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Selection for prolificacy in modern pig farming has resulted in increasing litter sizes. Since rearing large litters is challenging, artificial rearing of piglets with a milk replacer is an alternative strategy. It is hypothesized that the development of the piglets' mucosa-associated lymphoid tissues (MALT) is affected by these artificial conditions. Therefore, the stereologically estimated volumes of the tonsil of the soft palate, and the lingual, nasopharyngeal and paraepiglottic tonsils, as well as the jejunal and ileal Peyer's patches were statistically compared at day 21 postpartum between six conventionally reared piglets and six piglets that were artificially reared from day 7 onwards. In addition, six 7-day-old sow-fed piglets were examined to evaluate the effect of age. All tonsils and Peyer's patches significantly increased in volume with age. The rearing strategy had no significant effect on the volumes of the tonsil of the soft palate and the lingual tonsil. The former tonsil was by far the largest with a mean volume of 967.2 ± 122.4 mm 3 and 822.3 ± 125.4 mm 3 in the conventionally and artificially reared piglets, respectively. The lingual tonsil only measured 9.4 ± 6.4 mm 3 and 6.3 ± 2.6 mm 3 in conventionally and artificially reared groups, respectively. In contrast, the rearing strategy did affect the volumes of the nasopharyngeal and paraepiglottic tonsils, which had a mean volume of 137.1 ± 32.4 mm 3 and 84.4 ± 26.9 mm 3 , and 30.7 ± 7.8 mm 3 and 20.0 ± 3.9 mm 3 in conventionally and artificially reared piglets, respectively. The rearing strategy did not affect the development of the Peyer's patches. At day 21, the jejunal Peyer's patches of the conventionally and artificially reared piglets presented a volume of 1.6 ± 0.4 cm 3 and 1.3 ± 0.2 cm 3 , respectively. The volumes of the ileal Peyer's patch amounted to 15.1 ± 3.0 cm³ in conventionally reared piglets and 12.0 ± 2.6 cm³ in artificially reared piglets at day 21. The results showed that artificial rearing hampers the morphological development of the tonsils that are exposed to inhaled antigens, but the voluminous lymphoid tissues that sample oral antigens are not influenced. Since it is unlikely that the observed differences in both tonsils are due to the milk replacer, artificial rearing could be a valuable alternative for raising large litters. In addition, the presence of developing MALT in piglets allows for investigating the value of nasal and oral vaccination in this species for human or veterinary purposes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Quantitative evaluation of aggregated lymphoid follicles in various compartments of the large intestine was carried out in Wistar rats of different age: newborn (3-4 days), prepubertal (20-30 days), adult (2-3 months), and old (16-18 months). No aggregated lymphoid tissue was detected in the large intestinal mucosa of newborn animals. The cecum of prepubertal, adult, and old animals contained solitary patches with 7-9 follicles. Higher percentage of aggregated lymphoid tissue, associated with colonic mucosa, was explained by enlargement of the lymphoid patch area and of the number of solitary lymphoid follicles during the postnatal ontogenesis. The mean area of a patch in the distal part of the colon in prepubertal, adult, and old animals was 3.2, 2.5, and 2.2 times larger than in the medial part of the intestine, the number of follicles per patch was 2.8, 2.8, and 2.5 times higher, respectively. The differences were significant only for the two younger groups.

The structure and distribution of the mucosa-associated lymphoid tissue (MALT) throughout the large intestine of 10 Bactrian camels were comparatively studied by anatomical and histological methods. The results showed that Peyer's patches (PPs) were mainly located on the mucosal surfaces of the entire ileocecal orifice, the beginning of the cecum and the first third of the colon. The shape of PPs gradually changed from "scrotiform" to "faviform" along the large intestine with the scrotiform PP as the major type in the ileocecal orifice. The distribution density also gradually decreased from the ileocecal orifice to the colon. The histological observations further revealed that the MALT in the form of PPs or isolated lymphoid follicles (ILF) and lamina propria lymphocytes was mainly present in the lamina propria and submucosa from the entire ileocecal orifice, where the muscularis mucosa is usually incomplete, to the colonic forepart. In addition, lymphoid tissue was much more abundant in the lamina propria and submucosa of the ileocecal orifice as compared to the cecum and colon. Statistically, the MALT of the ileocecal orifice contained a higher number of lymphoid follicles (37.7/10 mm(2) ) than that of the cecum, colon, or rectum (P < 0.05). The germinal centers of the lymphoid follicles were clearly visible. Together, our data suggest that the ileocecal orifice constitutes the main inductive site for the mucosal immunity in the large intestine of the Bactrian camel; and that scrotiform PPs are likely to the result of long-term adaptation of the Bactrian camel to the harsh living environment., (© 2014 Wiley Periodicals, Inc.)

Tragulidae is a primitive ungulate family within the order Cetartiodactyla, suborder Ruminantia. Domestic ruminants such as cattle, sheep, and goat have two types of Peyer's patches (PP): jejunal and ileal PP, in which there are morphological and functional differences. In this study, lesser mouse deer (Tragulus javanicus) PP was studied by gross anatomical and histological procedures. At the fetal stage, both types of PP were formed in the small intestine. Ileal PP was observed as a single continuous aggregation of lymphatic follicles extending cranially from the ileo-caecal junction. However, jejunal PP was observed as multiple and discrete accumulations of lymphatic follicles. This study showed that the lesser mouse deer has two types of PP in the small intestine. In addition, the anatomical and histological characteristics of jejunal and ileal PP are quite similar to those of other ruminants' jejunal and ileal PP. Further studies are needed to analyze immune function of both PP in lesser mouse deer in order to determine the evolutionary process of Cetartiodactyla., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.

