Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum.

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.

Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends.

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach.

Oligofructose contributes to the protective role of bifidobacteria in experimental necrotising enterocolitis in quails.

Bifidobacteria are dominant in the gut of full-term infants, although colonisation by them is often delayed in preterm neonates. Bifidobacteria are recognised to have beneficial effects on digestive disorders and they might prevent neonatal necrotising enterocolitis (NEC), a gastrointestinal disease that predominantly affects premature infants. They have been shown to protect gnotobiotic quails against NEC-like lesions when the birds were inoculated with faecal flora from preterm infants, decreasing the clostridial population. The present study was designed to investigate whether oligofructose, which stimulates the activity of bifidobacteria, may enhance their protective role. Experiments were done in eight groups of germ-free quails for 28 days. The groups differed as to their bacterial status, diet and environment. Quails were inoculated with one of two flora from premature twins. The first flora included Bifidobacterium pseudo-catenulatum, Escherichia coli and no clostridia. The second flora included clostridial species and was associated with B. infantis-longum. Caecal bacterial population and metabolism changes were investigated with a lactose (6%) diet versus a lactose-oligofructose (3%-3%) diet, either in a gnotobiotic environment or in an ordinary environment permitting post-colonisation by exogenous bacteria. In both environments and with both flora, oligofructose significantly increased the level of bifidobacteria and this was associated with a decrease of E. coli or C. perfringens and C. ramosum. The bacterial changes in the ordinary environment depended on the initial composition of the microflora and the colonisation resistance against exogenous bacteria was more efficient with the flora that included B. pseudo-catenulatum. The changes in caecal pH and short-chain fatty acids were minimal. It was demonstrated that, irrespective of the environmental conditions, the use of oligofructose helped to prevent the overgrowth of bacteria implicated in necrotising enterocolitis in preterm neonates.

Altered patterns of DNA methylation on chromosomes from leukemia cell lines: identification of 5-methylcytosines by indirect immunodetection.

An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.

Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria.

The pathogenesis of neonatal necrotising enterocolitis (NEC) remains unclear. Gnotobiotic quails fed a lactose diet have been used to investigate the role of clostridial strains originating from faecal specimens of neonates through the intestinal lesions, the changes in microflora balance and the production of bacterial metabolites, i.e., short-chain fatty acids and hydrogen. Bifidobacteria are thought to exert various beneficial effects on host health, including interaction with the colonic microflora. Therefore, it was hypothesised that a protective role could be exercised through bifidobacterial colonisation. A Clostridium butyricum strain (CB 155-3) and a whole faecal flora including three clostridial species (C. butyricum, C. perfringens, C. difficile), each from premature infants suffering from NEC, caused caecal lesions in quails similar to those observed in man, i.e., thickening of the caecal wall with gas cysts, haemorrhagic ulceration and necrotic areas. Conversely, a whole faecal flora including bifidobacteria (identified as Bifidobacterium pseudo-catenulatum) and no clostridia, isolated from a healthy premature infant, was unable to produce NEC-like lesions. When the two clostridial groups were associated with a Bifidobacterium strain (B. infantis-longum, CUETM 89-215, isolated from a healthy infant), bifidobacterial colonisation suppressed all pathological lesions. This study is the first demonstration of a protective role for bifidobacteria against NEC via the inhibition of growth of C. butyricum or the disappearance of C. perfringens. C. difficile was not found to be responsible for the aetiology of the caecal lesions in quails. The main effect of bifidobacteria on lactose fermentation was either a dramatic decrease or a disappearance of butyric acid. The protective role was not associated with changes in H2 production. Therefore, a new step between colonic colonisation and its relevance to NEC is thought to involve the fermentation of unabsorbed lactose into butyric acid at the onset of the disease.

Effects of chair-restraint on gastrointestinal transit time and colonic fermentation in male rhesus monkey (Macaca mulatta).

The incidence of an 18 day chair-restraint on the digestive physiology of male rhesus monkey was investigated for space research purposes, comparing four trained restraint subjects with two vivarium controls. Chair-restraint induced a 2.5-fold acceleration of the gastrointestinal transit time, which persisted throughout the 7 day postrestraint period, and an increase of the fecal dry matter content, which mean value rose from 40.7% to 69.6%. Fecal pH remained unaltered throughout the experiment. Modifications of fermentative metabolites produced by the colonic microflora and excreted through the breath (hydrogen and methane) or in the feces (short chain fatty acids and ammonia) could not be reliably related to chair-restraint and probably involved side-stress factors. On the whole, alterations due to chair-restraint are shown to be different from those reported in the literature, following a modification of the dietary composition. These data may help to predict the alterations of digestive physiology likely to occur in immobilized human patients.

Effect of two levels of transgalactosylated oligosaccharide intake in rats associated with human faecal microflora on bacterial glycolytic activity, end-products of fermentation and bacterial steroid transformation.

