Intestinal Patches Research Articles

Coexistence of Granulomatous Enteric Inflammation and Neoplasia in an Adult Sheep

A 7-year-old dairy sheep suffering from chronic loss of weight without diarrhea or anorexia was euthanized after failing to respond to any treatment (antibiotic and antiparasitic). The main findings at the necropsy of this animal were multifocal miliary nodules in several organs, mainly in the Peyer's patches of the small intestine, and a segmental thickening of the jejunal wall. Histologic examination of the samples taken at the necropsy showed a multifocal chronic granulomatous inflammation, with mineralization and caseous necrosis at the core of the larger granulomas and scarce intrahistiocytic acid-fast bacilli consistent with a disseminated digestive tuberculosis. Polymerase chain reaction and bacteriological culture from these samples confirmed Mycobacterium avium subsp avium to be the etiologic agent of this infection. Histologically, the cause of the segmental thickening of the jejunal wall was found to be a small intestine adenocarcinoma, which in some areas coexisted with the granulomatous lesion.

Expression patterns of porcine Toll-like receptors family set of genes (TLR1-10) in gut-associated lymphoid tissues alter with age

The aim was to study the expression pattern of the porcine TLR family (TLR1-10) genes in gut-associated lymphoid tissues (GALT) of varying ages. A total of nine clinically healthy pigs of three ages group (1day, 2months and 5months old) were selected for this experiment (three pigs in each group). Tissues from intestinal mucosa in stomach, duodenum, jejunum and ileum and mesenteric lymph node (MLN) were used. mRNA expression of TLRs (1–10) was detectable in all tissues and TLR3 showed the highest mRNA abundance among TLRs. TLR3 expression in stomach, and TLR1 and TLR6 expression in MLN were higher in adult than newborn pigs. The western blot results of TLR2, 3 and 9 in some cases, did not coincide with the mRNA expression results. The protein localization of TLR2, 3 and 9 showed that TLR expressing cells were abundant in the lamina propria, Peyer’s patches in intestine, and around and within the lymphoid follicles in the MLN. This expressions study sheds the first light on the expression patterns of all TLR genes in GALT at different ages of pigs.

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%–60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.

Formulation and evaluation of oral microparticulate ovarian cancer vaccines

Ovarian cancer is the fifth most leading cause of cancer related deaths in women in the US. Customized immunotherapeutic strategies may serve as an alternative method to control the recurrence or progression of ovarian cancer and to avoid severe adverse effects of chemotherapy. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared with the use of a spray dryer. These particles were designed for oral delivery using enteric polymers such as methacrylic copolymer, Eudragit® FS30D and hydroxyl propyl methyl cellulose acetate succinate. These particles were targeted for uptake via microfold cell (M-cell) in Peyer's patches of small intestine using M-cell targeting ligand, Aleuria aurantia lectin. The interleukins (ILs) such as IL-2 and IL-12 were added to the vaccine formulation to further enhance the immune response. The particles obtained were of 1.58±0.62μm size with a charge of 12.48±2.32mV. The vaccine efficacy was evaluated by administering the particles via oral route to C57BL/6 female mice. At the end of vaccination, mice were challenged with live tumor cells. Vaccinated mice showed significant (around six-fold) retardation of tumor volume in comparison to non-vaccinated animals for 3 weeks after the tumor challenge (p<0.001). The serum IgG antibody levels were found to be elevated in case of vaccinated animals in comparison to non-vaccinated group (p<0.05). Analysis of IgG1 titers (indicative of Th2 response) and IgG2a titers (indicative of Th1 response) showed a mixed Th1 and Th2 immune response in case vaccine alone and Th2 response in case of vaccine with interleukins group. Moreover, CD8+ T-cell, CD4+ T-cell and B-cell populations in different lymphatic organs were elevated in case of vaccinated mice. Thus, whole cell lysate vaccine microparticles formulated by spray drying could trigger humoral as well as cellular immune response when administered orally. Such vaccine could potentially be an effective treatment for patients with residual tumor or high tumor-relapse probability.