The distribution, size, and appearance of Peyer's patches vary according to species. In order to determine the anatomical characteristics of Peyer's patches in small intestine of Bactrian camel, and age-related changes in the number of Peyer's patches, 40 Bactrian camels of the following four age groups were studied: young (0.5-2 years), pubertal (3-5 years), middle-aged (6-16 years), and old (17-20 years). The exact number of Peyer's patches was recorded, and the appearance of Peyer's patches was described in detail. The results indicated that Peyer's patches of Bactrian camels not only have a particular anatomical location and distinct appearance but also change with age. They were distributed in the whole small intestine and there were four distinct types of Peyer's patches: nodular, faviform, cup-shaped, and cystic form Peyer's patches. However, the nodular and cystic form Peyer's patches are specific to Bactrian camel, which have not been found in other animals including Dromedary camel. In addition, the distribution density of Peyer's patches in ileum was the maximum, then was jejunum and duodenum. Further statistical analysis showed that the number of Peyer's patches was altered with age. The number peaked in 5-year-old camels and declined subsequently with age. However, there was little change in the size of Peyer's patches in different age groups; no age-related macroscopic variations in the shape or size of the Peyer's patches were found. Results obtained from this study provide the basic information to further study on the gastrointestinal mucosal immunity of Bactrian camel.

The lymphoid tissue that is associated with the intestinal tract, the so-called gut-associated lymphoid tissue (GALT), is well developed in the chicken. Depending on the location, it is present as aggregations of lymphoid cells, or organized in lymphoid follicles and tonsils. From proximal to distal, the intestinal tract contains a pharyngeal tonsil, diffuse lymphoid tissue and lymphoid follicles in the cervical and thoracic parts of the oesophagus, an oesophageal tonsil, diffuse lymphoid tissue in the proventriculus, a pyloric tonsil, Peyer's patches, Meckel's diverticulum, two caecal tonsils, diffuse lymphoid tissue in the rectum, the bursa of Fabricius, and diffuse lymphoid tissue in the wall of the proctodeum. The lymphoid tissues are frequently covered by a lympho-epithelium that is infiltrated by lymphoid cells. Such an epithelium often contains M or microfold cells, which are specialized in antigen sampling and transport antigens to the underlying lymphoid tissue. A solid knowledge of the avian GALT could contribute to the development of vaccines to be administered orally. Additionally, immune stimulation via pre- and probiotics is based on the presence of a well-developed intestinal immune system.

The chicken bursa of Fabricius and calf ileal Peyer's patch are thought to be the primary lymphoid organs of B cell development. In the bursa, the existence of secondary lymphoid tissue, called the diffusely infiltrated area, has been recognized. Recently, we have found the presence of a region of secondary lymphoid tissue in the ileal Peyer's patch at the period of the most rapid growth of this organ. In this study, we compared the development of these secondary lymphoid tissue regions in the bursa and ileal Peyer's patch histologically. Before hatching, lymphatic follicle formation occurred in the bursa, but not in the diffusely infiltrated area, where only a small number of lymphoid cells were found. However, during fetal calf development, lymphatic follicle formation occurred not only in the primary lymphoid organ but also in the secondary lymphoid tissue regions. Therefore, the prenatal development of the secondary lymphoid tissue regions of the bursa and ileal Peyer's patch were distinct. After hatching, formation of the germinal center, which contained many CD4+ cells, was observed in the diffusely infiltrated area of the bursa. After birth, many CD4+ cells and IgG mRNA expression were observed in the lymphatic follicle of the secondary lymphoid tissue regions in the ileal Peyer's patch, but rarely in the ileal Peyer's patch lymphatic follicles. The change of character of these secondary lymphoid tissue regions at the postnatal stage might be dependent on external antigens., (Copyright 2009 Elsevier B.V. All rights reserved.)

Purpose: To validate Peyer's patch as an anatomical repair landmark for colorectal surgery in rats and to measure the collagen content in segments of the colon containing or not containing Peyer's patch., Methods: The distance between Peyer's patch and the peritoneal reflection was measured in forty-five Wistar rats. The colon and rectum were resected for quantification of collagen content by means of computer-assisted image analysis in regions of the colon with and without Peyer's patch., Results: There was great variation in the distance between Peyer's patch and the peritoneal reflection when the male and female rats were considered as a single group (p=0.04). Comparison between the genders showed that the distance between the patch and the peritoneal reflection was greater in female than in male rats (p=0.001). The colonic segment containing Peyer's patch was observed to have lower tissue collagen content than the segment in which this structure was not present (p=0.02)., Conclusion: Peyer's patch can be indicated as an anatomical repair landmark, and there is a need to study the healing of colorectal anastomoses in rats based on differing quantities of tissue collagen existing in the colonic wall with or without this structure.

Secondary lymphoid organs (SLOs) are tissues that facilitate the induction of adaptive immune responses. These organs capture pathogens to limit their spread throughout the body, bring antigen-presenting cells into productive contact with their cognate lymphocytes and provide niches for the differentiation of immune effector cells. Therefore, the microanatomy of SLOs defines the ability of an organism to respond to pathogens. SLO microarchitecture is, at the same time, extremely adaptable to environmental changes. In this Review, we discuss recent insights into the function and plasticity of the SLO microenvironment with regards to antimicrobial immune defence.

