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Stressful events result in the alteration of gut permeability and sensitivity. Lactobacillus paracasei NCC2461 (Lpa) therapy prevents antibiotic-induced visceral hyperalgesia in mice. This study aimed at evaluating the influence of 3 probiotic strains: Bifidobacterium lactis NCC362, Lactobacillus johnsonii NCC533, and Lpa on stress-mediated alterations of colorectal hyperalgesia, on gut paracellular permeability and whether bacteria and/or bacterial products present in the spent culture medium (SCM) were involved in the antinociceptive properties of the effective strain. Rat pups were separated from their mothers 3 h/d during postnatal d 2-14. At wk 13, gut paracellular permeability was determined as a percentage of urinary excreted (51)Cr-EDTA and visceral sensitivity to colorectal distension (CRD), assessed by abdominal muscle electromyography. Visceral sensitivity was also analyzed in adults rats subjected to partial restraint stress (PRS, 2 h restriction of body movements). Rats received either the probiotics resuspended in SCM or fresh growth medium as control for 2 wk. Maternal deprivation significantly increased colonic sensitivity in response to CRD and enhanced gut paracellular permeability compared with control rats. Only Lpa treatment significantly improved stress-induced visceral pain and restored normal gut permeability. Similarly, among the 3 probiotics tested, only Lpa prevented PRS-mediated visceral hyperalgesia. Both bacteria and bacterial products present in Lpa SCM were required for the antinociceptive properties against PRS. This study illustrates strain-specific effects and suggests a synergistic interplay between L. paracasei bacteria and bacterial products generated during fermentation and growth that confers the ability to suppress PRS-induced hypersensitivity in rats.

Helicobacter pylori infection, a highly prevalent pathogen, is a major cause of chronic gastritis and peptic ulcer and a risk factor for gastric malignancies. Antibiotics-based H. pylori eradication treatment is 90% effective. However, it is expensive and causes side effects and antibiotic resistance. Probiotics could present a low-cost, large-scale alternative solution to prevent or decrease H. pylori colonization. A literature search of the MEDLINE database (1966-2006) has been performed selecting all in vitro, animal, and human fully published English-language studies dealing with H. pylori and probiotics. Probiotics had an in vitro inhibitory effect on H. pylori. Animal studies demonstrated that probiotic treatment is effective in reducing H. pylori-associated gastric inflammation. Seven of 9 human studies showed an improvement of H. pylori gastritis and decrease in H. pylori density after administration of probiotics. The addition of probiotics to standard antibiotic treatment improved H. pylori eradication rates (81% vs. 71%, with combination treatment vs. H. pylori-eradication treatment alone; chi(2)test: P=0.03). Probiotic treatment reduced H. pylori therapy-associated side effects (incidence of side effects: 23% vs. 46%, with combination therapy vs. H. pylori-eradication treatment alone; chi(2)test: P=0.04). No study could demonstrate the eradication of H. pylori infection by probiotic treatment. So long-term intake of products containing probiotic strains of probiotics may have a favorable effect on H. pylori infection in humans, particularly by reducing the risk of developing disorders associated with high degrees of gastric inflammation.

Irritable Bowel Syndrome (IBS) is a common multifactorial intestinal disorder for which the aetiology remains largely undefined. Here, we have used a Trichinella spiralis (T. spiralis)-induced model of post-infective IBS, and the effects of probiotic bacteria on gut dysfunction have been investigated using a metabonomic strategy. A total of 44 mice were divided into four groups: an uninfected control group and three T. spiralis-infected groups, one as infected control and the two other groups subsequently treated with either Lactobacillus paracasei (L. paracasei) NCC2461 in spent culture medium (SCM) or with L. paracasei-free SCM. Plasma, jejunal wall and longitudinal myenteric muscle samples were collected at day 21 post-infection. An NMR-based metabonomic approach characterized that the plasma metabolic profile of T. spiralis-infected mice showed an increased energy metabolism (lactate, citrate, alanine), fat mobilization (acetoacetate, 3-D-hydroxybutyrate, lipoproteins) and a disruption of amino acid metabolism due to increased protein breakdown, which were related to the intestinal hypercontractility. Increased levels of taurine, creatine and glycerophosphorylcholine in the jejunal muscles were associated with the muscular hypertrophy and disrupted jejunal functions. L. paracasei treatment normalized the muscular activity and the disturbed energy metabolism as evidenced by decreased glycogenesis and elevated lipid breakdown in comparison with untreated T. spiralis-infected mice. Changes in the levels of plasma metabolites (glutamine, lysine, methionine) that might relate to a modulation of immunological responses were also observed in the presence of the probiotic treatment. The work presented here suggests that probiotics may be beneficial in patients with IBS.

