Björn Weström

A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells.

Background Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Methodology/Principal Findings The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4+CD25+ Tregs are involved in the suppressive effect induced by the lactobacilli mixture. Conclusions/Significance Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.

Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease?

BACKGROUND & AIMS Crohns disease (CD) is associated with a disturbed intestinal barrier. Permeability studies have focused on inert molecules, but little is known about transepithelial transport of macromolecules with antigenic potential in humans. The aim of this study was to quantify permeation and to characterize passage routes for macromolecules in ileal mucosa in CD. METHODS Noninflamed and inflamed ileal mucosa specimens from patients with CD (n = 12) and ileal specimens from patients with colon cancer (n = 7) were studied regarding transmucosal permeation of ovalbumin, dextran (mol wt, 40,000), and 51Cr-EDTA for 90 minutes in vitro in Ussing chambers. Transepithelial passage routes for fluorescent ovalbumin and dextran 40,000 were investigated by confocal microscopy. RESULTS Noninflamed ileum from CD patients showed increased permeation of ovalbumin compared with ileum from colon cancer patients (P < 0.05). Dextran permeation was equal in the three groups, whereas 51Cr-EDTA permeability was increased in inflamed ileum. Ovalbumin passed both transcellularly and paracellularly, but dextran followed a strictly paracellular route. Both markers were subsequently endocytosed by cells of the lamina propria. CONCLUSIONS Noninflamed ileal mucosa from patients with CD shows increased epithelial permeability to ovalbumin, probably by augmented transcytosis. This increase in antigen load to the lamina propria could be an initiating pathogenic event in CD.

Lactobacillus plantarum 299v inhibits Escherichia coli-induced intestinal permeability.

The purpose of this work was to investigate whether a probiotic bacterium, Lactobacillus plantarum 299v, could affect Escherichia coli-induced passage of mannitol across the intestinal wall. Sprague-Dawley rats were pretreated for one week by either tube feeding with L. plantarum 299v twice daily, free access to L. plantarum 299v by adding the bacterium in the drinking water, or negative control receiving regular feeding. Intestinal segments were mounted in Ussing chambers and the mucosa was exposed to control medium, E. coli, and L. plantarum 299v (alone or together). [14C]Mannitol was added as a marker of intestinal permeability and samples were taken from the serosal side. E. coli exposure induced a 53% increase in mannitol passage across the intestinal wall (P < 0.05). One week of pretreatment with L. plantarum 299v in the drinking water abolished the E. coli-induced increase in permeability. Tube feeding for one week or short-term addition of L. plantarum 299v in the Ussing chambers had no effect on the permeability provoked by E. coli challenge. Notably, L. plantarum 299v itself did not change the intestinal passage of mannitol. These data demonstrate that pretreatment with L. plantarum 299v, which is a probiotic bacterium, protects against E. coli-induced increase in intestinal permeability, and that L. plantarum 299v alone has no influence on the intestinal permeability. Thus, this study supports the concept that probiotics may exert beneficial effects in the gastrointestinal tract.

Mucosal in vitro permeability in the intestinal tract of the pig, the rat, and man: species- and region-related differences.

Background: The barrier properties of the gastrointestinal mucosa may be studied by measuring its permeability to different-sized marker molecules. Owing to difficulties in obtaining human tissue it is, however, often necessary to extrapolate findings from experimental animals to man. The aim of the present study was to compare regional intestinal mucosal permeability in man, the rat, and the pig, using the same marker molecules and in vitro technique. Methods: Segments from jejunum, ileum, colon, and rectum were mounted in Ussing diffusion chambers, and the mucosa-to-serosa passage of C-mannitol, fluorescein isothiocyanate (FITC)–dextran 4,400, a-lactalbumin, ovalbumin, and FITC–dextran 70,000 was studied.Results: Irrespective of species or intestinal region an inverse relationship between the molecular weight of the markers and the permeability was seen. The mannitol permeability was higher in the small intestine than in the colon in man, whereas the rat showed a higher permeability in the ileum than in the jejunum and colon. The FITC–dextran 4,400 permeability was higher in all intestinal regions in the rat than in man and the pig. The macromolecules showed low permeability with no regional differences. Conclusions: The results showed differences between intestinal regions and between species. Permeability data from the pig correlated fairly well with those of man, whereas the rat differed, making it difficult to extrapolate from the rat to man.BACKGROUND The barrier properties of the gastrointestinal mucosa may be studied by measuring its permeability to different-sized marker molecules. Owing to difficulties in obtaining human tissue it is, however, often necessary to extrapolate findings from experimental animals to man. The aim of the present study was to compare regional intestinal mucosal permeability in man, the rat, and the pig, using the same marker molecules and in vitro technique. METHODS Segments from jejunum, ileum, colon, and rectum were mounted in Ussing diffusion chambers, and the mucosa-to-serosa passage of 14C-mannitol, fluorescein isothiocyanate (FITC)-dextran 4,400, alpha-lactalbumin, ovalbumin, and FITC-dextran 70,000 was studied. RESULTS Irrespective of species or intestinal region an inverse relationship between the molecular weight of the markers and the permeability was seen. The mannitol permeability was higher in the small intestine than in the colon in man, whereas the rat showed a higher permeability in the ileum than in the jejunum and colon. The FITC-dextran 4,400 permeability was higher in all intestinal regions in the rat than in man and the pig. The macromolecules showed low permeability with no regional differences. CONCLUSIONS The results showed differences between intestinal regions and between species. Permeability data from the pig correlated fairly well with those of man, whereas the rat differed, making it difficult to extrapolate from the rat to man.