The Peyer's patches (PP) have been intensely investigated in several species because this is an important entry site for antigens and infectious agents. There are many PP in the jejunum, and in some species such as ruminants, carnivores, and omnivores, a different continuous PP is found in the terminal ileum. This PP disappears with age in these species studied. So far the ileal PP (IPP) has only been examined in the camel by light microscopy. Therefore, the localization of ileal Peyer's patches in the dromedary camel at different ages, as well as the histology and ultrastructures were now investigated. The IPP were characteristically seen as dark rose-colored isolated structures in the shape of a cup, arranged in three irregular rows. The central row was antimesenteric. Each patch was formed by several mainly elongated dome regions flanked by intestinal villi. In cross-sections these domes appeared as short, wide villi. The domes were formed from lymphoid follicles covered with a typical dome-associated epithelium of enterocytes and M cells without any goblet cells. The M cells showed variable appearance depending on the functional status. The lymphoid follicles expressed clear germinal centers. High endothelial venules were localized in the interfollicular region. In contrast to other species the IPP were still present with a comparable macroscopic and histological structure in camels of 25 years of age.

The indigenous microflora plays an integrative role in the maintenance of immunological homeostasis. Several studies reported that immunological tolerance is dependent on microbial colonization of the gut. In the present study, we investigated whether the absence of the microflora influences the sensitization process to an allergen as well as the ability to develop mucosal tolerance in a mouse model of birch pollen allergy. Germ-free or conventional BALB/c mice were intranasally or intragastrically pre-treated with the major birch pollen allergen Bet v 1 prior to sensitization with this allergen. Both germ-free and conventional mice displayed comparable Th2 biased immune responses after allergic sensitization. Oral as well as intranasal tolerization led to suppression of allergen-specific serum antibodies (IgG1, IgE, IgA) as well as cytokine production by splenocytes (IL-5, IFN-gamma) in both germ-free and conventional animals. Peyer's patches of germ-free animals were approximately 20 times smaller than in conventional animals, but the relative distribution of lymphocyte subpopulations was equal. We conclude that the absence of the microflora does not influence the ability to mount Th2 responses nor to establish tolerance towards the aeroallergen Bet v 1. Our findings may challenge the view that the commensal microflora is a key factor for breakdown of physiological tolerance and allergy development.

Intestinal microparticle uptake is important for drug delivery, environmental pollution and multiple organ dysfunction syndrome. This paper explores further whether uptake occurs at mucosa associated lymphoid tissue (MALT) via the microfold (M) cells of Peyer's patch domes or through villous epithelium. It does this by comparing the results of exposure of either severe combined immunodeficient (SCID) mice (lacking MALT) or normal BALBc mice, to oral gavage with 2 microm fluorescent latex microparticles. At 5 and 30 min after gavage, full circumference samples along the small intestine were processed for fluorescence microscopy and microparticle numbers were collected for surface and tissue sites. Uptake occurred in both BALBc and SCID mice within 5 min of particle administration and increased further in the following 25 min. In BALBc mice, almost all particles (96%) are in non-MALT sites in MALT circumference samples, with very few at the domes: uptake was also substantial in entirely villous samples. In SCID mice, particle numbers were only slightly lower than those of the BALBc mice, and occurred exclusively by the villous route. These findings confirm that the villous uptake route must be considered when assessing the extent of the dose delivered following pharmaceutical or toxicological oral exposure to microparticles.

Peyer's patches (PPs) are the most probable sites of intestinal uptake of the transmissible spongiform encephalopathy (TSE) agent. The amount of PP tissue varies considerably between different age groups of individuals, and whether this variation is related to susceptibility to TSE infection raises an intriguing possibility. The purpose of this study was to determine the surface area of PP tissue and the number of associated lymphoid follicles in different age groups of Neuropathogenesis Unit (NPU) Cheviot sheep. Terminal ilea were obtained from 33 sheep of different ages. Samples of ileal tissue were collected for immunocytochemistry and immunolabelled for prion protein (PrP). Specimens were then fixed in acetic acid, stained with methylene blue and transilluminated. Image analysis software was used to calculate the area of intestinal and PP tissue. The number of associated lymphoid follicles was determined using a dissecting microscope. Results showed a marked fall in surface area of PP tissue and lymphoid follicle density around puberty (about 8-9 months of age in NPU Cheviot sheep) and both measures remained low throughout adulthood. Using the Spearman's rank correlation coefficient, r(s), these two measures were found to be closely correlated (r(s)=0.899, n=33, P<0.0001). There was also a significant (negative) correlation between age and the two respective measures (surface area of PP tissue versus age, r(s)=-0.879 (n=33, P<0.0001); lymphoid follicle density versus age r(s)=-0.943 (n=33, P<0.0001). Immunolabelling for PrP was observed primarily in the light zone of lymphoid follicles. Results obtained from this study are useful for future oral pathogenesis studies of the NPU Cheviot flock. They may also offer a possible biological explanation for the apparent age-susceptibility relationship observed in natural cases of TSEs and might help to explain the young age-distribution of cases.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Freshly excised rat, ovine and bovine ileal Peyer's patch (PP) and non-Peyer's patch tissues (NPP) were mounted in modified horizontal polyethylene diffusion chambers with a range of window areas. Rat tissue was initially used to establish that barrier function and histology were maintained for up to 60 min. Horse-radish peroxidase (HRP) fluxes and S. Typhimurium adherence and invasion were significantly higher in rat PP over NPP. Particle uptake was shown to be a rapid, energy-, time-, and size-dependent process, occurring more readily in PP than NPP tissue in each species. In a kinetic analysis, particles were localized initially in the follicle-associated epithelium and then in the dome region. For NPP uptake, particles were initially localized to villous epithelium, and were then detected in the crypts and lamina propria. Electrophysiological parameters including pharmacologically-stimulated inward short-circuit current responses were determined in isolated PP and NPP from each species mounted under identical conditions in Ussing chambers. In conclusion, comparative functional and histological characteristics of PP from several species were demonstrated in horizontal diffusion chambers. Horizontal diffusion chambers are therefore a useful in vitro model in which a range of functions including transport of particulate formulations by PP may be examined.

