Structural robustness of the gut mucosal microbiota is associated with Crohn's disease remission after surgery.

OBJECTIVES: Preventing postoperative recurrence after ileocolonic resection (ICR) for Crohn's disease (CD) is challenging. Defining the disturbances of the microbial composition and community structure after ICR and their link with early disease recurrence is crucial. DESIGN: Microbiota composition (fingerprinting and 16S rDNA sequencing) and community structure (correlation networks of bacterial species) were assessed from ileal mucosa sampled in 20 patients undergoing ICR and 6 months later during endoscopy from above (neoterminal ileum) and below (subanastomotic colon) the surgical anastomosis. RESULTS: ICR had a dramatic effect on gut microbial ecosystem. At surgery, CD mucosa harboured a dysbiotic microbiota with high proportions of α/ß Proteobacteria and Bacilli. Six months later, half of the patients had recurrent lesions at ileocolonoscopy and presented higher numbers of Lachnospiraceae. Recurrence of endoscopic lesions was associated with enrichment in Enterococcus durans while patients in remission had increased proportions of Dorea longicatena and Bacteroides plebeius. Structural differences were striking between recurrence and remission microbiota; while the microbiota of patients with CD recurrence exhibited a loose community structure, the microbiota of patients in remission displayed communities that were robustly correlated to each other. Microbiota colonising the neoterminal ileum and subanastomotic colon 6 months after ICR only differed in patients with recurrence. CONCLUSIONS: ICR modifies the gut microbiome. Remission after 6 months was associated with homogenous bacterial distribution around the anastomosis. Community structure and bacterial networks highlight target species, including Faecalibacterium prausnitzii and Ruminococcus gnavus, which may allow precise modulations of the overall microbial ecosystem towards remission pattern.

Short-chain fatty acids and commensal microbiota in the faeces of severely malnourished children with cholera rehydrated with three different carbohydrates.

BACKGROUND: Short-chain fatty acids (SCFAs) liberated by fermentation of complex carbohydrates might stimulate water and salt absorption, and provide energy. The aim of the study was to assess the number and proportion of faecal bacteria and the concentration of SCFAs of severely malnourished children with cholera receiving oral rehydration solution (ORS) containing glucose, amylase-resistant starch (ARS) or rice. METHODS: Serial faecal samples were collected from 30 malnourished children with cholera until rehydration and partial nutritional recovery. SCFAs were identified and quantitated by high-performance liquid chromatography. In situ hybridization combined with flow cytometry was used to analyse the microbiota in the faeces. RESULTS: Before treatment the concentration of total SCFA in faecal sample of cholera children was found to be 4.7±0.6 mmol/kg and it increased steadily until 95.0±8.7 mmol/kg at day 28. Among different ORS groups, concentration was significantly higher in the Rice-ORS group at day 1 (P<0.011) and at day 2 (P<0.025). During recovery faecal output was significantly reduced and the number of bacteria also increased faster in the Rice-ORS group than in the glucose-ORS group at day 1 and day 2 (P<0.01), and a modest increase in bacterial number was observed in the glucose-ORS plus ARS group (day 1, P=0.07; day 2, P=0.09). CONCLUSION: Clinical recovery was associated with an increase in bacterial and SCFA concentrations with all three carbohydrates in ORS. However, the increases were significantly higher in children receiving Rice-ORS.

Amoxicillin treatment modifies the composition of Bifidobacterium species in infant intestinal microbiota.

OBJECTIVES: Amoxicillin is a beta-lactam antibiotic largely used in childhood. However only few studies described its impact on composition of children gut microbiota, in particular on Bifidobacterium populations considered as beneficial microorganisms. In this study, the impact on faecal Bifidobacterium species of a seven-day amoxicillin treatment was quantitatively and qualitatively assessed in infants during an episode of acute respiratory infection. METHODS: Faecal samples from 31 infants were obtained on day 0 (just before amoxicillin therapy) and on day 7 (the end of therapy). Total DNA was extracted and bifidobacteria were quantified using real-time PCR. Predominant Bifidobacterium species were then identified using specific PCR-TTGE. RESULTS: Bifidobacteria concentrations were not significantly altered by amoxicillin compared to the healthy group. However, amoxicillin treatment induced a complete disappearance of Bifidobacterium adolescentis species (occurrence rate of 0% versus 36.4% in healthy group, P < 0.001), a significant decrease in the occurrence rate of Bifidobacterium bifidum (23% versus 54.5% in healthy group, P < 0.05), but did not affect Bifidobacterium longum (93.5% versus 100% in healthy group) and Bifidobacterium pseudocatenulatum/B. catenulatum (about 55% in both groups). The number of Bifidobacterium species per microbiota significantly decreased from 2.5 +/- 1 for healthy group to 1.8 +/- 0.9 for treated infants (P < 0.05). CONCLUSIONS: This study showed that a 7 day amoxicillin treatment did not alter the counts of Bifidobacterium. However amoxicillin can have an impact by changing the microbiota at the species level and decreased the diversity of this population.

