Hodaka Suzuki

Development of apoptosis and changes in lymphocyte subsetsin thymus, mesenteric lymph nodes and Peyer's patches of mice orally inoculated with T-2 toxin

Development of apoptosis and changes in lymphocyte subsets were examined mainly by flow cytometer in thymus, mesenteric lymph nodes and Peyers patches of mice up to 24 hours after oral inoculation with T-2 toxin (10 mg/kg). T-2 toxin attacked Peyers patches first, then mesenteric lymph nodes, and finally thymus in relation to the course of enteric absorption of orally inoculated T-2 toxin. The degree of lymphocyte apoptosis was prominent in the thymus, moderate in the Peyers patches, and somewhat mild in the mesenteric lymph nodes, suggesting the difference in lymphocyte population susceptible to T-2 toxin. As to the changes in lymphocyte subsets, CD4+ CD8+ T cells were most sensitive to T-2 toxin, and CD4+ CD8- T cells were more severely depressed than CD4- CD8+ T cells in the thymus. In the mesenteric lymph nodes, CD3+ cells was more clearly affected than CD19+ cells, and the numbers of CD4+ and CD8+ cells were similarly decreased. In the Peyers patches, the numbers of CD3+, CD 19+, CD4+ and CD8+ cells were unexceptionally decreased. In addition, among IgM+, IgG+ and IgA+ B cells, the number of IA+ B cells which are more important in the mucosal immunity was most severely affected.

Ultrastructural study on the follicle-associated epithelium of nasal-associated lymphoid tissue in specific pathogen-free (SPF) and conventional environment-adapted (SPF-CV) rats

Membranous (M) cells in follicle‐associated epithelium (FAE) play an important role in the mucosal immunity through transport of a variety of foreign antigens to the underlying mucosa‐associated lymphoid tissue (MALT). We aimed to investigate the ultrastructure of M cells in the FAE covering nasal‐associated lymphoid tissue (NALT) both in specific pathogen‐free (SPF) rats and in conventional environment‐adapted (SPF‐CV) rats aged 8–38 wk. In NALT of both SPF and SPF‐CV rats, FAE included the nonciliated microvillous cell, which appears to be an analogue of M cell previously described in other MALT. In SPF rats, M cells increased in number only slightly with age, and they maintained morphological uniformity irrespective of age. In SPF‐CV rats, M cells selectively increased in number resulting in prominent expansion of FAE surface area in parallel with the duration of maintenance in a conventional environment. In addition, M cells in SPF‐CV rats showed heterogeneity in their surface morphology such as the length and number of microvilli and cell surface area and outline. In addition, the FAE was stratified by various subtypes of M cells, which were characterised by several subcellular alterations including the presence of many keratin filaments, homogeneous dark bodies and extensive cytoplasmic interfoliation with wide intercellular spaces filled with amorphous proteinaceous material. These characteristics of M cells in SPF‐CV rat were intimately related with a preferential influx of immunocompetent cells into the FAE, which was not seen or was very rare in SPF rats irrespective of age. The results suggest the possibility that NALT may effectively carry out the mucosal immune response against antigenic stimuli of different magnitude through the unique dynamics of M cells which seem to be influenced by the infiltration of immunocompetent cells.

Development of early apopotosis and changes in lymphocyte subsets in lymphoid organs of mice orally inoculated with nivalenol.