Abstract 5705: Orally delivery alginic acid-coated chitosan nanoparticles loaded with legumain DNA vaccine protect against murine breast cancer

Abstract Legumain DNA vaccine has been proven to be a potential treatment approach to protect against breast cancer. However, absence of safe and efficient oral delivery system highly restricts its clinical application. Here, we aim to develop a novel oral delivery system for legumain DNA vaccine with better biocompatibility and efficiency. We constructed alginic acid-coated chitosan NPs (A.C.NPs) and tested its anti-acid characteristics in vitro. DNA agrose electrophoresis data showed that compared to chitosan NPs (C.NPs), A.C.NPs significantly protected DNA from degradation in the condition of pH=1.5. Furthermore, size distribution anlysis showed that when pH&amp;lt;3.0, A.C.NPs aggregated to form the micrometer scale complex and dispersed to nanoparticle sized about 314 nm with the increased pH value. To visualize DNA expression, mice were treated by gavage with the A.C.NPs carrying EGFP expression plasmid. Significant EGFP expression can be detected in the intestinal peryer's patch of mice. Immunoflurescence staining showed that overlapping existed in EGFP and F4/80 (CD11b) positive cells. Full length legumain plasmids were loaded into different delivery systems including chitosan NPs (CL), Salmonella typhi (SL), A.C.NPs (ACL). PBS and A.C.NPs loaded with empty plasmid was also set as control groups (n=5 each group). Oral vaccination was performed in murine orthotropic 4T1 breast cancer model. Animals were scarified when the average tumor size in the PBS group reached 500 mm3. Our data showed that tumor volume was significantly smaller in ACL and SL groups compared to other groups. Further, we isolated splenocytes from mice and co-cultured with 4T1 cells pre-stimulated with hypoxia, which intrigued the translocation of legumain from cytoplasm to membrane. FACS assay showed that active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) in ACL group increased 4.5 folds compared to PBS group and 2.3 folds compared to CL group respectively. Our study suggests that chitosan NPs modified with alginic acid could be a potential safe and efficient tool for oral delivery plasmid DNA. Alginic acid-coated chitosan NPs loaded with legumain DNA vaccine can effectively improve auto-immune response and protect against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5705. doi:1538-7445.AM2012-5705

Spray-dried microparticles: a potential vehicle for oral delivery of vaccines

This study aims to formulate a microparticle-based system that protects the protein from the harsh gastric conditions and also provides appropriate uptake via M cells for desired immune response upon oral administration. The formulation was derived using a valid statistical model, analysed by JMP® (SAS). The average size and charge of the resulting microparticles were 1.51 ± 0.125 µm and + 15.7 ± 2.5 mV, respectively. Moreover, the particles provided a prolonged release over a period of 8 hrs which ensures M-cell uptake of intact particle with antigen (Kunisawa et al., 2011). This was further supported with in vivo studies where particle uptake was found in Peyer's patches of small intestine when observed for 8 h. Thus, these microparticles can be used as an efficient vaccine delivery vehicle upon oral administration.

Peyer's Patch Dendritic Cells Sample Antigens by Extending Dendrites Through M Cell-Specific Transcellular Pores

Peyer's patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo. We performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC. LysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED. We describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mucosa.

Intestinal patches with an immobilized solid-in-oil formulation for oral protein delivery

Oral administration of biomolecular drugs such as peptides, proteins, and DNA is an attractive delivery method because of the safety and convenience of delivery in contrast to injection administration. However, oral delivery of biomolecules has several potential barriers such as enzymatic degradation in the gastrointestinal tract and low permeability across an intestinal membrane. In this study, we proposed an intestinal patch system that included surfactant-coated insulin for oral delivery. The intestinal patches, which have mucoadhesive and drug-impermeable layers, induced sustained unidirectional insulin release toward intestinal mucosa and inhibition of insulin leakage from the patches. Moreover, the surfactant-coated insulin, which has high compatibility with cell membranes, enhanced insulin transport across the intestinal membrane. This study demonstrates that the intestinal patches might improve protein permeability in the intestinal mucosa, thereby offering an innovative therapeutic strategy.

Influence of Adding Pyrroloquinoline Quinone to Parenteral Nutrition on Gut‐Associated Lymphoid Tissue

