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

The effect of intestinal colonization withBifidobacterium 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 humanB. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes—lactase, alkaline phosphatase and γ-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 withB. 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 ofB. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase IV in immature gut of weaned mice than of 2-month-old ones.     

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.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation.

The postition of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and gamma-glutamyl-transferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Protective effects of nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ-free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ-free piglets. Three-week-old germ-free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole-body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non-irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush-border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM-pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.     

Activity of renal hydrolases in pre- and postnatal development of mice

The fetal and postnatal activity patterns of different hydrolytic enzymes (alkaline phosphatase, gamma-glutamyltransferase, trehalase, maltase, glucoamylase, lactase, and sucrase) have been examined in mouse renal homogenates. Alkaline phosphatase and gamma-glutamyltransferase activities presented approximately similar changes. They increased from 18 days of gestation up to 30 days after birth. These activities showed marked increases during the 3rd and 4th postnatal weeks. A similar important rise was observed for trehalase activity at the end of the suckling period. Maltase activity increased gradually after birth. Traces of lactase, sucrase, and glucoamylase activities were detected at each developmental stage.     

Vitamin A deficiency leads to severe functional disturbance of the intestinal epithelium enzymes associated with diarrhoea and increased bacterial translocation in gnotobiotic rats

A disturbance of the integrity of the intestinal epithelium with an increased risk for bacterial translocation is one of the suggested factors underlying the increased incidence of infections and septicaemia during vitamin A deficiency. In the present study the effects of vitamin A deficiency on the enzymic activity of enterocytes in response to bacterial colonization with a non-pathogenic Escherichia coli strain were studied in monocolonized and conventional Wistar rats. The monocolonized, but not the conventional, vitamin A-deficient rats had markedly reduced weight compared to their pair-fed controls and presented neurological symptoms, such as hind leg weakness, tremor and slow gait. Moreover, only in the monocolonized vitamin A-deficient rats were severe diarrhoea and bacterial translocation to extraintestinal sites-mainly kidneys-detected. Measurements of enterocyte brush-border enzyme activities revealed that lactase, sucrase, gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) were significantly reduced in the monocolonized vitamin A-deficient rats compared to the pair-fed controls, indicating a severe functional disturbance of the enterocytes. In conventional vitamin A-deficient rats only sucrase activity was markedly lower than in the respective controls. Our observation, that the deficient vitamin A status led to a strong reduction of enterocyte enzymic activities, associated with diarrhoea and increased bacterial translocation, mainly in the gnotobiotic rats, suggests that the composition of the bacterial flora, i.e. the colonization state, has a strong influence on triggering the severity of the functional disturbances of the intestinal epithelium, and adds to the clinical manifestations of vitamin A deficiency.     

The adhesive properties of a nonenteropathogenic strain of Escherichia coli in systems in vitro on isolated rat enterocytes and in vivo

The adhesive properties and colonizing capacity of E. coli strain O83, isolated from feces of healthy humans and marked according to its resistance to rifampicin and nalidixic acid, were studied. In vivo experiments on germ-free rats revealed that these bacteria were capable of colonizing intestinal mucosa; colonization increased from the small to large intestine and E. coli cells were mainly concentrated in the intestinal lumen and in mucin. In vitro studies showed that this nonenteropathogenic E. coli strain possessed pronounced adhesive properties with respect to the colonic cells of germ-free rats; these properties were considerably less pronounced with respect to the enteric cells of the small intestine. The electron microscopic study of E. coli cells revealed the presence of fimbriae and fibrillae on their surface.     

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

Cœliac 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.     

Acute dieldrin toxicity: effect on the uptake of glucose and leucine and on brush border enzymes in monkey intestine

Administration of a single oral dose of dieldrin (20 mg/kg body wt.) to rhesus monkeys considerably elevated the uptake of glucose and the activities of brush border sucrase, lactase, maltase and alkaline phosphatase in intestine compared to control animals. Leucine uptake and leucine amino peptidase activity was significantly depressed in pesticide-treated animals. Kinetic studies with brush border sucrase revealed that augmentation of enzyme activity in pesticide-fed animals was due to an increase in the disaccharidase content.     

Protection against rotavirus diarrhoea in mice by trypsin inhibitor

To investigate the role of soyabean trypsin inhibitor (TI) during rotavirus (RV) diarrhoea, changes in enzyme activities of six relevant mucosal enzymes (lactase, sucrase, maltase, trehalase, glucoamylase and alkaline phosphatase) were assayed following inoculation of suckling mice with EB rotavirus (serotype 3) along with the TI and compared with the age-matched healthy control mice. The animals were divided into three groups i.e. group 1 (controls), group 2 (RV inoculated) and group 3 (RV + TI inoculated and sacrificed under light anaesthesia on 0, 1, 3, 5, 7 and 10 day post inoculation (dpi). Then intestines were excised and divided into two parts (jejunum and ileum). They were separately homogenized in 0.9% cold normal saline and activities of mucosal enzyme were measured. Alkaline phosphatase and disaccharidases were found to be decreased significantly in RV inoculated animals in both the anatomical portions of small intestine of mice. These enzyme levels were restored with the administration of TI i.e. in group 3 and became comparable to the controls in both intestinal portions. These studies suggest that activity of intestinal enzymes which are important in digestive absorptive functions of small intestine were restored with the addition of TI whengiven to infant mice showing its protective efficacy during rotavirus infection.     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.     

