Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice.

One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.

Monocolonization with Bacteroides ovatus protects immunodeficient SCID mice from mortality in chronic intestinal inflammation caused by long-lasting dextran sodium sulfate treatment.

This study was aimed to evaluate the role of commensal Gram-negative bacterium Bacteroides ovatus in murine model of chronic intestinal inflammation. The attempt to induce chronic colitis was done in Bacteroides ovatus-monoassociated, germ-free and conventional mice either in immunocompetent (BALB/c) mice or in mice with severe combined immunodeficiency (SCID), using 2.5 % dextran-sodium sulfate (DSS) in drinking water (7 days DSS, 7 days water, 7 days DSS). Conventional mice developed chronic colitis. Some of germ-free BALB/c and the majority of germ-free SCID mice did not survive the long-term treatment with DSS due to massive bleeding into the intestinal lumen. However, monocolonization of germ-free mice of both strains with Bacteroides ovatus prior to long-term treatment with DSS protected mice from bleeding, development of intestinal inflammation and precocious death. We observed that though DSS-treated Bacteroides ovatus-colonized SCID mice showed minor morphological changes in colon tissue, jejunal brush-border enzyme activities such as gamma-glutamyltranspeptidase, lactase and alkaline phosphatase were significantly reduced in comparison with DSS-untreated Bacteroides ovatus-colonized mice. This modulation of the enterocyte gamma-glutamyltranspeptidase localized to the brush border membrane has been described for the first time. This enzyme is known to reflect an imbalance between pro-oxidant and anti-oxidant mechanisms, which could be involved in protective effects of colonization of germ-free mice with Bacteroides ovatus against DSS injury.

Interaction of mucosal microbiota with the innate immune system.

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m2, and the skin covers approximately 1.8 m2 surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.

Binding of the Galanthus nivalis agglutinin to thymocytes reveals alterations in surface glycosylation during T-cell development.

Surface binding of the Galanthus nivalis agglutinin (GNA) to thymocyte subsets has been studied in pigs and rodents by multicolour flow cytometry. In all the species examined, analogous staining profiles have been recorded. Counter-staining with anti-CD3epsilon, anti-CD4 and anti-CD8 monoclonal antibodies (MoAb) revealed that a significant increase of the GNA targets on the cell surface occurred during early thymocyte differentiation and reached its maximum at the level of the CD3loCD4+CD8+ small cortical thymocyte. This was followed by a decrease in the GNA binding capacity upon terminal maturation to the single positive thymocytes. PAGE analysis has revealed a dominant GNA-binding glycoprotein (molar mass approx. 90 kDa) present on thymocyte plasma membranes and absent on the surface of splenic lymphocytes, although both the whole cell lysates from both organs contained GNA ligands of the same size. Our findings are in agreement with previous data showing that immature thymocytes differ from their mature counterparts and peripheral T lymphocytes in the surface glycosylation pattern, and support the hypothesis that lectin-glycoprotein interaction plays a significant role in the cell-to-cell crosstalk in the thymic cortex.

Differential effect of Bacillus firmus on immune response and enterocyte brush-border enzyme levels in BALB/c and B10.BR mice.

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.

Bifidobacterium bifidum monoassociation of gnotobiotic mice: effect on enterocyte brush-border enzymes.

The effect of intestinal colonization with Bifidobacterium bifidum (Gram-positive anaerobic bacterium colonizing the intestine of healthy new-born mammals, exhibiting a probiotic effect, protecting the intestinal mucosa against colonization by pathogenic microflora) on enterocyte brush-border enzymes was examined in weaned 23-d- and in 2-month-old gnotobiotic inbred mice and compared with that in corresponding germ-free (GF) and conventional (CV) controls. The two groups of GF mice were associated with human B. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes--lactase, alkaline phosphatase and gamma-glutamyltranspeptidase was significantly higher in both age groups of GF mice in comparison with CV ones; on the other hand, sucrase and glucoamylase activities were higher in CV mice. Monoassociation with B. bifidum accelerates biochemical maturation of enterocytes resulting in a shift of specific activities of brush-border enzymes between the values found for GF and CV mice. This effect of B. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase i.v. in immature gut of weaned mice than of 2-month-old ones.

Differences in enterocyte brush border enzyme activities in ageing rats reared in germ-free and conventional conditions.

The aim of this study was to evaluate the levels of disaccharidase and dipeptidyl peptidase i.v. activities in rat jejunal enterocytes under the influence of long-term germ-free conditions. We found that the brush-border lactase and dipeptidyl peptidase i.v. activities were two to three times higher in 2-month-old germ-free rats in comparison with their conventional counterparts. The highest effect of germ-free condition was observed on lactase activity in 6-month-old and dipeptidyl peptidase i.v. in 2-month-old rats. No difference between germ-free and conventional rats in sucrase and glucoamylase activities was found in 2-month-old rats. The difference develops with increasing age, sucrase activity becoming significantly higher in 6- and 12-month-old rats and glucoamylase in 12-month-old germ-free rats.

Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human coeliac disease.

Coeliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+ BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.

The expression of epidermal growth factor and transforming growth factor-alpha mRNA in the small intestine of suckling rats: organ culture study.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are associated with regulation of various gastrointestinal functions. In order to better understand their role in developing small intestine EGF, TGF-alpha and EGF-R steady-state mRNA levels and transcript stability were determined. Reverse transcription (RT) competitive-polymerase chain reaction (PCR) revealed that intestinal TGF-alpha mRNA levels were 10-fold higher in comparison with EGF mRNA. The primary intestinal culture technique was used to evaluate mRNA stability. The stability of TGF-alpha mRNA was remarkably lower than the stability of EGF mRNA. High levels of TGF-alpha mRNA accompanied by high degradation rate of this mRNA suggested a rapid turnover of intestinal TGF-alpha mRNA.

Technetium-99m mercaptoacetyltriglycine clearance values in children with minimal renal disease: can a normal range be determined?

Use of technetium-99m labelled mercaptoacetyltriglycine (99mTc-MAG3) simplifies and improves the quantification of renal clearance in children by virtue of its permanent availability, good imaging properties and low radiation exposure. Due to the lack of reference values for 99mTc-MAG3 clearance in children, the Paediatric Task Group of the EANM initiated a multicentre study to evaluate 99mTc-MAG3 clearance values in children with minimal renal disease. One hundred and twenty-five children aged between 12 months and 17 years, classified as renally healthy using defined diagnostic criteria, were included in the study. 99mTc-MAG3 clearance was calculated using an algorithm on the basis of a single blood sample taken at any time between 30 and 40 min after tracer injection. In addition, the absolute 99m-Tc-MAG3 clearance values were normalized to body surface area. For further evaluation the children were classified into several groups according to age. There was a continuous increase in non-corrected 99mTc-MAG3 clearance values from the age of 1 year up to the age of 17 years (mean value <2 years: 98+/-57 ml/min; mean value >8 years: 208+/-66 ml/min). Normal clearance values for adults were achieved by the age of 8 years. Analysis of the relationship between non-corrected clearance and age yielded a correlation coefficient of r=0.7. When these absolute clearance values were normalized to body surface area, we found nearly constant clearance values for all age groups, with a mean clearance value of 315+/-114 ml/minx1.73 m2. The correlation coefficient for the relationship between normalized clearance and age was r=0.28. In conclusion, the clearance of 99mTc-MAG3 increases continuously throughout childhood into adolescence due to the maturation and growth of the kidney. After normalization of the absolute clearance to body surface area, no correlation between clearance and age could be proven.

[Immunoscintigraphy examination of bone marrow in patients with hematologic diseases]. / Imunoscintigrafické vysetrrení kostní drene u hematologických nemocných.

In 36 patients with different haematological diseases the authors examined by immunoscintigraphy the bone marrow using the 99mTc preparation Scintimun Granulozyt of Behring Co. They studied the distribution of haematopoietic tissue order to detect deviations from normal, focal defects, elongation of haematopoiesis into the periphery of long bones or extramedullary haematopoiesis. Distribution of haematopoietic tissue was evaluated visually and semiquantitatively.

The hydrocortisone-induced transcriptional down-regulation of beta-galactoside alpha2,6-sialyltransferase in the small intestine of suckling rats is suppressed by mifepristone (RU-38.486).

The progressive loss of sialic acids of the brush-border membrane glycoproteins is one of the major biochemical changes which occur in the rat small intestine during the transition from suckling to weaning, and this process is speeded up by an injection of glucocorticoids to the suckling animals. We used the rat liver beta-galactoside alpha2,6-sialyltransferase (ST6(N), EC 2.4.99.1) cDNA as a probe to examine the mRNA level of this enzyme in the small intestine of both suckling (13-day-old) and weaned (25-day-old) rats. In the ileum of suckling rats, the ST6(N) mRNA level was about four times higher than in the jejunum, whereas the membrane-bound enzyme activity was less than two times higher. In comparison with the controls, hydrocortisone treatment significantly decreased the level of this transcript and of the corresponding enzyme activity in both segments of the small intestine of suckling rats. Additionally, the antiglucocorticoid mifepristone (RU-38.486) suppressed the effect of hydrocortisone. The expression of ST6(N) mRNA in the small intestine of weaned (25-day-old) rats was several times lower than that in suckling (13-day-old) rats, and was unresponsive to hydrocortisone as well as to mifepristone. These results indicate that the glucocorticoid-induced transcriptional down-regulation of ST6(N) expression in the small intestine of suckling rats is mediated via the glucocorticoid receptor pathway, and support the notion that alterations in sialylation of brush-border membrane glycoconjugates occurring upon weaning are the result of a lower expression of ST6(N).