The ileal Peyer's patches (Pp), secondary gut-associated lymphoid tissue of the mucosal immune system, may serve as an important site for monitoring inflammatory and immunologic responses of the host against enteric pathogens. Chicken Pp are often difficult to observe grossly, and a simple technique to enhance visualization of the Pp is lacking. Therefore, we designed a novel staining method that is quick, easy, and accurate to aid in gross identification and recovery of the chicken Pp from fresh tissue specimens. Lower alimentary tracts were harvested from White Leghorn hens and commercial broilers. The ileocecocolic region was excised intact, flushed with deionized water to remove ingesta, and a dilute eosin-Y solution was infused. After 1 min, the eosin-Y was gently extruded. Modified-crystal violet (mCV) was then injected into the gastrointestinal segment, where on the lymphoid tissue area became apparent at the serosal surface. The distal ileal Pp was visible as a pale whitish pink ovoid-focalized area with surrounding gut tissue stained light purple. The exact Pp site could be delineated at the serosal and mucosal surface by gross assessment. Light microscopy evaluation of hematoxylin and eosin-stained tissue slides prepared from the excised Pp site revealed lymphoid tissue aggregations with multiple follicular units indicative of Pp. The novel eosin-Y + mCV staining technique promotes rapid identification and accurate recovery of chicken Pp lymphoid tissue from fresh tissue specimens.

Background: Epidemiological analyses indicate that the age distribution of natural cases of transmissible spongiform encephalopathies (TSEs) reflect age-related risk of infection, however, the underlying mechanisms remain poorly understood. Using a comparative approach, we tested the hypothesis that, there is a significant correlation between risk of infection for scrapie, bovine spongiform encephalopathy (BSE) and variant CJD (vCJD), and the development of lymphoid tissue in the gut., Methods: Using anatomical data and estimates of risk of infection in mathematical models (which included results from previously published studies) for sheep, cattle and humans, we calculated the Spearman's rank correlation coefficient, rs, between available measures of Peyer's patch (PP) development and the estimated risk of infection for an individual of the corresponding age., Results: There was a significant correlation between the measures of PP development and the estimated risk of TSE infection; the two age-related distributions peaked in the same age groups. This result was obtained for each of the three host species: for sheep, surface area of ileal PP tissue vs risk of infection, rs = 0.913 (n = 19, P < 0.001), and lymphoid follicle density vs risk of infection, rs = 0.933 (n = 19, P < 0.001); for cattle, weight of PP tissue vs risk of infection, rs = 0.693 (n = 94, P < 0.001); and for humans, number of PPs vs risk of infection, rs = 0.384 (n = 46, P = 0.008). In addition, when changes in exposure associated with BSE-contaminated meat were accounted for, the two age-related patterns for humans remained concordant: rs = 0.360 (n = 46, P = 0.014)., Conclusion: Our findings suggest that, for sheep, cattle and humans alike there is an association between PP development (or a correlate of PP development) and susceptibility to natural TSE infection. This association may explain changes in susceptibility with host age, and differences in the age-susceptibility relationship between host species.

Functional anatomy offers an attempt to exploit anatomical information as a platform from which to decipher mechanistic details of complex or multistep immunological processes. Immune function depends on structural organization, therefore this approach contributes to a comprehensive understanding of the immune system. Major advances in functional anatomy require progress in both experimental techniques and analytical equipment - largely synonymous to refinement of the anatomist's favorite tool, the microscope. Here, we describe how currently available techniques co-operate to gain new insights into the biology of secondary lymphoid organs.

B cell receptor (BCR)-mediated antigen recognition is thought to regulate B cell differentiation. BCR signal strength may also influence B cell fate decisions. Here, we used the Epstein-Barr virus protein LMP2A as a constitutively active BCR surrogate to study the contribution of BCR signal strength in B cell differentiation. Mice carrying a targeted replacement of Igh by LMP2A leading to high or low expression of the LMP2A protein developed B-1 or follicular and marginal zone B cells, respectively. These data indicate that BCR signal strength, rather than antigen specificity, determines mature B cell fate. Furthermore, spontaneous germinal centers developed in gut-associated lymphoid tissue of LMP2A mice, indicating that microbial antigens can promote germinal centers independently of BCR-mediated antigen recognition.

A defect in RelB, a member of the Rel/nuclear factor (NF)-kappa B family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-kappa B-inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/- x aly/+ compound heterozygous mice. After stimulation with alpha-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/- or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

The intestinal immune system has to discriminate between harmful and beneficial antigens. Although strong protective immunity is essential to prevent invasion by pathogens, equivalent responses against dietary proteins or commensal bacteria can lead to chronic disease. These responses are normally prevented by a complex interplay of regulatory mechanisms. This article reviews the unique aspects of the local microenvironment of the intestinal immune system and discuss how these promote the development of regulatory responses that ensure the maintenance of homeostasis in the gut.

Intestinal M cells deliver macromolecules, particles, and pathogens into the subepithelial dome (SED) region of Peyer's patch mucosa, an area rich in dendritic cells (DCs). We tested whether uptake of the mucosal adjuvant cholera toxin (CT) or live Salmonella bacteria can induce DC migration within Peyer's patches. Virus-sized, fluorescent polystyrene microparticles were efficiently transported by M cells and ingested by CD11c(+), CD11b(-), and CD8a(-) DCs in the SED region. DCs loaded with microparticles remained in the SED for up to 14 days. CT (but not the CT B subunit) and live attenuated Salmonella enterica serovar Typhimurium bacteria induced migration of the microparticle-loaded DCs from the SED region into underlying B-cell follicles and adjacent parafollicular T-cell zones. Our data provide the first demonstration that DCs move in response to enterotoxin adjuvants and live bacteria that enter the mucosa via M cells.