Autonomous biochemical and morphological differentiation in fetal rat intestine transplanted at 17 and 20 days of gestation

The morphological and biochemical development of fetal rat intestine was examined for up to 5 weeks following transplantation to syngeneic hosts at 17 and 20 days of gestation. In transplants of both ages, normal villi bearing mature enterocytes developed. In addition, the disaccharidases lactase, maltase, and sucrase, as well as alkaline phosphatase, underwent normal patterns of development. Lactase activity, initially high, fell significantly, while maltase and sucrase activities increased significantly in the interval between 2 and 5 weeks following transplantation. During this same period, alkaline phosphatase developed the proximally located, high-activity form. The transplanted intestine also developed normal topographical distributions of enzyme activities. Measurement of corticosterone levels demonstrated that, except for a transient upsurge at the time of operation, hormone levels did not change significantly during the period of transplant maturation. These data indicate that the brush-border enzymes of the small intestine develop according to an intrinsic program which is already established as early as 17 days of gestation.     

Effects of hydrocortisone on carbohydrase concentrations, de novo synthesis and turnover patterns in immature rat intestine

Hydrocortisone administration to infant rats enhanced cellobiase and maltase activities and induced precocious expression of sucrase and trehalase activities along the length of the small intestine. These activity changes reflected proportional concentration increases in the enzymes lactase (EC 3.2.1.23), maltase/glucoamylase (EC 3.2.1.20) and sucrase-isomaltase (EC 3.2.1.48/10). Administration of an equivalent tracer dose of [3H]leucine (by body weight) to control and hydrocortisone-treated infant rats resulted in greater accumulation of label in the carbohydrase pools of the treated rats, suggesting their increased de novo synthesis. The increased concentrations of lactase and maltase/glucoamylase induced by exogenous hydrocortisone were matched by the presence of corresponding greater amounts of label in their brush border pools. Accumulation of label in each of the lactase, maltase/glucoamylase and sucrase-isomaltase pools was generally similar in the hydrocortisone-treated rats, suggesting equivalent stimulation of their synthesis as a group by the humoral agent. The turnover rates of the carbohydrases as a group were found to be similar and did not appear to differ in control and hydrocortisone-treated rats. Total protein synthesis rates were slightly greater in the intestine of the hydrocortisone-treated group of rats.     

Applicability of a model for non-pathogenic Escherichia coli for predicting the growth of pathogenic Escherichia coli

A model was developed for the temperature dependence of growth rate of a non-pathogenic Escherichia coli strain. The suitability of that model for predicting the growth rate of pathogenic E. coli strains was assessed. Growth rates of pathogenic strains were found to be adequately described by the model. Model predictions were also found to describe sufficiently well-published growth rate data for non-pathogenic E. coli on mutton carcase surfaces and E. coli O157:H7 in ground roasted beef, milk, and on cantaloupes and water melons. In addition, E. coli O157:H7 was found to grow in the region of 44–45·5 °C.     

HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets

Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.     

Protective activity ofStreptococcus faecalis against pathogenic action ofEscherichia coli 055 in gnotobiotic pigs

Peroral administration of Streptococcus faecalis suppresses the lethal effect of simultaneous or subsequent infection with pathogenic Escherichia coli O55 in gnotobiotic colostrum-free piglets. The presence of S. faecalis in the intestine and mesenteric lymph nodes in artificially diassociated colostrum-free piglets influences neither multiplication of E. coli in the intestine nor its penetration into the organism.     

Metabolic/nutrition programming of the enzyme system in the offspring small intestine

Protein deficiency in female rats diet during pregnancy and lactation resulted in changes of the intestine enzymes activity in posterity in early and late periods of ontogenesis. In the former period, deceleration of sucrase induction, acceleration of lactase suppression and maltase induction, and an earlier occurrence of the adult-type distribution of the intestine alkaline phosphatase, were found. At 2 to 4-month age a reduction of the latter enzyme activity was revealed in the doudenum, jejunum and ileum. The changes in the intestine enzymes activities led to a disorder in intermediary metabolism and to occurrence of "risk diseases".     

小鼠小肠四种消化酶活力的胎后发育模式

为明确晚成型小鼠胎后发育肠道消化酶活力的建立过程和发育模式,探讨其与适应性调节假说的关系,测定了从出生后至27日龄小鼠小肠前、中、后段的乳糖酶、蔗糖酶、麦芽糖酶和氨基肽酶的酶活力。结果发现单位组织酶活力方面,乳糖酶活力先增后降,小肠前段在9日龄而中后段在12日龄达到最高,至27日龄时仅中段有微弱的酶活力;蔗糖酶活力12日龄始出现,前段和后段自15日龄迅速升高,至18日龄达最高,但随后显著降低,而中段在15日龄后持续升高至21日龄达到最高,此后维持在较高水平;麦芽糖酶出生时已具有活力,但在15日龄前维持较低水平,此后迅速升高,前后段在18日龄,中段在21日龄达到峰值,此后下降;小肠前段的氨基肽酶活力出生后至27日龄持续下降,而后段和中段从出生到断乳前则持续升高,断乳后略有下降。除乳糖酶总酶活力先增后降,在15日龄达峰值外,其余3种酶的总酶活力均持续增加。在小肠不同位置4种酶活力的分布具有显著差异,且日龄对不同位置酶活力的影响趋势不同。总之,小鼠小肠4种消化酶的酶活力随时间的变化能够与其食物转变的消化需求相匹配,部分地支持适应性调节假说。