Protease Inhibitors and their Relation to Protease Activity in Human Milk

Summary: Protease inhibitors and protease (caseinolytic, elastinolytic and esterolytic) activity were analysed in 190 milk samples from 94 mothers from day 1 to day 160 after delivery The main protease inhibitors in human milk are α1-antichymotrypsin and α1-antitrypsin. As measured by electroimmunoassay, the level of α1-antichymotrypsin in day 1 colostrum was higher than that in normal serum. Trace amounts of inter-α-trypsin inhibitor, α2-antiplasmin, α2-macroglobulin, antithrombin III, or antileukoprotease could be demonstrated. According to their protease inhibiting activity, the 53 milk samples from day 1–3 could be divided into two groups. (1) Presence of protease inhibiting activity (n = 35). Both α1-antitrypsin and α1-antichymotrypsin appeared intact and were able to form complexes with added trypsin or chymotrypsin although the major part of α1-antichymotrypsin showed a retarded electrophoretic mobility. The proteolytic activity was undetectable or low in these samples. (2) No protease inhibiting activity, in spite of the presence of immunoreactive inhibitors (n = 18). α1-Antichymotrypsin had a precipitate pattern similar to group 1, whereas α1-antitrypsin had a major fraction with slightly retarded mobility and two minor peaks in the α-1and β-regions. These precipitate patterns were unchanged on addition of human trypsin or chymotrypsin compatible with the presence of nonreactive inhibitor only. These samples had a caseinolytic and esterolytic activity with an electrophoretic mobility in the β-region.All samples from day 4 and later had a demonstrable protease inhibiting activity.Speculation: About two-thirds of colostral milk samples have a protease inhibiting activity, i.e., can inhibit proteolytic enzymes present in or added to human colostrum. One-third has no protease inhibiting activity, and possess free protease activtiy. These different properties of human colostrum may cause differences in the absorption of proteins in newborn infants.

Intestinal Transmission of Macromolecules (BSA and FITC-Dextran) in the Neonatal Pig: Enhancing Effect of Colostrum, Proteins and Proteinase Inhibitors

The effects of colostrum and constituents/factors in colostrum which may influence intestinal macromolecular transmission in the newborn preclosure pig were investigated. Unsuckled piglets were given, by use of a stomach tube, bovine serum albumin (BSA) and fluorescein-isothiocyanate (FITC)-labelled dextran 70,000 (FITC-D) as markers together with colostrum or the factors under study. The serum levels of BSA and FITC-D 4 h after feeding were then determined as a measure of the transfer. It was found that the two colostrums tested, bovine and especially porcine, markedly enhanced the transmission of both BSA and FITC-D. Furthermore, increasing amounts of the model proteins, BSA and bovine IgG (50-200 mg/ml), significantly increased the transfer of FITC-D, whereas unlabelled dextran 70,000 given in similar amounts did not. Proteinase inhibitors obtained from sow colostrum or soy bean also enhanced the transmission of both BSA and FITC-D while the inactive inhibitors, given as trypsin-inhibitor complexes, had no effect. On the other hand, addition of a proteinase, porcine trypsin, significantly decreased the transmission of FITC-D. These findings indicate that the intestinal transmission of macromolecules in the preclosure piglet is governed by the amount of protein available in the intestine. Therefore, feeding colostrum with a high protein content and proteinase inhibitors is likely to favour efficient intestinal transmission, although other colostrum factors may also be of importance.

Age, sex, and weight at weaning influence organ weight and gastrointestinal development of weanling pigs