A phase I study of CR002, a fully-human monoclonal antibody against platelet-derived growth factor-D.

OBJECTIVE: To investigate the safety, pharmacokinetics (PK), binding activity and immunogenicity of CR002, a human monoclonal antibody (mAb) directed against platelet-derived growth factor-D (PDGF-D), administered as a single intravenous (i.v.) infusion over a range of doses. SUBJECTS: 40 healthy male subjects received increasing doses of CR002 at 0.3, 1, 3, 10, 30 mg/kg or placebo. METHOD: This was a randomized, double-blind, placebo-controlled, dose-escalation Phase I study. The trial had a duration of 90 days, with dosing on Day 1 and follow-up visits on Days 2, 4, 7, 14, 21, 30, 45 and 90. Serum was collected for PK, binding activity and immunogenicity analysis at screening and up to Day 90. Safety was recorded throughout the study by performing laboratory tests, recording vital signs and electrocardiograms (ECGs), by monitoring the occurrence of adverse events (AEs). The use of concomitant medications was also recorded. RESULTS: All 40 subjects received CR002 or placebo, and completed the trial. No dose-limiting toxicities (DLTs) occurred, the maximum tolerated dose (MTD) was not reached and was estimated as > 30 mg/kg. There were no deaths during this study and no SAEs or other significant AEs reported. The most frequent drug-related treatment-emergent AE (TEAE) was headache in 4 of 30 subjects (13.3%) in the CR002 group vs. 0 of 10 subjects in the placebo group. CR002 exhibited linear PK parameters, had a long half-life (t1/2 in the range 15.5 â 48.1 days) and a volume of distribution at steady state in the range 4.7 â 6.5. Free PDGF-D in the serum bound to CR002 in a reversible manner, as shown in the lowest dose cohort. However, levels of total circulating PDGF-D remained constant throughout the study. There were no anti-CR002 antibodies detected in subjects dosed with CR002. CONCLUSIONS: CR002 was safe and well-tolerated at all doses tested as a single i.v. administration. The MTD was estimated to be above 30 mg/kg, the highest dose tested. CR002 had a long half-life, low clearance and a limited tissue distribution. Although total levels of PDGF-D at all dose levels remained relatively constant, there was no detectable circulating free PDGF-D after CR002 administration. At the lowest CR002 dose tested (0.3 mg/kg), PDGF-D was detectable again by Day 21 and the levels increased near to pre-infusion levels by Day 90. In this study, CR002 was not immunogenic during the 90-day study period.

Antioxidant effects of metronidazole in colonic tissue.

Reactive oxygen species, primarily generated in the mitochondria, contribute to tissue injury in inflammatory bowel diseases. The efficacy of metronidazole (MTZ) in some situations of inflammatory bowel disease may result not only from its antibiotic effect, but also from an antioxidant effect. We evaluated, under physiologic conditions, the antioxidant potential of MTZ on the biomarkers of oxidative damage to proteins (protein carbonyls), lipids (malondialdehyde), and the levels of antioxidant defense (glutathione) in the small bowel, large bowel, and liver of control and MTZ-treated rats. Basal levels of protein carbonyls and malondialdehyde were significantly higher in the colon compared with those in the small intestine, whereas glutathione levels were quite similar along the bowel. MTZ reduced significantly the colonic oxidative damage to proteins without any side effect in the liver. This is the first evidence of an antioxidant effect of MTZ on oxidative protein damage in the colon under physiologic conditions.

Review article: the role of bacteria in onset and perpetuation of inflammatory bowel disease.