Objectives Psychological stress during the neonatal period results in intestinal barrier dysfunction and growth alterations later in life. We aimed to restore impaired barrier function and growth rate by a nutritional intervention. Methods Male rat pups (n = 84) were assigned to 1 of 2 rearing conditions from postnatal day (PND) 2 to PND14: S, separated 3 h/d from their mothers, or H, 15 min/d handled controls. From PND15 to PND35, rats received a control diet or a similar diet adapted to contain arachidonic and docosahexaenoic acids, galacto- and fructo-oligosaccharides and Lactobacillus paracasei NCC2461. Results Maternal separation had only a minor impact on the measured gut barrier parameters at PND15, whereas it severely affected them at PND35. At this age, intestinal permeability to macromolecules was higher, mucin content in small intestinal tissues was lower and microbiota composition was altered in S compared with H animals. Feeding the adapted diet normalized the intestinal permeability, although it did not restore intestinal mucin content or microbiota. In addition, the adapted diet improved the growth rate recovery of the S animals after weaning and resulted in increased villus length in small intestine. Conclusion Our results suggest that an adapted diet containing specific long-chain polyunsaturated fatty acids, prebiotics and probiotics can revert the negative imprinting of neonatal stress on both intestinal barrier function and growth.

The gastrointestinal tract (GIT) of eukaryotes is colonized by a vast number of bacteria, where the commensal microbiota play an important role in defining the healthy gut. To investigate the influence of commensal bacteria on multiple regions of the host GIT transcriptome, the gene expression profiles of the corpus, jejunum, descending colon, and rectum of conventional ( n = 3) and germ-free mice ( n = 3) were examined using the Affymetrix Mu74Av2 GeneChip. Differentially regulated genes were identified using the global error assessment model, and a novel method of Gene Ontology (GO) clustering was used to identify significantly modulated biological functions. The microbiota modify the greatest number of genes in the jejunum (267 genes with an α < 0.001) and the fewest in the rectum (137 genes with an α < 0.001). Clustering genes by GO biological process and molecular function annotations revealed that, despite the large number of differentially regulated genes, the residential microbiota most significantly modified genes involved in such biological processes as immune function and water transport all along the length of the mouse GIT. Additionally, region-specific communication between the host and microbiota were identified in the corpus and jejunum, where tissue kallikrein and apoptosis regulator activities were modulated, respectively. These findings identify important interactions between the microbiota and the mouse gut tissue transcriptome and, furthermore, suggest that interactions between the microbial population and host GIT are implicated in the coordination of region-specific functions.

The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

An increased density of Helicobacter pylori in the gastric mucosa can be associated with more severe gastritis and an increased incidence of peptic ulcers. Therefore, people with asymptomatic gastritis would certainly benefit from a nutritional approach to help them manage the infection and therefore decrease the risk of development of associated pathologies. We analyzed the activities of 60 essential oils against H. pylori P1 and identified 30 oils that affected growth, with in vitro inhibition zones ranging between 0.7 and 6.3 cm in diameter. We further analyzed the effects of 16 oils with different activities on H. pylori P1 viability. Fifteen showed strong bactericidal activities, with minimal bactericidal concentrations after 24 h ranging from 0.02 to 0.1 g/liter at pH 7.4. Even though slight variations in activities were observed, the essential oils that displayed the strongest bactericidal potentials against H. pylori P1 were also active against other Helicobacter strains tested. Among the pure constituents of different essential oils tested, carvacrol, isoeugenol, nerol, citral, and sabinene exhibited the strongest anti- H. pylori activities. Although oral treatment of H. pylori SS1-infected mice with carrot seed oil did not result in significant decreases in the bacterial loads in the treated animals compared to those in the control animals, in all experiments performed, the infection was cleared in 20 to 30% of carrot seed oil-treated animals. Our results indicate that essential oils are unlikely to be efficient anti- Helicobacter agents in vivo. However, their effects may not be irrelevant if one plans to use them as food additives to complement present therapies.

