Hirokazu Tashiro

Morphological alteration of gut-associated lymphoid tissue after long-term total parenteral nutrition in rats

SummaryThe morphological alteration of gut-associated lymphoid tissue (GALT) induced by long-term absence of dietary stimulation was investigated. Male Wistar rats weighing ∼230 g were maintained with total parenteral nutrition (TPN). Control rats were allowed to have the same amount of the solution orally. After two weeks, the morphological alteration of GALT was examined. Although no significant difference in weight gain was noted between the two groups, the area comprised by Peyers patches was decreased in TPN rats. The number of transported lymphocytes and the ratio of helper T (Th) cells to suppressor/cytotoxic T (Ts/c) cells in intestinal lymph were lowered after TPN treatment. In an immunohistochemical study of the rat ileum, the number of T cells and the Th/Ts/c ratio were decreased both in the intraepithelial spaces and in the lamina propria of TPN rats. The percentage of interleukin-2 receptor-positive cells and the number of IgA-containing cells in the lamina propria were significantly reduced in TPN rats. These results suggest that dietary stimulation might play a role in the maintenance of GALT function and morphology.

Ischemic bowel necrosis induced by endothelin-1: an experimental model in rats.

Local intra-arterial administration of endothelin-1 induced small intestinal mucosal damage in rats in a dose-dependent manner. A remarkable decrease in mucosal blood flow (15% of control values) was observed by a laser Doppler flowmetry 10 min after injection of endothelin-1 (1 nmol/kg). Endothelin-1 at this dose induced significant hemorrhagic and necrotic lesions in the small intestinal mucosa 30 min after the injection. Decreased mucosal blood flow was attenuated to some extent by the pretreatment with platelet-activating factor (PAF) inhibitor, CV-6209, superoxide dismutase plus catalase or the calcium antagonist, nicorandil. All these inhibitors significantly prevented the endothelin-1-induced ischemic necrotic damage in the small intestine. These results suggest a potential role of endothelin-1 in the pathogenesis of ischemic bowel diseases in clinical situations and also the possibility that PAF and oxygen-derived free radicals may be involved as secondary mediators in endothelin-induced intestinal tissue damage.

Changes in intestinal absorption of nutrients and brush border glycoproteins after total parenteral nutrition in rats.

The effect of total parenteral nutrition on nutrients absorption and glycoprotein changes of brush border membrane was examined in rat small intestine. In total parenteral nutrition rats, a marked decrease in activity of brush border enzymes was observed mainly in the proximal and middle segments of the intestine. Galactose perfusion of jejunal segment showed that hexose absorption was significantly inhibited, while intestinal absorption of glycine or dipeptide, glycylglycine was not significantly affected by total parenteral nutrition treatment. When brush border membrane glycoprotein profile was examined by [3H]-glucosamine or [3H]-fucose incorporation into jejunal loops, significant changes were observed in the glycoprotein pattern of brush border membrane especially in the high molecular weight range over 120 kDa after total parenteral nutrition treatment, suggesting strong dependency of glycoprotein synthesis on luminal substances. Molecular weight of sucrase isomaltase in brush border membrane detected by specific antibody showed no significant difference, however, in total parenteral nutrition and control rats. Also, molecular weight of specific sodium glucose cotransporter of intestinal brush border membrane detected by selective photoaffinity labelling was not altered in total parenteral nutrition rats. It may be that prolonged absence of oral food intake may produce significant biochemical changes in brush border membrane glycoprotein and absorptive capacity of small intestine, but these changes were not observed in all brush border membrane glycoproteins.

Role of platelet activating factor on the fibrinolytic activation in the pathogenesis of gastric mucosal damage induced by endothelin-1.

We have examined the hypothesis that the release of tissue type plasminogen activator may play a prominent role in endothelin induced gastric mucosal injury. We determined tissue type plasminogen activator activity in the regional blood sample and the concentration of platelet activating factor in the gastric mucosa after the administration of endothelin-1 in a range of 50-500 pmol/kg into the left gastric artery of male Wistar rats. Endothelin-1 increased the tissue type plasminogen activator release and platelet activating factor formation, and induced subsequent gastric mucosal haemorrhagic change in a dose dependent manner. In addition CV-6209, a selective platelet activating factor blocker, attenuated the activation of regional tissue type plasminogen activator and the development of mucosal damage induced by endothelin-1. The results of this study showed that tissue type plasminogen activator activation may play an important role in the pathogenesis of endothelin induced mucosal injury of rat stomach, and suggest that the platelet activating factor may be involved in the process of regional fibrinolytic activation induced by endothelin-1.