We review the evidence that strongly suggests a role of the intestinal microbiota in the onset and perpetuation of inflammatory bowel disease (IBD). Experimental studies consisted of suppressing micro-organisms from the microbiota (using germ-free or gnotoxenic animals or antibiotics), introducing new micro-organisms or microbial components (e.g. probiotics, CpG-DNA) or selectively increasing some endogenous bacteria (e.g. using prebiotics). Intervention studies were performed in patients or animal models of spontaneous or chemically-induced colitis. Information was also obtained from observational studies that described the composition of the faecal and mucosal microbiota at various stages of the disease process and in controls. Many have used culture-independent techniques that identify bacteria based on the nucleic acid sequence of ribosomal RNA molecules. Microbiota in patients with IBD seem to be characterized by high concentrations of bacteria in contact with the mucosa, instability, the presence of high numbers of unusual bacteria and sometimes a reduction in the biodiversity. Studies searching for a generalized or localized dysbiosis in IBD are discussed, as well as those trying to identify bacterial molecules and receptors, which may be implicated in triggering the inflammatory process.

Review article: gut flora and inflammatory bowel disease.

The pathogenesis of inflammatory bowel disease involves interactions between the host susceptibility, mucosal immunity and intestinal microflora. There is therefore great interest in the changes in the endogenous flora in inflammatory bowel disease patients and in the establishment of potential genetic variations in host responses to endogenous bacteria. In this review, we summarize the modifications in the various regional ecosystems in the gastrointestinal tract during inflammatory bowel disease (luminal bacteria in faeces or inside the gastrointestinal tract, bacteria in mucus and bacteria directly attached to the mucosa). Results were obtained following a 'candidate microorganism strategy' and, as is occurring increasingly frequently, following a 'full description strategy', which has progressed largely due to the development of culture-independent techniques. The possibility of modifying the ecosystem using prebiotics or probiotics offers hope for new treatment developments, particularly in the prevention of relapse.

A protein interaction map of Drosophila melanogaster.

Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon.

BACKGROUND AND AIMS: The colonic microflora is involved in the pathogenesis of Crohn's disease (CD) but less than 30% of the microflora can be cultured. We investigated potential differences in the faecal microflora between patients with colonic CD in remission (n=9), patients with active colonic CD (n=8), and healthy volunteers (n=16) using culture independent techniques. METHODS: Quantitative dot blot hybridisation with six radiolabelled 16S ribosomal ribonucleic acid (rRNA) targeting oligonucleotide probes was used to measure the proportions of rRNA corresponding to each phylogenetic group. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. RESULTS: Enterobacteria were significantly increased in active and quiescent CD. Probe additivity was significantly lower in patients (65 (11)% and 69 (6)% in active CD and quiescent CD) than in healthy controls (99 (7)%). TTGE profiles varied markedly between active and quiescent CD but were stable in healthy conditions. CONCLUSION: The biodiversity of the microflora remains high in patients with CD. Enterobacteria were observed significantly more frequently in CD than in health, and more than 30% of the dominant flora belonged to yet undefined phylogenetic groups.

Comparative study of bacterial groups within the human cecal and fecal microbiota.

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.

Pharmacokinetics of Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363 in the human gastrointestinal tract.

OBJECTIVES: Genetically modified lactic acid bacteria may be a way to deliver vaccinal epitopes in the gastrointestinal tract. AIM: Three strains of lactic acid bacteria were studied for their pharmacokinetics in the human gastrointestinal tract. METHODS: The survival of the strains was studied up to the ileum in six subjects each, after ingestion of 150 g of fermented milk. The strains and their concentrations in the products were Lactobacillus fermentum KLD (107 cfu/g), Lactobacillus plantarum NCIMB 8826 (108 cfu/g), and Lactococcus lactis MG 1363 (108 cfu/g). Ileal fluid was aspirated by intestinal intubation and immediately cultured. L. plantarum NCIMB 8826, which was found in high concentrations in the ileum, was studied for its survival in the faeces after consumption of 150 g of fermented milk three times daily for 7 days. Faecal samples were collected for culture. RESULTS: The concentration of L. plantarum NCIMB 8826 in the ileum reached 108 cfu/mL after a single dose, with a survival of 7%. L. fermentum KLD and Lc. lactis MG 1363 had lower (0.5 and 1.0%, respectively) and shorter (4 h) survival in the ileum. During the 7-day ingestion period, L. plantarum NCIMB 8826 reached high concentrations (108 cfu/g) in the faeces, with a survival of 25 +/- 29%. None of the strains colonized. CONCLUSIONS: L. plantarum NCIMB 8826 has a promising pharmacokinetic profile as a candidate vaccine vehicle.

Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization.

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% +/- 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% +/- 7% and 14% +/- 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

Intestinal barrier function and cow's milk sensitization in guinea pigs fed milk or fermented milk.