Summary Background : Lactobacillus johnsonii (Lj1) had an in vitro and in vivo inhibitory effect on Helicobacter pylori. Fermented milk containing Lj1 (LC1), coadministered with antibiotics had a favourable effect on H. pylori gastritis. Aim : Evaluate the effect of LC1 intake without antibiotics on H. pylori gastritis. Methods : Fifty H. pylori positive healthy volunteers were randomised in a double-blind study to LC1 or placebo. Gastric biopsies from the antrum and corpus were obtained before, and after 3 and 16 weeks of treatment, for histology and quantitative cultures. Results : Severity and activity of antral gastritis was reduced after 16-week LC1 intake (pretreatment and 16-week inflammatory cell score: 6.0 ± 0.8 vs. 5.3 ± 0.1; P = 0.04). H. pylori density decreased in the antrum after LC1 intake (3-week: 4.4 ± 0.6; 16-week: 4.3 ± 0.5 log10 colony forming units (cfu) vs. pretreatment 4.5 ± 0.4 log10 cfu; P = 0.04, respectively). Mucus thickness increased after 16 weeks of LC1 consumption (change of mucus thickness with LC1 and placebo in the antrum: 0.6 ± 1.3 vs. -0.2 ± 1.0, P = 0.01; in the corpus: 0.3 ± 1.1 vs. -0.6 ± 1.5, P = 0.03). Conclusion : LC1 intake had a favourable, albeit weak, effect on H. pylori associated gastritis, particularly in the antrum. Regular ingestion of fermented milk containing L. johnsonii may reduce the risk of developing disorders associated with high degrees of gastric inflammation and mucus depletion.

Objective The supernatant of Lactobacillus johnsonii La1 culture was shown to be bactericidal and to have a partial, acid-independent suppressive effect on Helicobacter pylori in humans. The aim of the present study was to investigate the effect of L. johnsonii La1 -acidified milk (LC-1) on H. pylori infection. Design and methods Fifty-three volunteers infected with H. pylori as determined by positive 13 C-urea breath test and positive serology were randomized to receive either LC-1 or a placebo 180 ml twice a day for 3 weeks. All subjects also received clarithromycin 500 mg bid during the last two weeks of acidified milk therapy. Oesophagogastroduodenoscopy and biopsies were performed at inclusion and repeated 4-8 weeks after the end of the treatment. H. pylori infection was confirmed by urease test and histology. H. pylori density and inflammation were scored using a modified Sydney classification. Results LC-1 ingestion induced a decrease in H. pylori density in the antrum (P= 0.02) and the corpus (P= 0.04). LC-1 also reduced inflammation and gastritis activity in the antrum (P = 0.02 and P = 0.01, respectively) and of activity in the corpus (P=0.02). Clarithromycin eradicated H. pylori in 26% of the subjects; LC-1 did not improve the antibiotic effect. Conclusion These results suggest that H. pylori infection and gastritis can be down-regulated by LC-1.

Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire displayed on filamentous phage. Aft

SUMMARY Homeostasis between indigenous intestinal flora and host response may be broken in inflammatory bowel disease. The present study explores whether repeated oral administration of intestinal flora antigens can protect mice against dextran sodium sulphate (DSS)-induced colitis. Sonicates of Gram-positive, Gram-negative, or anaerobic resident bacteria isolated from mouse intestinal flora were fed to BALB/c mice by gastric gavage, with or without cholera toxin. After four weekly doses of 1 mg of these antigen preparations (or of PBS as control), DSS colitis was induced. One week later colitis was evaluated by clinical scores and histology. Mice fed a pool of the three sonicates had decreased inflammation scores (5 (1–14); median (range)) compared with PBS-fed control animals (15 (7–19); P

Mucosal immunization with Helicobacter heilmannii urease B or Helicobacter pylori urease, given nasally with cholera toxin, protects BALB/c mice against H. heilmannii infection and significantly reduces a preexisting infection. However, immunization aggravates gastric corpus atrophy. Our results underline the necessity of defining immunization regimens that do not enhance mucosal damage.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

Helicobacter pylori colonises the gastric mucosa, but can also be found within the oral cavity. The presence of H. pylori was monitored in the oral cavity of 22 patients with duodenal ulcer, before and after antibiotic treatment and of 24 hospital employees who were or were not professionally exposed to H. pylori. Gastric infection was determined by breath test. Bacteria in the oral cavity were detected by nested PCR of samples containing saliva and dental plaque, using primers specific for 16S rRNA and ureC genes. Before treatment, 9 out of 22 infected ulcer patients harbored H. pylori in their oral cavity. Bacteria disappeared from the oral cavity of 3 of 7 cured patients. Twelve of 17 exposed subjects harbored H. pylori in their oral cavity, while no bacteria could be detected in the mouths of the 7 nonexposed subjects. Presence of bacteria in the oral cavity reflects handling of contaminants; it does not correlate with gastric infection and does not seem to promote it.