Experimental intravenous (IV) parenteral nutrition (PN) diminishes gut-associated lymphoid tissue (GALT) cell number and function. PN solution cannot maintain GALT at the same level as a normal diet, even when delivered intragastrically (IG). Previous studies demonstrated pyrroloquinoline quinone (PQQ)-deficient mice to be less immunologically responsive. Because standard (STD) PN solution lacks PQQ, PQQ supplementation may prevent PN-induced GALT changes. This study was designed to determine the influence of adding PQQ to PN on GALT. In experiment 1, mice (n = 32) were randomized to chow, IV-STD-PN, and IV-PQQ-PN groups. The chow group was fed chow with the same caloric content as PN. The IV-STD-PN group received STD-PN solution, whereas the IV-PQQ-PN group was given PQQ (3 mcg/d)-enriched PN by the IV route. After 5 days of feeding, lymphocytes were isolated from the Peyer's patch (PPs), intraepithelial space (IE), and lamina propria (LP) of the small intestine. GALT lymphocyte number and phenotype (αβTCR+, γδTCR+, CD4+, CD8+, B220+ cells) and intestinal immunoglobulin A (IgA) level were determined. In experiment 2, mice (n = 28) were randomized to IG-STD-PN or IG-PQQ-PN group. After IG nutrition supports, GALT mass and function were determined as in experiment 1. The IV-PQQ-PN group showed increased PP lymphocyte number and PP CD8+ cell number compared with the IV-STD PN group. The IG-PQQ-PN group had significantly greater PP lymphocyte number and PP CD4+ cell numbers than the IG-STD-PN group. Neither IV nor IG PQQ treatment raised IgA level. PQQ added to PN partly restores GALT mass, although its effects on GALT function remain unclear.

Age-related changes in the anatomical characteristics of Peyer's patches in small intestine of Bactrian camels (Camelus bactrianus)

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.

Peyer's Patch-Deficient Mice Demonstrate ThatMycobacterium aviumsubsp.paratuberculosisTranslocates across the Mucosal Barrier via both M Cells and Enterocytes but Has Inefficient Dissemination

Mycobacterium avium subsp. paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested that M. avium subsp. paratuberculosis interacts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa by M. avium subsp. paratuberculosis and Mycobacterium avium subsp. hominissuis, a pathogen known to interact with intestinal cells, was compared. M. avium subsp. paratuberculosis was capable of invading the mucosa, but it was significantly less efficient at dissemination than M. avium subsp. hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate that M. avium subsp. paratuberculosis enters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated that M. avium subsp. paratuberculosis had equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice. M. avium subsp. paratuberculosis was also shown to interact with epithelial cells by an alpha(5)beta(1) integrin-independent pathway. Upon translocation, dendritic cells ingest M. avium subsp. paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary, M. avium subsp. paratuberculosis interacts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently.

Liposomal oral DNA vaccine (mycobacterium DNA) elicits immune response

Tuberculosis is one of the leading causes of mortality from an infectious disease worldwide, however, the efficacy of BCG vaccine against adult pulmonary tuberculosis still remains instability. Therefore, it is an urgent work to develop both safe and effective vaccine to TB.To clarify the liposome encapsulated DNA vaccine was effectively working as a vaccine delivery system to evoke mucosal intestinal immune responses. A Mycobacterium pcDNA3.1+/Ag85A DNA was constructed and encapsulated into liposome. Ag85A protein antigen was observed to substantially express in the epithelium, microfold cells (M cells), dendritic cells (DCs) and Peyer's patches (pp) of the small intestine, respectively, after oral administration into C57BL/6 mice 3 times each 14 days interval. Furthermore, levels of IL-2 and IFN-γ in the IELs isolated from the small intestine were markedly increased, IL-4 level was not significantly changed as compared to those in control group after oral administration of Ag85A DNA encapsulated in liposome, together with the augmented Ag85A-specific cytotoxicity of IELs, indicating a local Th1 dominant cellular immune response was elicited, and thus enhanced cytotoxicity of IELs as compared to those in control mice. Furthermore, sIgA level was also elevated in liposomal encapsulated Ag85A DNA immunized mice. These data indicated that oral vaccination with the liposomal-pcDNA 3.1+/Ag85A DNA is able to induce antigen specific mucosal cellular and humoral immune responses. Especially, cellular compartment in the epithelium of small intestine plays a key role on the regulation of immune response to eliminate TB. These findings have important understanding and possible implications for the design of new strategies based on oral DNA vaccine on regulation of immune response in protection against TB. Further study is clearly necessary to improve the effectiveness of Ag85A DNA vaccines against TB as compared with BCG.

