Timothy P. King

Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-gamma and RelA.

The human gut microflora is important in regulating host inflammatory responses and in maintaining immune homeostasis. The cellular and molecular bases of these actions are unknown. Here we describe a unique anti-inflammatory mechanism, activated by nonpathogenic bacteria, that selectively antagonizes transcription factor NF-κB. Bacteroides thetaiotaomicron targets transcriptionally active NF-κB subunit RelA, enhancing its nuclear export through a mechanism independent of nuclear export receptor Crm-1. Peroxisome proliferator activated receptor-γ (PPAR-γ), in complex with nuclear RelA, also undergoes nucleocytoplasmic redistribution in response to B. thetaiotaomicron. A decrease in PPAR-γ abolishes both the nuclear export of RelA and the anti-inflammatory activity of B. thetaiotaomicron. This PPAR-γ-dependent anti-inflammatory mechanism defines new cellular targets for therapeutic drug design and interventions for the treatment of chronic inflammation.

The nutritional toxicity of Phaseolus vulgaris lectins.

In rats fed on beans (Phaseolus vulgaris) the poorly digestible lectins were shown to react with intestinal cells in vivo and to cause a disruption of many of the brush borders of duodenal and jejunal enterocytes. Although depressed to a certain extent, absorption still occurred, probably through the non-disrupted cells of the small intestine. In addition, abnormal absorption of potentially harmful substances, lectin-related or of bacterial origin, could also occur, possibly as a direct effect of the disruption caused by the lectins on the enterocytes. It is suggested that toxicity was the result of ensuing systemic effects, such as for example the observed high N excretion possibly through increased tissue catabolism.

Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine: 1. Light microscope studies

Abstract The inclusion in rat diets of raw kidney beans (Phaseolus vulgaris, variety “Processor”), containing high levels of lectins, resulted in severe disruption and abnormal development of microvilli in the small intestine. Similar damage occurred when rats were fed on diets containing purified lectin preparations obtained from the same beans. Few or no microvillus abnormalities were detected when the beans fed to rats were of a low-lectin variety (“Pinto III”). It is concluded that the observed depression of appetite, growth and dietary protein utilization of rats fed on diets containing high-lectin kidney beans may be at least partly due to malabsorption of nutrients. However, as many bacteria were associated with the damaged intestinal microvilli of rats which had been fed for several days on the high-lectin beans, the observed decline in the condition of the animals may also be attributed to the effects of bacterial endotoxaemia.

Immunocytochemical localization of ingested kidney bean (Phaseolus vulgaris) lectins in rat gut

SummaryThe binding of ingested kidney bean (Phaseolus vulgaris) lectins to the luminal surface of the rat gut was investigated by an indirect immunofluorescence method in which the primary antiserum was monospecific for kidney bean globulin lectins. The major lectin-positive sites were found to be the non-crypt regions of villi in the proximal region of the small intestine; exactly the same regions displayed extensive disruption of microvilli. Lectins were not localized in the brush border regions of ileal villi and no microvillus abnormalities were detected in this region of the gut. The luminal surface of the caecum was strongly lectin-positive and here also microvillus disruption was evident. Low levels of lectins were detected on the luminal surface of the colon; no microvillus abnormalities were detected in this region.

Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine: 3. Ultrastructural studies

Abstract Ultrastructural investigations revealed the extent of disruption of intestinal microvilli which occurred when rats ingested kidney bean lectins. Intracellular changes associated with the disrupted microvilli included disorganization of the terminal web, swelling of the apical cytoplasm and increase in numbers of lysosomes. It is suggested that these changes reflected disturbances in one or more membrane-associated transport processes. Microvilli were often present on the lateral and basal surfaces of enterocytes of rats fed on diets containing kidney bean lectins. Intracellular microvilli-lined cysts were also seen in some of these enterocytes.

Immunogold localization of ingested kidney bean (Phaseolus vulgaris) lectins in epithelial cells of the rat small intestine

SummaryThe interactions between dietary kidney bean (Phaseolus vulgaris) lectins and the epithelial cells of the rat small intestine were investigated by immunogold electron microscopy. The results demonstrated that the lectins bind to the glycocalyx of duodenal and jejunal microvilli and that some of these dietary constituents are endocytosed into lysosomal pathways within both absorptive and secretory gut cells. It is concluded that the lysosomal response serves to limit the absorption of nutritionally significant levels of these dietary toxins.