Live Salmonella typhimurium phoP c bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens. The genes encoding the A and B subunits of H. pylori urease were introduced into S. typhimurium phoP c and expressed under the control of a constitutive tac promoter (tac- ureAB ) or a two-phase T7 expression system (cT7- ureAB ). Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7- ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer’s patches of immunized mice, while the plasmid carrying tac- ureAB was rapidly lost. Spleen and Peyer’s patch CD4 + lymphocytes from mice immunized with S. typhimurium phoP c cT7- ureAB proliferated in vitro in response to urease, whereas cells from mice given S. typhimurium phoP c alone did not. Splenic CD4 + cells from mice immunized with phoP c cT7- ureAB secreted gamma interferon and interleukin 10, while Peyer’s patch CD4 + cells did not secrete either cytokine. Specific H. pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized with S. typhimurium phoP c cT7- ureAB were found to be resistant to infection by H. pylori , while all mice immunized with phoP c tac- ureAB (15 of 15) or phoP c (15 of 15) were infected. Our data demonstrate that H. pylori urease delivered nasally by using a vaccine strain of S. typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection.

We have developed a 14C-urea breath test to follow the course of Helicobacter felis infection in mice. Peak 14CO2 production occurred approximately 8 min after substra

Background/Aims: Because Helicobacter pylori is a potentially dangerous human pathogen, the protective potential of oral immunization with H. pylori urease and its subunits was evaluated in an animal model. Methods: Mice were orally immunized with H. pylori sonicate, urease, or recombinant enzymatically inactive urease subunits and then challenged with Helicobacter felis . Control mice were sham-immunized. Results: H. felis colonization was present 5 days after challenge in 9 of 10 sham-immunized, 6 of 9 sonicate-immunized, and 3 of 10 urease-immunized animals ( P = 0.031 vs. sham-immunized). Twelve days after challenge, urease B-immunized mice had a weaker colonization than sham-immunized controls, whereas urease A had no effect. After 70 days, most urease A- and urease B-immunized mice had cleared the colonization (1017: P=0.0019; 1620: P=0.00002 vs. sham-immunized). In urease B-immunized animals, protection was often associated with corpus gastritis. Conclusions: Oral immunization with H. pylori urease protects mice against H. felis infection. Enzymatically inactive urease A and B subunits contain protective epitopes. It is unclear whether protection depends on the development of a mononuclear inflammatory response in the gastric corpus. Our observations should encourage the development of a human vaccine.

BACKGROUND & AIMS: Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. METHODS: AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. RESULTS: T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor- and interferon-, as well as the kynurenine and kynurenine/ tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. CONCLUSIONS: Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve.

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn's disease patients, we identified genes that might be biomarkers of Crohn's and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.

The intestinal mucus layer and endogenous microbiota are strongly intertwined and this contributes to the maintenance of the epithelial barrier and ultimately of gut homeostasis. To understand the molecular foundations of such relationship, we investigated if the nature of the microbiota transcriptionally regulates mucus layer composition in vivo. We found that the expression of mucins 1 to 4 and trefoil factor 3 was down-regulated in the ileum and colon of conventional and reconventionalized mice compared with germ-free animals. Conversely, very limited colon-restricted changes in transmembrane mucins were detected in mice colonized with human adult or baby microbiota. Moreover, by microarray analysis, the murine endogenous microbiota was found to modulate genes putatively involved in mucin secretion. These findings show that a well-established microbial community participates in the regulation of the gut mucus layer and that its composition and adequacy to the host are key factors in this process.

Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium,Lactobacillus johnsoniiLa1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed inEscherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogenHelicobacter pylori. In addition, La1 rGroEL mediates the aggregation ofH. pyloribut not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregateH. pylori.