BACKGROUND: The respective effect of milk and fermented milks on intestinal barrier capacity and on sensitization to beta-lactoglobulin was studied using a guinea pig model of cow's milk allergy. METHODS: Guinea pigs were fed a control diet or the same diet supplemented with milk, fermented milk (Streptococcus thermophilus and Bifidobacterium breve), or dehydrated fermented milk. Intestinal barrier capacity to macromolecules was assessed in an Ussing chamber, and sensitization to cow's milk proteins was measured by systemic anti-beta-lactoglobulin immunoglobulin G1 titers and by intestinal anaphylaxis, the latter assessed by the beta-lactoglobulin-induced increase in short-circuit current of jejunal fragments (deltaIsc(beta-LG)). RESULTS: The electrical resistance of jejunum was similar in the four groups (approximately 80 omega/cm2) suggesting the same paracellular permeability. The transport of 14C-beta-lactoglobulin from mucosa to serosa was significantly decreased in the animals fed dehydrated fermented milk (403+/-131 ng / hr x cm2) compared with that in control animals or animals fed milk (767+/-250 ng / hr x cm2 and 749+/-475 ng / hr x cm2, respectively; p < 0.05). Milk fermentation did not modify native beta-lactoglobulin concentration but anti-beta-lactoglobulin immunoglobulin G1 titers were higher in fermented milk and dehydrated fermented milk (log10 titer = 2.86 and 2.79, respectively) than in guinea pigs fed milk (log10 titer = 2.5; p < 0.007). However, beta-lactoglobulin-induced intestinal anaphylaxis remained the same in the three groups (deltaIsc(beta-LG), 9.6+/-4.1 microA/cm2, 8.5+/-4.3 microA/cm2, and 8.5+/-3.4 microA/cm2 in milk-fed, fermented milk-fed, and dehydrated fermented milk-fed guinea pigs, respectively). CONCLUSIONS: The intestinal barrier capacity to milk proteins seems to be reinforced by dehydrated fermented milk, but milk and fermented milks are equally efficient in inducing cow's milk allergy in guinea pigs.

Short-chain fructo-oligosaccharide administration dose-dependently increases fecal bifidobacteria in healthy humans.

Short-chain fructo-oligosaccharides (SC-FOS) are a mixture of oligosaccharides consisting of glucose linked to fructose units (Gfn; n =

Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides populations.

Colonic Bacteroides include several species which, by their population level and activities, are significant contributers to the metabolic activity and health of man and animals. Yet, the understanding of their ecology has been hampered by the lack of highly specific and reliable enumeration techniques. Based on 16S rRNA sequence comparisons within the available database, we have designed an 18-mer oligonucleotide that targets a region common to-and specific for the Bacteroides-Porphyromonas-Prevotella group. We have tested the specificity of the probe and its usefulness for studies of human faecal samples. Under experimentally optimized hybridization conditions, the probe was shown to similarly recognize the rDNA obtained from 40 strains representing 8 species of the Bacteroides-Porphyromonas-Prevotella group. Importantly, it did not recognize 31 strains of microorganisms representing 8 genera of the dominant human faecal microbiota. Among selected colonies of dominant microorganisms of the faecal flora of two human individuals, strains identified as B. vulgatus by immunoblots using a species-specific monoclonal antibody were all detected by the probe. Colony hybridization was used to enumerate total Bacteroides-group microorganisms in faecal specimen from children and adults. The probe described therein was further used in quantitative RNA blots to monitor fluctuations of the Bacteroides-group versus Bifidobacterium genus in frozen faecal samples from a child between 85 and 125 days of age. It will be applicable to similar investigations of other anaerobic environments.

Conserved properties between functionally distinct MutS homologs in yeast.

In the yeast Saccharomyces cerevisiae there are five nuclear MutS homologs that act in two distinct processes. MSH2, 3, and 6 function in mismatch repair in both vegetative and meiotic cells, whereas MSH4 and MSH5 act specifically to facilitate crossovers between homologs during meiosis. Coimmunoprecipitation as well as two-hybrid experiments indicate that the Msh4 and Msh5 proteins form a hetero-oligomeric structure similar to what is observed for the Msh proteins involved in mismatch repair. Mutation of conserved amino acids in the NTP binding and putative helix-turn-helix domains of Msh5p abolish function but are still capable of interaction with Msh4p, suggesting that NTP binding plays a role downstream of hetero-oligomer formation. No hetero-oligomers are observed between the mismatch repair MutS proteins (Msh2p and Msh6p) and either Msh4p or Msh5p. These results indicate that one level of functional specificity between the mismatch repair and meiotic crossover MutS homologs in yeast is provided by the ability to form distinct hetero-oligomers.