The effects of two levels of transgalactosylated oligosaccharide (TOS) intake on bacterial glycolytic activity, end products of fermentation and bacterial steroid transformation were studied in rats associated with a human faecal flora. Rats were fed a human-type diet containing 0, 5 or 10% TOS. Caecal pH decrease correlated with the amount of TOS in the diet. Intake of the TOS diet induced a decrease in blood cholesterol and a strong increase in beta-galactosidase activity in the hindgut. TOS fermentation led to production of hydrogen and short chain fatty acids, whereas ammonia and branched-chain fatty acids were decreased. A diet containing 10% TOS increased caecal lactic acid concentrations and reduced beta-glucuronidase activities and steroid transformation.

Effects of galacto-oligosaccharide and bacterial status on mucin distribution in mucosa and on large intestine fermentation in rats.

The purpose of the present paper was to study the effects of a dietary undigestible carbohydrate and intestinal microflora on mucin distribution (neutral, acid, sulphonated), glycolytic activities: beta-D-galactosidase (EC 3.2.1.23), N-acetyl-beta-D-galactosaminidase (EC 3.2.1.43), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alpha-L-fucosidase (EC 3.2.1.51) and bacterial metabolism (gas production, short-chain fatty acids (SCFA) and lactic acid caecal concentration) in germ-free (GF), conventional (CV) and heteroxenic (HE) rats (GF rats associated with a human flora). Rats were fed on either a control diet or a diet containing 40 g trans-galactosylated oligosaccharide (TOS)/kg. In GF rats fed on the control diet caecal pH was almost neutral and glycolytic activities negligible. The number of mucus-containing cells increased from the caecum to the colon for the three types of mucin. TOS had no effect in the caecum but it modified mucin cell repartition in the colon. In CV and HE rats fed on the control diet caecal pH was similar (6.8), but caecal SCFA and lactic acid concentrations (mumol/g) and gas production (ml/24 h) were higher in CV (70, 5.9 and 2.3 respectively) than in HE rats (32, 4.6 and 0.4 respectively). In CV, as in HE rats, acid-mucin-containing cells increased from the caecum to the colon and glycolytic activities were similar. TOS reduced acid-mucin-containing cells in the caecum of CV rats by twofold but had no effect in either the caecum or the colon of HE rats. TOS strongly increased beta-galactosidase activity and slightly modified the other glycolytic activities. Its effect on bacterial metabolites depended on bacterial status. However, comparison between CV and HE rats showed no evident relationship between the number of mucus-containing cells and measured bacterial metabolites. Differences between CV and HE rats might be due to bacterial microflora specificity. TOS had an intrinsic effect on mucus cell distribution in the colon of GF rats. In CV and HE rats the presence of the flora abolished this effect.

Production of volatile fatty acids as a result of bacterial interactions in the cecum of gnotobiotic rats and chickens fed a lactose-containing diet.

Volatile fatty acid (VFA) productions from lactose and lactate by a Clostridium butyricum and a Veillonella alcalescens strain, alone or in combination with a Lactobacillus acidophilus strain, were determined both in vitro in culture media and in vivo in the ceca of gnotobiotic animals. Gnotobiotic rats, which possess intestinal lactase, and chickens, which are devoid of it, were used. Both animal species were fed a diet containing 4% lactose. The in vitro results showed that the C. butyricum strain fermented lactose and D-lactic acid to butyric and acetic acids, whereas L-lactic acid was not fermented. The V. alcalescens strain did not ferment lactose and fermented L better than D-lactic acid. The in vivo results showed that high VFA concentrations were obtained in the ceca of chickens either disassociated with V. alcalescens or C. butyricum and Lactobacillus strains or monoassociated with C. butyricum. VFA concentrations in the ceca of rats were low, whatever strain the rats harbored. In addition, an antagonistic effect of the C. butyricum strain against the Lactobacillus strain was evidenced both in rats and chickens. It is suggested that the absence of a host lactase makes the chick a good model for lactose intolerance studies in human newborns.

[Comparative effect of a single or continuous administration of "Saccharomyces boulardii" on the establishment of various strains of "candida" in the digestive tract of gnotobiotic mice]. / Effet comparé de l'administration unique ou en continu de Saccharomyces boulardii sur l'établissement de diverses souches de Candida dans le tractus digestif de souris gnotoxéniques.

Saccharomyces boulardii became established in the digestive tract of monoxenic mice; the number of viable cells ranged around 10(7.5) per gram faeces. This yeast was drastically eliminated from the digestive tract of gnotoxenic mice harbouring a complex flora of human origin. In monoxenic mice harbouring S. boulardii, Candida albicans became established at a level equivalent to that observed in monoxenic mice harbouring C. albicans alone. If gnotoxenic mice received a concentrated suspension of viable S. boulardii cells so as to steadily maintain a population level close to 10(9) viable cells, C. albicans then became established at a level 10 to 50 times lower than that reached by the yeast strain alone. The antagonistic effect exerted in vivo by S. boulardii was preventive and curative. It was active against C. albicans, C. krusei and C. pseudotropicalis strains, but ineffective against C. tropicalis. This antagonistic effect disappeared when S. boulardii cells were killed by heating.