The postnatal maturation of the gut, partially modulated by bacterial colonization, ends up in the establishment of an efficient barrier to luminal antigens and bacteria. The use of broad-spectrum antibiotics in pediatric practices alters the gut bacterial colonization and, consequently, may impair the maturation of the gut barrier function. To test this hypothesis, suckling Sprague-Dawley rats received a daily intragastric gavage of antibiotic (Clamoxyl; an amoxicillin-based commercial preparation) or saline solution from postnatal day 7 (d7) until d17 or d21. Luminal microbiota composition and global gene expression profile were analyzed on samples from small intestine and colon of each group. The treatment with Clamoxyl resulted in the almost-complete eradication of Lactobacillus in the whole intestine and in a drastic reduction of colonic total aerobic and anaerobic bacteria, in particular Enterobacteriacae and Enterococcus. The global gene expression analysis revealed that Clamoxyl affects the maturation process of 249 and 149 Affymetrix probe sets in the proximal and distal small intestine, respectively, and 163 probe sets in the colon. The expression of genes coding for Paneth cell products (defensins, matrilysin, and phospholipase A2) was significantly downregulated by the Clamoxyl treatment. A significant downregulation of major histocompatibility complex (MHC) class Ib and II genes, involved in antigen presentation, was also observed. Conversely, mast cell proteases expression was upregulated. These results suggest that early treatment with a large-spectrum antibiotic deeply affects the gut barrier function at the suckling-weaning interface, a period during which the gut is challenged by an array of novel food-borne antigens.

Current interest in probiotics is motivated not only by the clinical data showing efficacy of some probiotic bacteria but also by the increasing antibiotic resistance of pathogenic bacteria (particularly in hospitals) and the rise of consumers’ demand for natural substitutes of drugs. Only few randomized, double-blind placebo-controlled human trials are available, and some involved only small numbers of patients. They are difficult to compare because of differences in probiotic strains employed, doses and formulation. Among probiotic applications, reduction of diarrhea is probably the best-documented effect confirmed by recent meta-analyses. Literature on Helicobacter pylori indicates that probiotics are unable to eradicate the infection but could be useful in decreasing infection levels and as adjuvants of therapy-associated side effects. Studies performed in inflammatory bowel disease suggest that high doses of probiotics and most likely a combination of different lactobacilli and bifidobacteria are more effective in decreasing inflammatory score and maintaining patients in remission than a single probiotic strain. Probiotic studies evaluating amelioration of symptoms in irritable bowel syndrome would require more sustained patient numbers. However, accumulated data is encouraging and suggests that efficacy is strain-dependent. Finally, too few probiotic intervention trials have been reported on colon cancer to allow any firm conclusion.

The interaction between the human body and nutrition is an extremely complex process involving multi-organ physiology with molecular mechanisms on all levels of regulation (genes, gene expression, proteins, metabolites). Only with the recent technology push have nutritional scientists been able to address this complexity. Both the challenges and promises that are offered by the merge of 'biomics' technologies and mechanistic nutrition research are huge, but will eventually evolve in a new nutrition research concept: nutritional systems biology. This review describes the principles and technologies involved in this merge. Using nutrition research examples, including gene expression modulation by carbohydrates and fatty acids, this review discusses applications as well as limitations of genomics, transcriptomics, proteomics, metabolomics, and systems biology. Furthermore, reference is made to gene polymorphisms that underlie individual differences in nutrient utilization, resulting in, e.g., different susceptibility to develop obesity. Copyright © 2005 S. Karger AG, Basel/ILSI Europe.

Background & Aims: The effects of probiotics on gut dysfunction in postinfective irritable bowel syndrome are unknown. We tested whether probiotics influence persistent muscle hypercontractility in mice after recovery from infection with Trichinella spiralis and analyzed the underlying mechanisms. Methods: Mice were gavaged with Lactobacillus paracasei, Lactobacillus johnsonii, Bifidobacterium longum , or Bifidobacterium lactis in spent culture medium from days 10 to 21 after infection. Additional mice received heat-inactivated Lactobacillus paracasei, Lactobacillus paracasei -free spent culture medium, or heat-inactivated Lactobacillus paracasei -free spent culture medium. Lactobacilli enumeration, immunohistochemistry, and cytokine detection (enzyme-linked immunosorbent assay) were performed. Mice were also treated with Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from days 18 to 28 after infection. Contractility was measured on days 21 and 28 after infection. Results: Lactobacillus paracasei , but not Lactobacillus johnsonii, Bifidobacterium lactis , or Bifidobacterium longum , attenuated muscle hypercontractility. This was associated with a reduction in the Trichinella spiralis -associated T-helper 2 response and a reduction in transforming growth factor-β1, cyclooxygenase-2, and prostaglandin E 2 levels in muscle. Attenuation of muscle hypercontractility by Lactobacillus paracasei -free spent culture medium was abolished after heat treatment. Improvement of muscle hypercontractility at day 28 after infection was also observed after the administration of Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from day 18 after infection. Conclusions: Probiotics show strain-dependent attenuation of muscle hypercontractility in an animal model of postinfective irritable bowel syndrome. This likely occurs via both a modulation of the immunologic response to infection and a direct effect of Lactobacillus paracasei or a heat-labile metabolite on postinfective muscle hypercontractility. Lactobacillus paracasei may be useful in the treatment of postinfective irritable bowel syndrome.