Administration of transgalacto-oligosaccharides increases fecal bifidobacteria and modifies colonic fermentation metabolism in healthy humans.

Transgalacto-oligosaccharides are a mixture of oligosaccharides consisting of glucose and galactose; they are not digested in the human small intestine. In vitro, they specifically stimulate the growth of bifidobacteria. The aim of the present work was to assess tolerance of transgalacto-oligosaccharides and the effects of their prolonged administration on bifidobacteria and fermentative activity of colonic flora. Eight healthy subjects were given 10 g of transgalacto-oligosaccharides per day for 21 d in two daily doses. A breath test and stool sample collection were carried out on d 1, 7, 14 and 21 of transgalacto-oligosaccharides ingestion. The stools of three subjects were collected and mixed before the study, and then inoculated in vitro into a fermentor to which 10 g transgalacto-oligosaccharides was added daily for 14 d. In the eight volunteers, administration of transgalacto-oligosaccharides led to a significant decrease in breath hydrogen excretion (P < 0.01) and a significant increase in fecal concentrations of bifidobacteria from (means +/- SEM) 8.6 +/- 0.6 to 9.7 +/- 0.5, 9.7 +/- 0.6 and 9.5 +/- 0.6 log colony-forming units (CFU)/g on d 1, 7, 14 and 21, respectively (P < 0.05). Fecal concentrations of enterobacteria, as well as stool weight, fecal water and pH did not change during the study. In vitro, transgalacto-oligosaccharides fermentation became more efficient and faster with time. In addition, metabolic alterations such as a rise in acetate proportion and lactate formation after 7 d of fermentation were observed, indicating the transformation of the inoculated fecal flora into an acid-resistant lactic flora. Prolonged administration of transgalacto-oligosaccharides, at a dose which does not induce digestive symptoms, increases the number of bifidobacteria and alters the fermentative activity of colonic flora in humans.

Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans.

OBJECTIVES: Link-Amster reported an increase in serum IgA when healthy subjects ingested a fermented dairy product containing Lactobacillus johnsonii La1. We aimed to assess the effects of La1 on the jejunal secretions and serum concentrations of total and specific immunoglobulins and proteins. METHODS: Twelve healthy volunteers ingested a fermented milk containing La1 or a control from day 1 till day 28, following a randomised double blind protocol. At days 0 and 28, the jejunum was successively perfused with a control solution and with a La1 suspension. The serum concentrations and jejunal secretions of albumin, orosomucoid, transferrin, alpha 2-macroglobulin, m-IgA, p-IgA, IgG, IgM, secretory component, and specific antibodies against La1 were assessed. RESULTS: Serum concentrations of IgA slightly increased between d0 and d28 in the group receiving La1 (1.85 +/- 0.64 g/L vs 1.76 +/- 0.76; P = 0.02). The other parameters were not altered. CONCLUSION: This study shows that the immunomodulating effects of La1 ingestion in man are not due to modification of jejunal protein permeability.

Improved clinical tolerance to chronic lactose ingestion in subjects with lactose intolerance: a placebo effect?

BACKGROUND: Uncontrolled studies of lactose intolerant subjects have shown that symptom severity decreases after chronic lactose consumption. Adaptation of the colonic flora might explain this improvement. AIMS: To compare the effects of regular administration of either lactose or sucrose on clinical tolerance and bacterial adaptation to lactose. METHODS: Forty six lactose intolerant subjects underwent two 50 g lactose challenges on days 1 and 15. Between these days they were given 34 g of lactose or sucrose per day, in a double blind protocol. Stool samples were obtained on days 0 and 14, to measure faecal beta-galactosidase and pH. Symptoms, breath H2 excretion, faecal weight and electrolytes, and orofaecal transit time were assessed. RESULTS: Except for faecal weight, symptoms were significantly milder during the second challenge in both groups, and covariance analysis showed no statistical difference between them. In the lactose group, but not in the sucrose group, faecal beta-galactosidase activity increased, pH dropped, and breath H2 excretion decreased. CONCLUSION: Bacterial adaptation occurred when lactose intolerant subjects ingested lactose for 13 days, and all symptoms except diarrhoea regressed. Clinical improvement was also observed in the control group which displayed no signs of metabolic adaptation. This suggests that improved clinical tolerance may be just a placebo effect.