Development of early apoptosis and changes in lymphocyte subsets were examined in lymphoid organs of female BALB/c mice after oral administration of 15 mg/kg b.w. of nivalenol (NIV), the major type B trichothecene mycotoxin, by FACS analysis. Judging from the results of viable cell count and apoptotic cell index, NIV attacked Peyers patches first and thymus most severely. In thymus, selective damage in CD4(+)CD8(+) cells was observed at 12 and 24 h after inoculation (HAI), following the peak of apoptosis at 9 HAI. CD4(+) cells were clearly suppressed at 3 HAI in Peyers patches, at and after 9 HAI in mesenteric lymph nodes, and 3 to 12 HAI in spleen, respectively. CD8(+) cells were also suppressed at 24 HAI in mesenteric lymph nodes and at 12 HAI in spleen, respectively. As to changes in B cell subsets, IgG(+) cells significantly decreased from 3 to 12 HAI and all B cell subsets at 24 HAI in mesenteric lymph nodes. In spleen, IgM(+) cells were suppressed at 9 HAI. On the other hand, in Peyers patches, following clear decrease in the numbers of pan-T and pan-B cells and viable cells at 3 HAI, all B cell subsets, especially IgA(+) cells, showed a significant increase in their numbers at 9 HAI, and the numbers of IgA(+) and IgM(+) cells remained higher values than controls thereafter. Taken together, in the course of recovery from NIV-induced prominent damage in Peyers patches at 3 HAI, interaction of NIV with Peyers patches might result in in vivo stimulation of interleukin production at this site and result in increased proliferation and differentiation of IgA-secreting B cells at and after 9 HAI.

Differences in Intraepithelial Lymphocytes in the Proximal, Middle, Distal Parts of Small Intestine, Cecum, and Colon of Mice

We have previously reported the regional differences in the intraepithelial lymphocytes (IELs) present in the small intestine of mice. In this study, we further investigated these differences on the basis of our previous findings and studied the entire intestine, including the cecum and colon. Most of the significant differences in phenotypic compositions were found between the small and large intestines, although some differences were found among the different parts of the small and large intestines. In particular, the composition of the subsets in αβ T cells and γδ T cells clearly differed between the small and large intestines. For example, in αβ T cells, the percentages of double negative (DN) and CD8αα+ cells were higher in the large intestine, that of CD8αβ+ cells was higher in the small intestine, and those of CD4+ and CD4+ CD8αα+ double positive (DP) cells were higher in the distal part of the small intestine. In γδ T cells, the percentage of CD8αα+ cells was higher in the small intestine and that of DN cells was higher in the large intestine. These results indicate that the differences between IELs in the small and large intestines are discontinuous.

Age-related changes in the regional variations in the number and subsets of intraepithelial lymphocytes in mouse small intestine.

Previously, we reported regional variations in the number and subsets of the small intestinal IELs of mice. In this study, we examined the age-related changes in the regional variations of IELs in mice from 2 to 11 weeks old. IELs were isolated from the proximal, middle and distal parts of the small intestine and analysed by flow cytometry. The total number of IELs gradually increased with age and reached a plateau at 8 weeks old. As to IEL subsets, the percentage of alpha beta T cells was higher in the distal part at and after 2 weeks of age (before weaning). The percentage of the alpha beta T cell subset of extrathymic origin was higher in the proximal part while the percentages of alpha beta T cell subsets of thymic origin were higher in the distal part at and after 3 weeks (just after weaning). It appears that regional variations in IELs may be formed before the weaning period in mice.

Comparison of Toxicity between Saxitoxin and Decarbamoyl Saxitoxin in the Mouse Bioassay for Paralytic Shellfish Poisoning Toxins

ABSTRACT The mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins has been used in the AOAC Official Method and the official Japanese method. In the AOAC Official Method, the saxitoxin (STX) standard provided by the U.S. Food and Drug Administration (FDA) is used, but no standard is used in the official Japanese method. The objective of this study was to compare the toxicity of decarbamoyl STX (dcSTX), one of the derivatives of STX and a candidate standard for the MBA for PSP toxins in Japan, to that of FDA STX in the MBA platform. In this study, the toxicity of dcSTX was 918.0 ± 44.9 mouse units/µmol, and the relative toxicity ratio of dcSTX to FDA STX based on moles was 0.478.

REGIONAL VARIATIONS IN THE DISTRIBUTION OF SMALL INTESTINAL INTRAEPITHELIAL LYMPHOCYTES IN ALYMPHOPLASIA (aly/aly) MICE AND HETEROZYGOUS (aly/+) MICE

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in alymphoplasia mutant (aly/aly) mice, which are characterized by the systemic absence of lymph nodes and Peyers patches, and heterozygous (aly/+) mice. The small intestines were taken from 10 to 12-week-old mice and divided equally into 3 parts (the proximal, middle and distal parts). IELs were isolated from each part of the intestine and analyzed with a flow cytometer. The number of IELs in the distal part was significantly fewer in aly/aly mice compared with aly/+ mice, although the total number of small intestinal IELs were comparable between them. As to the IELs subsets, regional variations in αβ T cells and γδ T cells were observed in aly/+ mice, but they disappeared in aly/aly mice. However, regional variations in composition of αβ T cell subsets were similarly observed in both aly/aly mice and aly/ + mice. This indicates that, although not essential, mesenteric lymph nodes (MLN) and/or Peyers patches may modify the regional variations in IELs.