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyer's patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTalpha1beta2/LTbetaR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2(-/-) mice. NALT organogenesis is initiated following the adoptive transfer of CD3(-)CD4(+)CD45(+) cells into Id2(-/-) mice, constituting direct evidence that CD3(-)CD4(+)CD45(+) inducer cells can provide an IL-7R-, LTalpha1beta2/LTbetaR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.

Lectin staining pattern in Peyer's patches of porcine ileum was studied using twenty one biotinylated-labeled lectins as cell markers which were visualized with avidin-biotin-peroxidase complex method (ABC). WGA appears to be a selective marker for tingible body macrophages in the porcine germinal centers. ConA may be a positive marker for the lymphoid tissues, whereas 9 lectins (DBA, SBA, SJA, s-WGA, PNA, ECL, UEA-I, PHA-E, and PHA-L) may be negative markers for the lymphoid tissues in all areas.

Peyer's patch and nasal-associated lymphoid tissue (NALT) are mucosal lymphoid tissues that appear similar in structure and function. Surprisingly, we found that NALT, unlike Peyer's patch, was formed independently of lymphotoxin (LT)alpha. Furthermore, using mice deficient in the retinoic acid receptor-related orphan receptor-gamma, we found that NALT was formed in the absence of CD4+CD3- cells, which are thought to be the embryonic source of LTalpha. However, we also found that NALT of LTalpha-/- animals was disorganized and lymphopenic, suggesting that the organization and recruitment of lymphocytes within NALT remained dependent on LTalpha. Finally, we demonstrated that both the structure and function of NALT were restored in LTalpha-/- animals upon reconstitution with normal bone marrow. These results demonstrate that the organogenesis of NALT occurs through unique mechanisms.

Leukocyte populations present in the discrete Peyer's patches (PP) of the pig were characterized from birth (Day 0) to day 35 after birth by immunohistochemistry and image analysis. Immediately after birth, cell membrane expression of CD2 and CD3, major histocompatibilty complex (MHC) class 11 (both SLA (swine leukocyte antigen) -DQ+ and SLA-DR+), CD21, 74-22-15 and surface immunoglobulin (sIg) were all demonstrable. Computer assisted morphometric techniques were used to confirm the significant expansion of these cell populations from birth onwards. The distribution of the cell types was not random but suggested a preferential retention of cells at specific sites. This implies a degree of organization of immunological cells within the discrete PP, enhancing the potential to mount immune responses in the most efficient manner.

We investigated the ability of pig ileal Peyer's patch segments to transport intestinal poly (D,L-lactide-co-glycolide) microspheres (PLGA MS) from intestinal lumen across the mucosae using in situ and ex vivo segments with confocal laser scanning microscopy (CLSM) and transmission electronic microscopy (TEM). From a global aspect, CLSM suggested that PLGA MS were translocated by M cells labelled with a FITC-conjugated anti-cytokeratin peptide 18, and transported through the follicle-associated epithelium (FAE) in the dome area in both types of experiments. At the ultrastructural level, TEM showed the traffic of PLGA MS throughout M cells, their transport into the basolateral invaginations of the M cells and their subsequent migration into the dome area and the follicular area in contact with macrophages and lymphatic vessels. Although in situ experiments allowed following the migration of PLGA MS until mesenteric lymph nodes, an ex vivo model could be used as a useful tool to study the targeting ability of PLGA MS formulations to the gut-associated lymphoid tissue (GALT).

The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK(-/-) mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK(-/-) mice also exhibited a marked deficiency of B cells in the spleen. RANK(-/-) mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

We investigated the role of IL-7 receptor alpha (IL-7Ralpha) signal in the formation of Peyer's patch (PP) anlage. Although pan-lymphopenia is a common phenotype of rag2-/- and il7ralpha-/- mice, a close inspection revealed nodules corresponding to PP in the adult rag2-/- but not in the il7ralpha-/- mouse. In our previous study, three histologically distinct steps in the formation of PP were identified. The first is the appearance of VCAM-1 + spots in the intestine, which probably represents an initial stage of the formation of the PP anlage. Accumulation of cells bearing IL-7Ralpha, CD4 or Ia in this region then follows and eventually entry of mature lymphocytes expressing CD3 or B220 occurs just before birth. Based on this criterion, we next investigated which of these events is defective in mice with severe combined immunodeficiency. Formation of VCAM-1 + spots and cluster formation of IL-7Ralpha+ cells proceed normally in the rag2-/- mouse which completely lacks mature lymphocytes. In contrast, no VCAM-1+ spots were detected in the embryonic nor neonatal il7ralpha-/- mice, suggesting that IL-7Ralpha signal is involved in the early phase of PP anlage formation. The same defect was found in the jak3-/- mouse. In addition to the appearance of VCAM1+ spots, the clustering of IL-7Ralpha+ cells was absent in the jak3-/- mouse, though IL-7Ralpha+ cells are found to scatter over the intestine. These results indicate that IL-7Ralpha is an essential signal for an early step of PP anlage formation, without which the subsequent processes cannot be initiated.