The objective of this study was to examine the effect of breast-feeding by immunized dams on Helicobacter colonization in newborns. Urease-based immunization regimens failed to protect nursing pups against H. felis , whereas H. felis lysate-cholera toxin resulted in protection. This observation correlated with a high recognition of cell surface-expressed bacterial antigens by milk antibodies. Protection lasted until weaning, indicating that infection is maintained at undetectable levels by passive immunity but then resumes when breast-feeding stops.

Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

Circumstantial evidence suggests that “ Helicobacter heilmannii ” infection is an example of zoonosis. The presence of “ H. heilmannii ” strains in a human subject with acute gastric erosions, in his two cats, and in two unrelated cats was analyzed, and the genetic relatedness of the human and feline strains was assessed. A 580-bp, PCR-amplified sequence of “ H. heilmannii ” urease B gene ( ureB ) obtained from biopsies from the human subject and his two cats was restricted with Alu I and cloned for sequencing. Analysis of the restriction fragment length polymorphism of the ureB -amplified product suggested the presence of different individual “ H. heilmannii ” strains in the cats and of three distinct strains in the human subject. One of the “ H. heilmannii ” ureB sequences amplified from the human subject’s biopsies was identical to that derived from one of his cats. The degree of similarity between the other “ H. heilmannii ” human and feline nucleotide sequences was higher than 97%. Most of the base substitutions were conservative. We conclude that human and animal “ H. heilmannii ” strains are closely related and that humans can be infected by more than one “ H. heilmannii ” strain, as has been observed for Helicobacter pylori.

The presence of spiral bacteria in the feline stomach has been recognized for over a century, but the identities and degrees of prevalence of such organisms in privately owned cats are still poorly documented. The aims of this study were (i) to adapt different diagnostic tools and evaluate their practicality for diagnosing feline gastric Helicobacter colonization, (ii) to determine the prevalence of gastric Helicobacter -like organisms in pet cats, (iii) to identify the feline species, and (iv) to correlate the presence of a Helicobacter infection with gastritis. Biopsy samples were taken gastroscopically from the antra and the corpora of clinically healthy pet cats. Helicobacter -like organisms were detected by Gram staining, Warthin-Starry staining, and rapid urease testing in biopsy specimens and by [ 13 C]urea breath testing in 79, 77, 78, and 85% of cases, respectively. PCR analysis revealed that 78% of the cats (38 of 49) were infected by Helicobacter heilmannii ; however, none of them was harboring Helicobacter pylori or Helicobacter felis . Culture was positive for one cat; the organism was identified as Helicobacter pametensis by dot blot DNA hybridization. By a combination of the detection methods, 91% of the pet cats were found to be Helicobacter positive. For 46 cats (79%) diagnostic tests were concordant. All cats showed mild to moderate gastritis in either the antrum or the corpus, regardless of the presence or density of gastric bacteria. In summary, pet cats are frequently colonized by H. heilmannii without a significant correlation between infection and degree of gastritis.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

An immunological approach or a vaccine approach to clear chronic Helicobacter pylori infection was initially rejected by many investigators and clinicians, based on the observation that natural immunity was unable to cure or prevent Helicobacter infection and chronic atrophic gastritis. Recent animal studies, however, have established that immunization with Helicobacter whole cell extracts or purified components is efficient for the prevention of infection, and more importantly for the treatment of pre-existing infections. Therefore there is now considerable interest in the design of Helicobacter vaccines, which represent a promising alternative to conventional therapies, in which bad compliance and antibiotic resistance represent serious drawbacks1.

Background & Aims: Eradication of Helicobacter pylori infections in humans results in the healing of gastritis and gastric ulcers. This study used a mouse model to test whether oral vaccination can cure Helicobacter infection and gastritis. Methods: Mice were infected with Helicobacter felis . Three weeks after infection, the mice were orally immunized with H. pylori urease B subunit. Control mice were simultaneously infected but sham immunized. Results: Three to 8 weeks after oral immunization of H. felis —infected mice with recombinant H. pylori urease B subunit, the infection cleared and there was no evidence of gastritis. Vaccinated mice remained protected against two consecutive H. felis challenges. Conclusions: These results show that the lack of natural immunity against Helicobacter can be overcome by oral immunization and that vaccination offers a novel therapeutic approach to Helicobacter -induced gastritis.

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.