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In this issue

Transcriptional analysis of Toll-like receptors expression in M cells

M cells are located in the follicle associated epithelium (FAE) of Peyer's patches (PPs) in the small intestine, where they mediate the uptake and transcytosis of luminal antigens to the underlying lymphoid tissue. Toll-like receptors (TLRs) have emerged as key mediators in the innate immune response by recognising pathogen associated molecular patterns (PAMPs) expressed by microorganisms. TLRs have previously been shown to be differentially expressed in the gastrointestinal tract. In this study PP were harvested from BALB/c mice. Ulex europaeus agglutinin 1 (UAE-1) positive M cells were isolated from FAE and the expression of TLR1–9 transcripts in M cells, FAE and villus epithelium (VE) was compared by quantitative real-time PCR. Transcripts for TLR1, TLR2 and TLR4 were found to be expressed at a high level in M cells in comparison to VE, with no transcripts being detected in the FAE. TLR3 and TLR6 were not found to be expressed in M cells or in the FAE. TLR5 and TLR7 were found to be expressed at a higher level in FAE compared to M cells. TLR9, which recognises unmethylated CpG DNA of bacteria and viruses and TLR8, which recognises ssRNA, were found to be preferentially expressed in M cells compared to FAE and VE.

Donor single nucleotide polymorphism in the CCR9 gene affects the incidence of skin GVHD

The interactions between chemokines and their receptors may have an important role in initiating GVHD after allogeneic hematopoietic SCT (allo-HSCT). CCL25 and CCR9 are unique because they are exclusively expressed in epithelial cells and in Peyer's patches of the small intestine. We focused on rs12721497 (G926A), one of the non-synonymous single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the SNP of donors in 167 consecutive patients who received allo-HSCT from an HLA-identical sibling donor. Genotypes were tested for associations with acute and chronic GVHD in each organ and transplant outcome. Multivariate analyses showed that the genotype 926AG was significantly associated with the incidence of acute stage > or =2 skin GVHD (hazard ratio: 3.2; 95% confidence interval (95% CI): 1.1-9.1; P=0.032) and chronic skin GVHD (hazard ratio: 4.1; 95% CI: 1.1-15; P=0.036), but not with GVHD in other organs or with relapse, non-relapse mortality or OS. To clarify the functional differences between genotypes, each SNP in retroviral vectors was transfected into Jurkat cells. In chemotaxis assays, the 926G transfectant showed greater response to CCL25 than the 926A transfectant. In conclusion, more active homing of CCR9-926AG T cells to Peyer's patches may produce changes in Ag presentation and result in increased incidence of skin GVHD.

Induction of intestinal lymphoid tissue formation by intrinsic and extrinsic signals

Since the discovery of inducer cells as a separate lineage for organogenesis of Peyer's patches in the small intestine of fetal mice, a lot of progress has been made in understanding the molecular pathways involved in the generation of lymphoid tissue and the maintenance of the lymphoid architecture. The findings that inducer cells also exist in adult mice and in humans, have a lineage relationship to natural killer cells, and can be stimulated during infections highlight their possible role in establishing innate and adaptive immune responses. Novel concepts in the development of intestinal lymphoid tissues have been made in the past few years suggesting that lymphoid organs are more plastic as previously thought and depend on antigenic stimulation. In addition, the generation of novel lymphoid organs in the gut under inflammatory conditions indicates a function in chronic diseases. The present review summarizes current knowledge on the basic framework of signals required for developing lymphoid tissue under normal and inflammatory conditions.

Análise imuno-histoquímica de cães naturalmente infectados pelo parvovírus canino

Noventa e seis cães com lesões macroscópicas sugestivas de parvovirose canina foram necropsiados no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul no período de março de 2005 a novembro de 2006. Tecidos destes caninos foram analisados através de histologia e imuno-histoquímica. Aumento das placas de Peyer do intestino delgado e hiperemia da mucosa e serosa intestinal foram os achados macroscópicos mais observados. Microscopicamente, foi visualizada enterite necrótica em 77% dos cães. Em 17,7% as alterações histológicas do intestino delgado ficaram prejudicadas pela autólise, dificultando a interpretação. O teste de imuno-histoquímica em cortes de intestino delgado, linfonodo mesentérico, timo, baço, tonsila, língua e medula óssea de todos os 96 casos, foi positivo em 91,6% (88/96) dos casos. O intestino delgado demonstrou o melhor resultado, obtendo-se marcações em 77% (74/96) dos casos. A análise final do exame paramétrico de Fisher demonstrou uma fraca associação entre autólise intestinal e resultado positivo da imuno-histoquímica onde as chances de um intestino delgado autolisado histologicamente apresentar resultado positivo na imuno-histoquímica é 0,33 vezes menor (OR=0,33; 95%IC: 0,10-1,17) quando comparada a um intestino delgado não autolisado.