The present study was designed to determine the interrelationships between sex, weaning age, and weaning weight on aspects of physiological and gastrointestinal development in pigs. Forty-eight Large White x Landrace pigs were used in a factorial arrangement with the respective factors being: age at weaning ( 14 or 28 days), weight at weaning ( heavy or light), sex ( boar or gilt), and time after weaning ( 1, 7, and 14 days). At weaning, 48 pigs were removed from the sow; 16 pigs were then fasted for 24 h before euthanasia for determination of organ weights, gut histology, and enzymology, and 32 pigs were offered a high quality pelleted weaner diet ad libitum for subsequent assessment of organ weights, histology, and enzymology at 7 and 14 days after weaning. On Day 6 and 13 after weaning, 2 pigs from each group had their feed removed, and 24 h later were euthanased and similar measurements were taken. In general, the data highlighted the overall gastrointestinal underdevelopment of pigs weaned at 2 weeks of age and of pigs weaned light-for-age at either 2 or 4 weeks. Heavier body organs, gastrointestinal organs, and accessory digestive organs observed after weaning, except for the spleen, presumably reflected the increase in substrates available for cellular growth as feed intake increased after weaning, and the development of organs required to process this feed. Interestingly, the relative weights (% of liveweight) of the stomach and small intestine and, to a lesser extent, the caecum and colon, were greater in the light, 14-day-old weaned pigs, but these differences diminished with increasing time after weaning. Consistent effects due to age, weight, and sex were not observed for villous height and crypt depth, or for the specific activities of the brush-border and pancreatic enzymes measured. However, increases (P < 0.001) in the activities of maltase (P < 0.001), glucoamylase ( P < 0.001), and sucrase (P = 0.020) ( all expressed per gram of mucosa), and that of trypsin ( per gram of pancreas), occurred by 14 days after weaning. This most likely reflected the inducible nature of these enzymes in response to the increasing intake of substrates provided in the diet. In contrast, the specific activity of lactase declined (P = 0.012) in the first 14 days after weaning. These data suggest that pigs weaned at 2 weeks of age and pigs weaned light-for-age at either 2 or 4 weeks have a less developed gastrointestinal tract, and that its development after weaning might proceed differently to that of pigs weaned older and heavier. (Less)

Development of exocrine pancreas function in chronically cannulated pigs during 1-13 weeks of postnatal life

The development of exocrine pancreas function was studied in Swedish Landrace pigs surgically fitted with a chronic pancreatic duct catheter and a duodenal re-entrant cannula. The juice secretion and output of total protein and trypsin activity were followed before (basal secretion) and after feeding (postprandial secretion) during the first 1–13 weeks of life. The results showed that throughout the suckling period, up to 4–5 weeks of age, the basal pancreas function remained low and the secretory response to feeding, i.e., nursing sow milk, was also low. After weaning, the pancreatic juice secretion as well as the output of protein and trypsin activity markedly increased with respect to both basal and postprandial levels. Furthermore, the enzyme composition of the pancreatic juice changed qualitatively during this period. During the first 2 weeks of life, the intravenous administration of cholecystokinin (CCK) and secretin did not stimulate exocrine function, but a significant effect was achieved from 3–4 weeks of age. These results showed that there was both an increase in exocrine pancreas function and a qualitative change in the hydrolytic enzyme pattern during porcine postnatal ontogeny, apparently correlated with the changes in diet around weaning. An increase in the response of the pancreas to hormonal stimulation was also observed during the suckling period.

Intestinal permeability in humans is increased after radiation therapy

PURPOSE: Irradiation inflicts acute injuries to the intestinal mucosa with rapid apoptosis induction and subsequent reduction in epithelial surface area. It may therefore be assumed that the intestinal barrier function is affected. The aim of this study was to compare the mucosal permeability in irradiated rectum and nonirradiated sigmoid colon from patients subjected to radiation therapy before surgical treatment for rectal cancer. METHODS: Segments from sigmoid colon and rectum obtained from irradiated and nonirradiated patients were stripped from the serosa-muscle layer and mounted in Ussing diffusion chambers. The mucosa-to-serosa passage of the marker molecules14C-mannitol, fluorescein isothiocyanate-dextran 4,400, and ovalbumin was followed for 120 minutes. RESULTS: The permeability to the markers was size-dependent and increased linearly across time in all specimens. The passage of all markers was increased in irradiated rectum compared with nonirradiated sigmoid colon, whereas in specimens from nonirradiated patients there were no differences between rectum and sigmoid colon. Histologic signs of crypt and mucosal atrophy were found in the irradiated rectal specimens. CONCLUSIONS: Early gastrointestinal complications after radiation therapy may be the result of mucosal atrophy in addition to mucosal damage, with a loss of barrier integrity.

Levels of immunoreactive insulin, neurotensin, and bombesin in porcine colostrum and milk.

High concentrations of insulin (411 ± 214 μU/ml), neurotensin-like (265 ± 72 pg/ml), and bombesin-like immunoreactivities (1995 ± 288 pg/ml) were detected in porcine colostrum using radioimmunoassay, as compared to the levels found in sow blood serum at farrowing (5 μU/ml, <12 pg/ml, and 17 pg/ml, respectively). After 72 h of lactation, the levels of insulin and neurotensin-like immunoreactivities had decreased to 28 ± 17 μU/ml and 89 ± 23 pg/ml, respectively, while the bombesin-like activity remained constant. Characterization with reversed-phase high performance liquid chromatography showed that the insulin immunoreactivity eluted at the same position as the insulin standard, while the elution patterns of the neurotensin-like and bombesin-like immunoreactivities (eluted in three separate peaks) did not correspond to that of their respective standards. The biological function of the peptide hormones in colostrum/milk may be as triggers of the developmental changes taking place in the nursing neonate, especially in the gastrointestinal tract.