Coordinate expression of beta-galactoside alpha 2,6-sialyltransferase mRNA and enzyme activity in suckling rat jejunum cultured in different media: transcriptional induction by dexamethasone.

In attempting to elucidate the molecular basis of the expression of alpha 2,6-sialyltransferase (alpha 2,6-ST) in jejunal explants of 7-day-old rats during cultivation, the total jejunal RNA was analysed by hybridization using a cDNA clone encoding rat liver alpha 2,6-ST. Under cultivation in both serum-free and serum-containing media jejunal alpha 2,6-ST mRNA closely paralleled the bound (100,000 g pellet) as well as the soluble (100,000 g supernatant) alpha 2,6-ST activity, the correlation coefficients being 0.976 and 0.816, respectively. Dexamethasone (Dx) treatment enhanced alpha 2,6-ST mRNA and membrane-bound alpha 2,6-ST activity in close correlation. Jejunal alpha 2,6-ST mRNA is sensitive to actinomycin D and is lost with apparently identical kinetics in Dx-stimulated and control explants, suggesting that regulation by Dx may be exerted by altering the rate of mRNA synthesis. Dx-dependent activation resulted in elevation of the 4.3-Kb mRNA and can be inhibited by the antiglucocorticoid onapristone, demonstrating the participation of the glucocorticoid receptor pathway.

Early and delayed effects of hydrocortisone and onapristone on intestinal brush-border enzymes and their sialylation and on thymus growth in suckling rats.

The antigestagen-antiglucocorticoid onapristone (ZK 98.299) was tested on three glucocorticoid-sensitive systems after hydrocortisone (HC) administration to suckling male rats, by determining onapristone (ZK)-induced inhibition of HC-provoked (1) increase of activities of intestinal brush-border enzymes, (2) desialylation of brush-border components and (3) thymolysis. HC acetate (75 mg/kg body weight (b.w.)) was injected s.c. on postnatal days 9 and 10, and ZK (150 mg/kg b.w.) on days 9, 10 and 11. The animals were killed on day 12 for assessing the early effect, or on days 15-17 for determining the delayed effect of HC and ZK. In all three systems the glucocorticoid effects were antagonized by ZK. The most sensitive to HC were systems 1 and 3, which exhibited both the early and the delayed effects. The most sensitive to the counteraction of ZK against administered HC was system 1, where HC was antagonized in both its early and delayed effects, whereas only delayed antagonistic action against administered HC was found in system 2. ZK alone had an early inhibitory effect on the activities of several brush-border enzymes and produced an early increase in thymus weight, accompanied by an increased DNA-protein ratio. No delayed effects of ZK alone on the three systems were observed.

Molecular mimicry as a possible cause of autoimmune reactions in celiac disease? Antibodies to gliadin cross-react with epitopes on enterocytes.

Structural similarities between external antigen and self components are believed to be one of the possible causes of autoimmunity. This study describes the presence of similar structures shared by gliadin and enterocyte surface molecules recognized by antigliadin mAbs. The reactivity of mAbs to gliadin was followed by ELISA using fixed enterocytes, their brush-border membranes, or purified enterocyte antigen. The specificity of reaction was confirmed by ELISA inhibition studies and by immunohistochemical staining of rat tissue sections using biotin-avidin-peroxidase technique. Immunoprecipitation analysis of 125I-labeled intestinal epithelial cells using antigliadin mAb revealed the presence of two main cross-reactive molecules of 28 and 62 kDa. The 62-kDa and an associated 66-kDa protein were isolated by affinity chromatography. Immunoblotting analysis showed that a 28-kDa protein detected by immunoprecipitation also reacted with IgA of celiac disease patient sera.

The effect of an anti-glucocorticoid (ZK 98299) on thymus evolution and on hydrocortisone-induced thymolysis, intestinal brush-border enzymes and their desialylation in suckling rats.

1. The action in Onapristone infant male rats displays short-term and delayed effects. 2. Suppression of intestinal brush-border enzymes and increase of thymus mass were observed only immediately after 3-day treatment with Onapristone. After an additional 3 days its effect disappeared. There was no immediate or delayed effect of Onapristone on the desialylation of brush-border enzymes. 3. In the short-term and delayed effects, Onapristone suppressed the HC-provoked induction of several intestinal brush-border enzymes, especially alpha-glycosidases. In the delayed effect the drug also suppressed thymolysis induced by the exogeneously given glucocorticoid, and suppressed the HC-induced desialylation of a brush-border enzyme DP IV, which serves as a marker of the desialylation process. 4. These experiments seem to support a conclusion that the postnatal development of intestinal brush-border enzymes and the development of thymus in infant rats are controlled by endogeneously secreted glucocorticoids. 5. The control of sialylation of intestinal brush-border proteins by endogeneously secreted glucocorticoids during the postnatal development of the rat remains debatable.