Peyer's patches contains M cells that deliver pathogens to organized lymphoid tissue. Development of Peyer's patches, FAE, and M cells was found to be impaired in mice that had no B cells. Transgenic expression of membrane-bound immunoglobulin M restored B cells and FAE development. The lack of M cells abrogated infection with a milk-borne retrovirus. Thus, in addition to secretion of antibodies and presentation of antigens, B cells are important for organogenesis of the mucosal immune barriers.

Priming of Peyer's patch lymphoid cells by hemorrhagic shock and resuscitation to produce superoxide radical.

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.

Cholera toxin B subunit as an immunomodulator for mucosal vaccine delivery

This chapter discusses cholera toxin B subunit as an immunomodulator for mucosal vaccine delivery. Efforts to exploit oral immunization for the development of vaccines against the great majority of infections, which directly afflict or invade through the mucosal surfaces, were formerly limited by the modest responses usually generated by the mucosal immune system. In part, this is probably because its functions include the maintenance of homeostasis despite being continuously challenged by large quantities of mainly harmless environmental antigens and commensal microorganisms. In addition, most nonviable immunogens have no affinity for the M cells overlying mucosal inductive sites, such as the intestinal Peyer's patches, and are readily digested within the gut lumen. Thus, oral immunization with conventional nonviable immunogens typically requires the repeated administration of large doses, and yields modest responses that do not persist. However, numerous developments during the past decade have addressed these problems. Notably, the exceptionally potent mucosal immunogenic and adjuvant properties of cholera toxin (CT) and related enterotoxins have been exploited by coupling other antigens to the nontoxic B subunit of CT, or by seeking to detoxify the heat-labile toxin (LT) of Escherichia coli while preserving its adjuvanticity.

Effect of Alcohol on Bacterial Translocation in Rats

Bacterial translocation has been proposed to be important in the pathophysiology of sepsis, as well as to be a consequence of sepsis. To study the effect of alcohol on bacterial translocation from the gut, normal Sprague-Dawley rats were administered alcohol by gavage by two regimens: Acute (3.7 g/kg, one dose) or Subacute (1 of 2 doses, 2.4 or 3.7 g/kg/day once daily for 14 days). Mesenteric lymph node cultures were performed, and portal venous blood was assayed for endotoxin. Ileal and cecal permeability studies were performed in the Acute and Subacute groups using fluorescein isothiocyanate-labeled dextrans of either 4,000 or 70,000 kDa size. As an index of the effect of systemic endotoxin, tissues from mesenteric lymph nodes, liver, and intestinal Peyer's patches were assayed for the presence of mRNA for tumor necrosis factor. Additionally, because extrapulmonary sepsis has been shown to suppress pulmonary antibacterial defenses, animals in the Subacute group were challenged by aerosol inoculation with Pseudomonas aeruginosa to determine bacterial clearance and alveolar cellular responses. The results show that neither of the alcohol regimens resulted in bacterial growth from mesenteric lymph nodes or portal blood. Animals in the Subacute group had more endotoxin present in portal blood than did the Control group (92.9 pg/ml vs. 40.2 pg/ml; p < 0.02). None of the animals had demonstrable mRNA for tumor necrosis factor in any of the tissues assayed. There were no demonstrable increases in ileal or cecal permeability for either the small or large molecular weight dextran in either alcohol group. Furthermore, there was no delay in the clearance of P. aeruginosa from the lung in the Subacute group, but these animals recruited fewer neutrophils into the airspaces in response to this challenge than did the Control animals. Thus, alcohol intoxication does not result in bacterial translocation from the gut in this model. Despite higher levels of portal venous endotoxin in the animals in the Subacute alcohol group, no adverse systemic consequences of this phenomenon could be demonstrated.