The function of Peyer's patches as antigenic sampling sites involves the complex interplay of a variety of mechanisms that aim to recognize luminal antigens, induce an immunological response and decrease the incidence of antigen translocation across the mucosal epithelium. This is achieved by M cells, which facilitate the uptake of luminal antigens, a vascular architecture that promotes the retention of absorbed antigens within the patch interstitium (allowing for maximal antigenic activation of lymphocytes) and the presence of lymphoid follicles that contain antigen-presenting cells and lymphocytes. Lymphocytes encountering antigen in the Peyer's patches proliferate, differentiate into fully mature antigen-specific effector cells and migrate to the mesenteric lymph nodes where they undergo final maturation. The mature lymphocytes then enter the systemic circulation and migrate throughout the other mucosa-associated lymphoid tissues of the body and "home' into the gut via high endothelial venules and gut-associated lymphoid tissue-specific adhesion molecules, providing antigen-specific lymphocytes at sites likely to re-encounter the antigen.

Using microscopic methods and statistic analysis lymphoid structures of large and small intestine walls and appendix were studied in 4-7 years children. The highest cell reproductive activity was detected in duodenal walls being slightly lower in appendix and group lymphoid vessels. The highest ratio of plasma cells was found in duodenum, ileal lymphoid patches and cecum, where the index was 7-fold higher than index of appendix and ileum. Lamina propria of intestinal mucosa is characterized by small fraction of mitotically dividing cells and undifferentiated cell forms, especially in cecum walls. However, the latter shows the greatest percentage of plasma cells.

Purpose: To investigate the effect of microparticle size on gastrointestinal tissue uptake., Methods: Biodegradable microparticles of various sizes using polylactic polyglycolic acid (50:50) co-polymer (100 nm, 500 nm, 1 micron, and 10 microns) and bovine serum albumin as a model protein were formulated by water-in-oil-in-water emulsion solvent evaporation technique. The uptake of microparticles was studied in rat in situ intestinal loop model and quantitatively analyzed for efficiency of uptake., Results: In general, the efficiency of uptake of 100 nm size particles by the intestinal tissue was 15-250 fold higher compared to larger size microparticles. The efficiency of uptake was dependent on the type of tissue, such as Peyer's patch and non patch as well as on the location of the tissue collected i.e. duodenum or ileum. Depending on the size of microparticles, the Peyer's patch tissue had 2-200 fold higher uptake of particles than the non-patch tissue collected from the same region of the intestine. Histological evaluation of the tissue sections demonstrated that 100 nm particles were diffused throughout the submucosal layers while the larger size nano/microparticles were predominantly localized in the epithelial lining of the tissue., Conclusions: There is a microparticle size dependent exclusion phenomena in the gastrointestinal mucosal tissue with 100 nm size particles showing significantly greater tissue uptake. This has important implications in designing of nanoparticle-based oral drug delivery systems, such as an oral vaccine system.

The histological appearance and distribution of T cells, B cells and plasma cells were investigated for oropharyngeal tonsils, small intestinal lymphoid aggregations (Peyer's patches), caecocolic lymphoid patches and mesenteric lymph nodes of koalas (Phascolarctos cinereus), common brushtail possums (Trichosurus vulpecula) and common ringtail possums (Pseudocheirus peregrinus). The histological organisation and distribution of lymphoid cell subpopulations of these tissues were similar to those described in eutherian mammals, although some differences were found in comparison with previous descriptions of American opossum tissues. The main variation among the 3 species was in the structural organisations of the oropharyngeal tonsil and the caecocolic lymphoid patch which were more complex in the koala than in possums. In the koala the extensive crypts of the oropharyngeal tonsils and folding of the mucosa of the caecocolic lymphoid patch increased their surface area and, in addition, both structures had areas of epithelium heavily infiltrated with T and B cells. These features could indicate that these structures are important in immunological surveillance of orally presented antigens in koalas.

The anatomical arrangement of organised lymphoid tissues of the alimentary tract for 3 Australian marsupials, the koala (Phascolarctos cinereus), the common brushtail possum (Trichosurus vulpecula and the common ringtail possum (Pseudocheirus peregrinus), was determined by gross dissection and acetic acid treatment. Oropharyngeal tonsils were consistently found in the dorsolateral wall of the caudal oropharynx in all 3 species and additionally in the ventral soft palate of the koala. Aggregated lymphoid nodules (Peyer's patches) were present in the small intestine of koalas, ringtail possums and brushtail possums and were of similar appearance for all 3 species. Bilateral large intestinal lymphoid patches were detected in the caecocolic lateral wall adjacent to the termination of the ileum for all 3 species. Caecocolic patches were more complex in koalas and had mucosal folds and a central recess. In addition, solitary and grouped large intestinal lymphoid nodules were variably present in the proximal colon and caecum of the koala. In contrast, possums had solitary and grouped large intestinal lymphoid nodules present in the proximal colon and rectum but not the caecum. Aggregated lymphoid tissue was not detected in the tongue, oesophagus or stomach for all 3 species. In contrast to a previous report, this study did not find a paucity of lymphoid tissue associated with the gut of the koala. The appearance and distribution of gut-associated lymphoid tissue in koalas and possums was found to be similar to that described in other marsupials and eutherian mammals, although some variations in appearance and anatomical location were observed.

Ileal Peyer's patches of rhesus macaques were investigated by light and electron microscopy (scanning-electron microscopy, transmission-electron microscopy). The results were compared with findings in other species. Differences were found concerning the shape of the dome area, the composition of the dome epithelium and the apical membrane of M cells: in the rhesus monkey, hemispherical domes bulge into the intestinal lumen. The dome epithelium is composed of three populations of gut epithelial cells; absorptive enterocytes as the predominant cell type; goblet cells; and M cells ("microfold bearing' or "membraneous' gut epithelial cells). The apical membrane of M cells forms irregular protrusions.