Pancreatic atrophy in copper-deficient rats: Histochemical and ultrastructural evidence of a selective effect on acinar cells

SummaryCopper deficiency had a differential effect between tissues in the rat pancreas. There was marked loss and atrophy of acinar cells, in which both hypertrophied and degenerating mitochondria were present. Cytochrome oxidase activity in acini was greatly depleted while monoamine oxidase activity was enhanced. Atrophy of acinar cells was accompanied by extensive degeneration of the rough endoplasmic reticulum, and by a failure of zymogen granule synthesis. These changes contrasted strongly with the appearance of non-acinar tissues, in which hypertrophy and degeneration of mitochondria were rarely observed. Islet tissue, pancreatic ducts and blood vessels showed no atrophic changes. Cytochrome oxidase activity in islet tissue, and in the epithelium of the main pancreatic ducts, appeared unaffected.

Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine: 2. Microbiological studies

Abstract The inclusion in rat diets of raw kidney beans ( Phaseolus vulgaris variety “Processor”) containing high levels of lectins resulted in a dramatic overgrowth of Escherichia coli in the small intestine. No overgrowth occurred when the beans fed to rats were of a low-lectin variety. The hypothesis is presented that kidney bean lectins may indirectly or directly enhance the virulence of coliform strains either through aggregation and elimination of competitive strains or by agglutination of certain strains of E. coli to one another and to the mucosal surfaces of the gut. Severe disruption of microvilli precedes and accompanies the proliferation of E. coli and it is possible that malabsorption provides a suitable growth substrate for intestinal bacteria, augmented by cell fragments in the form of microvilli, and possibly also by cellular exudates due to loss of polarization in membrane transport systems.

Characterization of IGF-I receptors in the porcine small intestine during postnatal development

Abstract Receptors for insulin-like growth factor I were characterized on intestinal membranes of jejunum from newborn, suckling, and weaned pigs. 125I-labelled insulin-like growth factor I (IGF-I) binding was time-dependent, saturable, linearly related to membrane protein, and reversible. Analysis of saturation curve data from membrane homogenates revealed a single class of receptors, with receptor number declining during development. In vitro autoradiography localized receptors to the small intestinal submucosa and mucosa and further immunohistochemical localization identified receptors on the apical (microvillar membrane) and basolateral surfaces of enterocytes. After affinity cross-linking of 125I-labelled IGF-I to microvillar membranes, 135 and 260 kilodalton bands were visualized on polyacrylamide gels under reducing conditions. Parallel incubations with excess unlabeled IGF-I, -II, or insulin demonstrated that these bands correspond to the α subunit and an incompletely reduced dimeric α complex of the IGF-I receptor. Receptor number on microvillar membranes increased dramatically during the suckling period and was negatively correlated with maltase and sucrase activities. The functional activity of receptors on microvillar membranes was demonstrated using an in vitro phosphorylation assay. The expression of functional IGF-I receptors during development on both apical and basolateral membranes suggests a regulatory role for this receptor and its ligands.

Effect of lactation on the decline of brush border lactase activity in neonatal pigs.

It has been shown that during the early phase of lactation porcine milk contains high concentrations of hormones and growth factors. The aim of the present investigation was to examine the hypothesis that the temporal coordination of intestinal maturation in piglets can be extrinsically regulated through changes in the composition of milk during the suckling period. Gut morphology and the ontogeny of brush border lactase activity were investigated in piglets reared on two suckling regimens designed to expose the animals to compositionally distinct milk. The first group of animals were cross-fostered onto postcolostrum sows and thereafter suckled normally for up to eight weeks. These normally suckled (N) animals consequently received both early and late lactation products. The second group of piglets were cross-fostered each week, for up to eight weeks, onto newly farrowed sows which were postcolostrum. As a result of this repeated cross-fostering (CF) these animals received only early lactation products. Animals were sacrificed at one, three, five, seven, and eight weeks postpartum. Biochemically active lactase decreased significantly (p less than 0.001) in both groups over eight weeks, but the rate of loss of activity was greater in the CF animals than in the N pigs by approximately 50% at week 3 and 25% at week 8. Quantitative histochemical analysis of lactase activity corroborated the biochemical data. At three weeks maximal enzyme activity was observed approximately 400 microns from the villus/crypt junction. Histochemically detected lactase decreased throughout the suckling period, but the intensity of reaction product was consistently weaker over the entire villus surface in the CF animals. Immunocytochemically detectable lactase was identified at the same sites as the histochemical reaction products. In addition, immunofluorescence microscopy showed the presence of histochemically undetectable enzyme on the basolateral and brush border membranes of both villus and crypt cells. Villus/crypt ratios were significantly lower (p<0.001) in the CF animals than in the N pigs between weeks 3 and 5. The results of this study suggest that lactation products can accelerate the loss of brush border lactase activity. The observed decline in biochemically and histochemically detected lactase was considered to be a consequence of reduced enterocyte lifespan, decreased synthesis of enzyme protein, or altered post-translational modification of enzyme protein, or a combination of there.