Faecal clearance of α1-antitrypsin reflects disease activity and correlates with rapid turnover proteins in chronic inflammatory bowel disease

Abstract Faecal clearance of α1‐antitrypsin was measured in patients with ulcerative colitis and Crohns disease and compared with disease activity and markers of protein‐calorie malnutrition. Patients with active ulcerative colitis and Crohns disease showed elevated clearance of α1‐antitrypsin and clearance declined in most patients with induction of remission. However, even with inactive disease, elevated protein loss persisted in some patients, presumably reflecting residual inflammation in the intestinal mucosa. There was a significant correlation between clearance of α1‐antitrypsin and serum levels of retinol‐binding protein and transferrin in patients with ulcerative colitis and with retinol‐binding protein in patients with Crohns disease. Clearance of α1‐antitrypsin reflects disease activity in inflammatory bowel disease and correlates with serum levels of rapid‐turnover proteins such as retinol‐binding protein and transferrin, which are markers for the presence of protein‐calorie malnutrition.

Active oxygen species in formation of acute gastric mucosal lesions induced by thermal injury in rats

Active oxygen species generated by circulating leukocytes and released from the gastric mucosa were measured in the process of acute gastric mucosal lesion formation after thermal injury in rats. Alterations of luminol-dependent chemiluminescence activities generated by leukocytes obtained from the gastric vein and the inferior vena cava were approximately same. A decrease in chemiluminescence activity 15 min after thermal injury and a significant increase in chemiluminescence activity 5 hr after thermal injury were observed in leukocytes from both veins. From 15 min to 12 hr after thermal injury, luciferin-dependent chemiluminescence activities were significantly higher than that of the control group. Oral administration of rebamipide resulted in decreased mucosal lesion formation. Rebamipide, an antiulcer agent that protects the mucosa from damage in various animal models decreased chemiluminescence activities only released from the gastric mucosa but not from circulating leukocytes. These results suggest that two different pathways of active oxygen species formation may exist in the pathogenesis of acute gastric mucosal lesions after thermal injury.

In vivo effect of chronic administration of vasoactive intestinal peptide on gut-associated lymphoid tissues in rats

The in vivo effects of chronic administration of vasoactive intestinal peptides (VIP) on the lymphoid cell traffic and the population and function of cells in intestinal lymph and gut-associated lymphoid tissues were examined in rats. VIP was continuously infused from the superior mesenteric artery in rats at a dose of 10 ng/min/kg body weight for 96 h. Lymphocyte transport through intestinal lymph was significantly reduced by VIP without any changes in lymph flow. When lymphocyte subpopulation was examined in intestinal lymph, T cell subsets were decreased with a dominant reduction in the population of helper T cells. T cell subsets were also decreased in mesenteric lymph nodes, but in this case suppressor/cytotoxic T cell subsets were mainly reduced. Despite of the decrease in lymphocyte transport through intestinal lymph and changes of lymphocyte subpopulation, proliferative response of lymphocytes from intestinal lymph and mesenteric lymph nodes to phytohemagglutinin did not show any significant alteration after administration of VIP. By histochemical study on the lamina propria of the small intestine, the population of pan T cells, especially helper T cells, was demonstrated to be significantly decreased after VIP treatment. There was also a marked decrease in the number of immunoglobulin (Ig) A-containing cells in the lamina propria of the small intestine in VIP-treated rats, while no significant changes were seen in the number of IgG and IgM-containing cells. Our present results showed the possibility that a long-term alteration of serum VIP levels could affect the dynamics of immune effector cells and IgA production in gut-associated lymphoid tissues.