Despite the fact that the Peyer's patch (PP) is the primary site for antigen uptake in the intestine, the cellular basis of antigen handling after transport into the PP is poorly understood. We performed immunohistology of murine PPs using the dendritic cell (DC)-reactive monoclonal antibodies N418, NLDC-145, M342, and 2A1, as well as antibodies to other T cell, B cell, and macrophage markers. N418+, 2A1+, NLDC-145-, M342- cells form a dense layer of cells in the subepithelial dome (SED), just beneath the follicle epithelium, and are scattered throughout the follicle, sparing the germinal center. In contrast, N418+, 2A1+, NLDC-145+, and M342+ DCs are present in the interfollicular T cell regions (IFR). CD3+ and CD4+, but no CD8+ T cells were present in the SED and the follicle, including the germinal center, while CD3+, CD4+, and CD8+ T cells were present in the IFR. B cells and macrophages were poorly represented in the SED as no B220+ cells, only few Mac-1lo cells, and no F4/80+ cells were present at this site. In contrast, Mac-1hi cells were found in the IFR and lamina propria of intestinal villi, while F4/80+ cells were found only in the latter. In further phenotypic studies, we analyzed surface molecules of PP and spleen DCs by flow cytometry and found that these cells had similar fluorescence profiles when stained with N418, NLDC-145, and 33D1 DC-reactive antibodies, and antibodies to the costimulatory molecules B7-1 (1G10) and B7-2 (GL1). In contrast, PP DCs expressed 5-10-fold higher levels of major histocompatibility complex class II antigens (IEk) than spleen DCs. Finally, in functional studies, we demonstrated that both PP and spleen DCs process soluble protein antigens during overnight culture and induce similar levels of proliferation in CD3+ T cells, and CD4+/Mel 14hi T cells from T cell receptor transgenic mice. The in vivo relevance of such presentation was shown by the fact that PP DCs isolated from Balb/c mice after being fed ovalbumin stimulated proliferation in ovalbumin T cell receptor T cells. Taken together, our data suggest that DCs in the SED of the PP are uniquely positioned for the processing of antigens passed into the PP from the overlying M cell, and that PP DCs are effective at processing and presenting oral antigens to naive T cells.

The developmental biology of sheep ileal and jejunal Peyer's patches (PP) was investigated using corticosteroids to deplete immature B lymphocytes. During a 7-day treatment with dexamethasone, ileal PP follicular (iPf)B-cell proliferation was arrested and most iPfB-cells died. This resulted in follicular involution with the survival of mesenchymal cells. No iPfB-cell proliferation was detected in follicular remnants for 4 weeks postdexamethasone treatment, and during a subsequent 3-month period, there was limited iPfB-cell proliferation that resulted in a partial regeneration of follicles. Ileal PP involution was also associated with a severe B lymphopenia that persisted for over 14 weeks and was characterized by the survival of primarily isotype-switched and CD5+ sIgM+ B-cells in blood. In contrast, the size of jejunal PP follicles was reduced following dexamethasone treatment, but intrafollicular B-cell proliferation was not arrested. Furthermore, within 4 weeks, the jejunal PP follicles had recovered in size and cellularity and there was no disruption in IgA plasma-cell production. Thus, dexamethasone selectively depleted iPfB-cells and revealed that the ileal and jejunal PPs contain functionally distinct B-cell populations. The partial regeneration of the iPfB-cell population indicated that either an intrafollicular, corticosteroid-resistant B-stem cell existed or that ileal PP follicles can be repopulated by circulating B-cells. Finally, the association between ileal PP involution and the absence of circulating, CD5- B-cells confirmed that this lymphoid tissue provides an essential environment for conventional sIgM+ B-cell development.

A combination of immunohistochemical techniques, a panel of monoclonal antibodies, and computer-assisted morphometric analysis was used to examine the response of the ileal Peyer's patch of fetal lambs 7 days after treatment with ferritin per os. Consistent with previous studies in fetal lambs that have reported the ileal Peyer's patch to be indifferent to antigen, the present study did not find any significant changes in the size of the predominantly B-cell dome/follicle compartment or the predominantly T-cell interfollicular area, nor were differences identified in the distribution of IgM-positive (+), CD4+, and CD8+ cells in these two compartments. However, both compartments showed a significant increase (p < 0.05) in the percentage of area occupied by MHC II+ cells and a significant decrease (p < 0.05) in the percentage of area occupied by CD44+ and B5+ cells. These changes show that the ileal Peyer's patch of fetal lambs is not indifferent to antigen and may represent the transition of a purely primary lymphoid organ to an organ that has both primary and secondary lymphoid functions.

The interleukin 2 receptor gamma chain (IL-2R gamma) is a component of the receptors for IL-2, IL-4, IL-7, and IL-15. Mutations in IL-2R gamma in man appear responsible for the X chromosome-linked immunodeficiency SCIDX1, characterized by a defect in T-cell and natural killer (NK)-cell differentiation with the presence of poorly functioning B cells. To explore at which level IL-2R gamma affects lymphoid development in vivo, we have analyzed mice derived from embryonic stem (ES) cells with mutant IL-2R gamma loci generated by Cre/loxP-mediated recombination. In the peripheral blood of chimeric animals, lymphoid cells derived from IL-2R gamma- ES cells were not detected, although control ES cells carrying an IL-2R gamma gene with embedded loxP sites gave rise to T-, B-, and NK-cell lineages. Germline IL-2R gamma-deficient male animals, however, developed some mature splenic B and T cells, although the absolute number of lymphocytes was almost 10-fold reduced. In contrast, there was a complete disappearance of NK cells (over 350-fold reduction). Development of gut-associated intraepithelial lymphocytes was also severely diminished, and Peyer's patches were not detected. In vitro mitogenic responses of thymocytes, IL-4-directed immunoglobulin class switch of splenocytes, and NK activity were defective. Thus, IL-2R gamma facilitates mainstream B- and T-cell generation and function and also appears to be essential for NK-cell development.