Picryl chloride-induced allergic dermatitis in IQI/Jic female mice.

IQI/Jic (IQI) mice are an ICR-derived inbred strain developed in Japan, and it is known that aged females of this strain develop allergic dermatitis of spontaneous nature. In the present study, young IQI female mice which were sensitized with picryl chloride (PCL) to the shaved skin of abdomen and then topically applied with PCL to the ear at 4, 11, 18 and 25 days after the sensitization were examined. The ear swelling response increased rapidly after the 1st application, peaked after the 2nd one, and then gradually decreased. Histopathologically, edema with inflammatory cell infiltration developed after the 1st application and progressed after the 2nd one. The number of mast cells, CD4-positive cells and MHC class II-positive cells became prominent accompanied with epidermal thickening and dermal fibroplasia after the 4th application when clear elevation of total serum IgE levels was observed in many mice. Compared with the dermatitis induced in the same way in BALB/c female mice, the nature was similar with each other but the degree was obviously severer in IQI female mice. IQI female mice are considered to be a useful laboratory animal for the investigation of allergic dermatitis.

Growth of Listeria monocytogenes in refrigerated ready-to-eat foods in Japan

The ability of L. monocytogenes to grow in a series of Japanese ready-to-eat (RTE) foods, including boiled baby sardine and Japanese pickle, was tested at two different refrigeration temperatures. In RTE foods in which L. monocytogenes can grow, growth was significantly higher at 10°C than that at 4°C during their shelf lives and growth patterns varied extensively among the different types of foods. However, growth did not occur at 4°C within the shelf life of certain RTE foods, such as broiled squid. The patterns of growth were varied extensively with different sample types. These results suggest that some types of traditional Japanese RTE foods stored at 10°C may be potential sources of listeriosis. To reduce the risk of food-borne listeriosis, studies to determine the contamination levels in RTE foods and the effects of storage temperature on their shelf lives are needed.

Sequence of busulfan-induced neural progenitor cell damage in the fetal rat brain.

The sequence of neural progenitor cell (NPC) damage induced in fetal rat brain by transplacental exposure to busulfan, an antineoplastic bifunctional-alkylating agent, on gestational day 13 was examined by immunohistochemical and real-time RT-PCR analyses. Following busulfan treatment, pyknotic NPCs first appeared in the medial layer and then extended to the dorsal layer of the ventricular zone (VZ) of the telencephalon. Pyknotic NPCs that were immunohistochemically positive for cleaved caspase-3, i.e. apoptotic NPCs, began to increase at 24 h after treatment, peaked at 48 h, and returned to the control levels at 96 h. On the other hand, the index (%) of phospho-histone H3-positive NPCs, i.e. mitotic NPCs, and that of BrdU-positive NPCs, i.e. S-phase cells, decreased in accordance with the increase in the index of apoptotic NPCs. Prior to the peak time of apoptotic NPCs, the indices of p53- and p21-positive NPCs peaked at 36 h. In addition, the expression levels of p21 and Puma (p53-target genes) mRNAs were elevated in real-time RT-PCR analysis. These findings indicated that busulfan not only induced apoptosis through the p53-mediated intrinsic pathway but also inhibited cell proliferation in NPCs, resulting in a reduction of the width of the telencephalon. On the other hand, in spite of up-regulation of p21 expression, the expression of cyclin D1, part of the cell cycle machinery of the G1/S transition, and the expression levels of Cdc20 and cyclin B1 which are involved in G2/M transition, showed no changes, giving no possible information of busulfan-induced cell cycle arrest in NPCs.