STUDIES ON USE OF MATERIAL COLON AS AN IMPLANT ON THE THIN INTESTIN AT DOGS

In this study the authors present a method to delay intestinal transit. It is well known that after resection on the thin intestine, on a significant length appears the malabsorption effect, due to the surface lack of absorption. In the given circumstances it is necessary for a method of delaying the intestinal transition, which compensates for the surface lack of absorption with a delay in the process of transition of the intestinal chyle. Therefore a patch of the thick intestine was thought as an anti-peristaltic wave for the thin intestine in an auto-transplant on the thin intestine

Donor Single Nucleotide Polymorphism in the CCR9 Gene Affects the Incidence of Acute Skin Graft-Versus-Host Disease

Chemokines are a group of small molecules that regulate trafficking of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled chemokine receptors. Their interactions may play an important role in initiating acute graft-versushost disease (GVHD) after allogeneic hematopoietic stem cell transplanation. CCR9 is a very unique receptor because, outside of the thymus, it is almost exclusively expressed by epithelial cells and Pyer's patches in the small intestine. We focused on rs12721497 (G926A), one of the single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the impact of its genotype on the onset of organ-specific acute GVHD and transplant outcome. Included in the study were consecutive patients who had received allogeneic bone marrow or peripheral blood stem cell transplantation from an HLA-identical sibling donor at the Nagoya University Hospital and the Japanese Red Cross Nagoya First Hospital between 1987 and 2006. Of these, a total of 167 patients who received T cell replete transplantation with short-term methotrexate and cyclosporine as a GVHD prophylaxis were analyzed. Informed consent was obtained from all patients and donors, and the study was approved by the ethics committees at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. The CCR9 G926A genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism method using genomic DNA obtained from donor cells. The impact of CCR9 genotype on the incidence of acute GVHD in each organ, overall survival rates, relapse rates and nonrelapse mortality rates were analyzed using the Cox proportional hazard regression model. Age, intensity of conditioning, total body irradiation, disease risk, remission state and graft source were used as covariates for adjustment. Median age was 38 years old (range 15–62). Diseases were malignancies (n=148) and non-malignancies (n=19). The incidences of acute GVHD were 22% (grade I), 12% (grade II) and 6.6% (grade III-IV). The frequencies of the AA, AG and GG genotype were 94%, 6% and 0%, respectively. CCR9 926AG genotype was significantly associated with the incidence of stage³2 skin GVHD (hazard ratio 3.2; 95% CI, 1.1–9.1), but not associated with the incidence of liver GVHD, intestinal GVHD, grade II-IV GVHD, overall survival rates, relapse rates and nonrelapse mortality rates. In conclusion, the frequencies of each genotype were compatible with the reports in HapMap database. It was notable that donor CCR9 SNPs affected the incidence of skin GVHD, but did not affect that of intestinal GVHD. Beilhack et al. ( Blood . 2008; 111:2919–2928) recently reported redundancy of secondary lymphoid organs at different anatomical sites in GVHD initiation. They suggested that primed T cells could initiate GVHD at different sites from their original priming sites. Since Pyer's patches are important as sites of antigen presenting, T cell homing to Pyer's patches may be different between each CCR9 926AG genotype, resulting in different incidences of skin GVHD. We are now investigating the functional differences between each genotype.

Characterization of invasion pattern of Shigella flexneri in the small intestine of infant mice

Shigella spp. invades the large intestinal epithelium and promotes a strong inflammatory response in humans. A recent study revealed that 4‐day‐old mice were infected with invasive Shigella strains but not 5‐day‐old mice. We therefore attempted to clarify the underlying mechanism that induces inflammation in the small intestine of infant mice but not of adult mice. Balb/c mice of different ages were orally administered 5 × 109 CFU of Shigella flexneri 5a (M90T) strain and analyzed for colonization and physiopathological features. Unexpectedly, we found that 4‐, 7‐, and 14‐day‐old mice, but not 21‐day‐old mice, showed tissue destruction and PMN infiltration in the small intestine at 6 h following Shigella infection, mimicking human shigellosis. Interestingly, Shigella mainly invaded epithelial cells and crypt region but not the M cells in the small intestine. Further, high numbers of Shigella were mainly colonized in the lamina propria but not in Peyer's patches in the small intestine, and the Shigella colonization gradually decreased with ageing. We are now attempting to determine the crucial signaling mechanism of Shigella infection in the epithelium and crypt region of the small intestine of infant mice.