Background: The lymphatics of Peyer's patches disseminate immunological information from the gut and thus play a key role in protection of the body against environmental pathogens. The aim of this project was to describe the lymphatic pathways of these Peyer's patches in pigs, and the mucosal intercellular spaces which lead to these lymphatics., Methods: Ileal tissue from living or freshly killed pigs was examined by light microscopy or electron microscopy, or was injected with Mercox (CL-2B, Japan Vilene Hospital, Tokyo) for scanning electron microscopy of corrosion casts., Results: Intercellular fluid between intestinal epithelial cells passes through pores in the basal lamina to mix with that in the intercellular spaces and prelymphatic intercellular channels of the lamina propria and follicle domes. From there, lymph enters lacteals in the villi, or a branching network of vessels within the lamina propria. Small lymphatics penetrate the muscularis mucosae and are continuous with (1) lymphatic vessels which pass directly to the deep submucosa between follicles, or (2) lymphatic sinuses which lie adjacent to the follicles. This differs from the situation in sheep and rabbits. Basal lymphatics beneath the follicles convey lymph to vessels which leave the surface at the serosa., Conclusion: The differences in the structure and arrangement of the lymphatics of Peyer's patches between pigs, sheep, and rabbits will require further investigation to determine if such variation between species has an effect on the distribution of immune products to effector sites.

Background/aims: In the small intestine, lymphocytes migrate through Peyer's patches. The distribution of lymphatic microvessels in rat Peyer's patches and lymphocyte traffic through them were studied., Methods: Vital dyes were injected via a micropipette into the Peyer's patches tissue to fill lymphatic microvessels and to stain lymphocytes in lymphatic microvessels., Results: Direct microscopic observation revealed a dense plexus of lymphatic microvessels in the perifollicular and interfollicular areas. Injection of the dyes into the germinal center failed to delineate lymphatic microvessels. The lymphatic microvessels in the perifollicular area were filled with lymphocytes. Most lymphocytes in the perifollicular lymphatics stayed in the lymphatic microvessels. Some lymphocytes became detached and drained into lymphatic microvessels in the interfollicular areas. Lymphocytes then moved toward the submucosal lymphatics beneath the villi around the Peyer's patches. The interfollicular lymphatics did not display contractile activity but had valves. Opening and closing of valves was synchronized with the respiration and the back and forth flow of lymphocytes., Conclusions: There are numerous lymphocytes in a dense lymphatic network in the perifollicular and interfollicular areas of Peyer's patches. This well-developed lymphatic network has the potential capacity for storage of lymphocytes and modulation of lymphocyte migration.

In previous studies, chemically induced colonic carcinomas were found to originate preferentially from crypts adjacent to lymphoid tissue. Proliferative parameters and mucosecretion were analyzed in proximal and distal rat colon in relation to the proximity of lymphoid patches. Animals received an intraperitoneal pulse of bromodeoxyuridine 1-hr before death. In both proximal and distal colon, crypts located at the immediate proximity of the lymphoid formations contained fewer mucous cells (P less than 0.001), but a higher percentage of proliferative epithelial cells (P less than 0.001) than the crypts far from lymphoid formations. The labeling index was higher in crypts adjacent to lymphoid patches compared to crypts distant from lymphoid patches only in the lower third of the crypts. The association of an increased proliferative activity and a decrease in differentiated mucosecreting cells in colonic crypts adjacent to lymphoid patches could be related to the particular sensitivity of these crypts cells to the effects of mutagens and carcinogens.

Canine Peyer's patches were examined by light microscopy and immunohistochemistry for possible variations depending on the location within the small intestine and for similarities and dissimilarities to PPs from other species. The duodenal and jejunal PPs were characterized by relatively large domes and interfollicular areas. In contrast, the ileal PP had small domes and poorly developed interfollicular areas and very large follicles. T cells were found in the interfollicular area and corona and in lesser numbers in the dome and germinal centers. The ileal PP contained far fewer T cells than the proximal PPs. Domes of canine PPs contained some cytoplasmic IgA+ (cIgA+) and many cIgG+ cells. Peanut agglutinin (PNA) stained germinal center cells in a selective but not-uniform way and did not stain T cells.

The following stages in lymphoid patches (LP) development have been revealed: I-1-6, II-7-14, III-15-21, IV-22-30 days; during these periods lymphoid noduli with germinative centers, cupolae and internodular areas are formed. Amount of all kinds of cells in the LP parenchyma and their rearrangement in the zones takes place. In one-month-old rats blast forms predominate in the LP germinative centers, and small lymphocytes--in the zone of small lymphocytes of the noduli and internodular areas. Under conditions of antenatal effect of an antibiotic on the system mother--fetus there is no disturbances in formation of the anatomical structures. However at each stage of development the small intestine LP are not mature, concerning their cellular composition. Amount of cells decreases, their interrelationship changes. Lympho- and plasmocytopoiesis decreases.

This review discusses experimental and clinical evidence for the existence of immunologically mediated damage to the intestinal mucosa. The discussion of mechanisms of gastrointestinal immunoregulation and lymphocyte recirculation via recently described homing receptors is preceded by a summary of the basic structures of the gut-associated lymphoid tissues (GALT). The relevance of food-induced systemic hyporesponsiveness (oral tolerance) to the prevention of immunologically mediated damage to intestinal mucosa is discussed in the light of anaphylactic (Type I) and delayed type (Type IV) hypersensitivity reactions. Other, less well defined, mechanisms of allergic injury are also briefly discussed. It is postulated that the abrogation and/or modulation of orally induced tolerance is the pathogenetic mechanism underlying the induction of food-sensitive enteropathies.