Alteration of Mucosal Immunity After Long-term Ingestion of an Elemental Diet in Rats

The effects of an elemental diet on lymphocyte transport in intestinal lymph and immune responses of gut-associated lymphoid tissue were investigated in rats. The control animals were fed a conventional diet. After 4 week of feeding, the total calorie intake and body weight gain showed no differences between the two groups. The number and total area of Peyers patches and the ratio of height of villi to height of crypt showed no significant differences between the two groups. The rate of lymph flow in intestinal lymphatics showed no significant change in treated animals compared with the control rats. However, an elemental diet induced a significant decrease in lymphocyte flux in intestinal lymphatics compared with that in control rats. Lymphocyte subsets in intestinal lymph revealed a significant decrease in CD3-positive cells, especially CD4-positive cells in the elemental diet-treated group. A significant decrease in the number of immunoglobulin A-containing cells and a decreased CD4/CD8 ratio in T-cell subsets were observed in the lamina propria of ileal mucosa in the elemental diet-treated group by morphometric analysis in the immunohistochemical study. Specific antibody-secreting cells in intestinal lymph were also investigated after rats were intraduodenally primed with cholera toxin and challenged with the same toxin after an interval of 2 weeks. No significant difference was seen between the two groups in any of the numbers of anti-cholera toxin immunoglobulin-secreting cells in any immunoglobulin A, immunoglobulin G, or immunoglobulin M class as determined by the enzyme-linked immunospot assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Verotoxin induces hemorrhagic lesions in rat small intestine - Temporal alteration of vasoactive substances

E. coli O157:H7 produces a cytotoxin active against Vero cells that has been termed verotoxin. In this study, we demonstrated that local intraarterial injection of verotoxin induced a decrease in blood flow and an increase in hemorrhagic lesions in rat small intestine. Significant increases in the area of hemorrhagic lesions were observed at 120 min after verotoxin injection. These lesions were produced by either verotoxin 1 or 2, but verotoxin 2 produced more extensive lesions. The temporal alteration of vasoactive substances in microcirculatory beds was determined after the administration of culture filtrate ofE. coli O157:H7. Tissue-type plasminogen activator activity in regional plasma was significantly elevated as early as 30 min, suggesting that local fibrinolytic activation mediated by microvascular endothelium occurred. There was also early elevation of platelet-activating factor content in the ileal mucosa and its level remained significantly elevated thereafter. Intestinal blood flow, as determined by a laser Doppler flowmeter, started to decrease at about 45 min. The platelet-activating factor antagonist CV6209 was shown to attenuate the decrease in blood flow as well as the development of hemorrhagic lesions, demonstrating that platelet-activating factor is an important mediator for the microcirculatory damage. Accumulation of neutrophils demonstrated by myeloperoxidase activity in the intestinal mucosa and overproduction of oxygen-radicals from neutrophils of the mesenteric veins determined by the luminol-dependent chemiluminescence assay were observed at 60 min, corresponding with the decreased blood flow. Platelet-activating factor may be closely involved in the process of leukocyte accumulation and increased oxygen radical generation, because CV6209 also significantly attenuated these changes. Verotoxin is considered to induce vascular endothelial cell damage and elicit the accumulation of neutrophils in intestinal microvascular beds, leading to hemorrhagic lesions via mediators including tissue-type plasminogen activator, platelet-activating factor, and oxygen-derived free radicals.

Significant changes in intestinal lymphatic system and immune response elicited by Peyer's patch excision in adult rats

Abstract The effect of deprivation of Peyers patches (PP) on transport of lymphocytes through intestinal lymph and intestinal mucosal immune responses was investigated in rats. All visible PP in the rat small intestine were excised in order to examine the roles of PP in the intestinal lymphatic system and mucosal immune responses of the intestine. Two weeks after the experimental excision of PP, lymphocyte transport in intestinal lymph was significantly decreased in PP‐excised rats without significant changes in lymphocyte subsets as compared with sham operated control rats. Lymphocyte subsets as determined morphometrically in the intestinal mucosa showed no significant alteration in PP‐excised rats. There was a significant decrease in the number of immunoglobulin A (IgA) containing cells in the intestinal mucosa of PP‐excised rats, while IgM and IgG containing cells showed no statistically significant changes in number. Conversely, the macrophages in the intestinal mucosa increased in number, suggesting the enhanced accessory functions of these macrophages. Antigen‐specific immune response was further studied in PP‐excised rats using intraduodenal priming and challenge with cholera toxin (CT). Both the determinations of cells producing antigen‐specific antibody in the intestinal mucosa using anti‐CT antibody and those of cells secreting anti‐CT Ig in the intestinal lymph by enzyme‐linked immunospot (ELISPOT) assay showed a significant reduction of CT‐specific antibody production in PP‐excised rats compared with controls. Peyers patches appear to have an important role in lymphocyte transportation through intestinal lymph and also in mucosal immune responses.