Synergistic effect of anti-Helicobacter pylori urease immunoglobulin Y from egg yolk of immunized hens and Lactobacillus johnsonii No.1088 to inhibit the growth of Helicobacter pylori in vitro and in vivo.

Helicobacter pylori is a pathogenic bacterium that infects the stomach, causing chronic gastritis; and it is also considered to be related to the occurrence of gastric cancers. Although some eradication regimens including multiple antibiotics have been developed, the emergence of resistance to antibiotics becomes problematic. Therefore, other approaches to compensate or augment the effects of standard regimens are needed. In this study, we examined the possible synergistic effects of anti-H. pylori urease IgY and Lactobacillus johnsonii No.1088 (LJ88) both in vitro and in vivo. Anti-H. pylori urease IgY was purified from egg yolks laid by the hens immunized with urease purified from H. pylori. LJ88 is a unique strain of lactic acid bacterium isolated from human gastric juice, and it has been reported to inhibit H. pylori both in vitro and in vivo. The in vitro mixed culture study showed that anti-H. pylori urease IgY augmented the anti-H. pylori activity of LJ88 against both clarithromycin-sensitive and -resistant H. pylori strains. In a germ-free mice infection model, combined administration of daily anti-H. pylori urease IgY and weekly living LJ88 significantly reduced H. pylori infections, whereas either monotherapy did not. In an in vivo human gut microbiota-associated mice model, not only daily administration of living LJ88 but also heat-killed one significantly reduced an H. pylori infection in the stomach when combined with anti-H. pylori urease IgY. The extent of reduction of the stomach H. pylori by such a combination therapy was larger than that reported for LJ88 monotherapy. These results taken together revealed a synergistic effect of anti-H. pylori urease IgY and living or heat-killed LJ88, thus suggesting that such a combination might be a promising therapy to possibly compensate and/or augment standard anti-H. pylori regimens.

Regulation of gut luminal serotonin by commensal microbiota in mice.

Gut lumen serotonin (5-hydroxytryptamine: 5-HT) contributes to several gastrointestinal functions such as peristaltic reflexes. 5-HT is released from enterochromaffin (EC) cells in response to a number of stimuli, including signals from the gut microbiota. However, the specific mechanism by which the gut microbiota regulates 5-HT levels in the gut lumen has not yet been clarified. Our previous work with gnotobiotic mice showed that free catecholamines can be produced by the deconjugation of conjugated catecholamines; hence, we speculated that deconjugation by bacterial enzymes may be one of the mechanisms whereby gut microbes can produce free 5-HT in the gut lumen. In this study, we tested this hypothesis using germ-free (GF) mice and gnotobiotic mice recolonized with specific pathogen-free (SPF) fecal flora (EX-GF). The 5-HT levels in the lumens of the cecum and colon were significantly lower in the GF mice than in the EX-GF mice. Moreover, these levels were rapidly increased, within only 3 days after exposure to SPF microbiota. The majority of 5-HT was in an unconjugated, free form in the EX-GF mice, whereas approximately 50% of the 5-HT was found in the conjugated form in the GF mice. These results further support the current view that the gut microbiota plays a crucial role in promoting the production of biologically active, free 5-HT. The deconjugation of glucuronide-conjugated 5-HT by bacterial enzymes is likely one of the mechanisms contributing to free 5-HT production in the gut lumen.

Anti-Helicobacter pylori activity of non-living, heat-killed form of lactobacilli including Lactobacillus johnsonii No.1088.

Some strains of lactic acid bacteria are reported to inhibit the growth of Helicobacter pylori and proposed to be useful to support so-called triple therapy for H. pylori. Although most strains must be alive to exert their anti-H. pylori activity, some lactobacilli strains are effective even when dead. One possible underlying mechanism of such an activity of non-living lactobacilli is reportedly co-aggregation with H. pylori. In this study, we found that a non-living heat-killed form of Lactobacillus johnsonii No.1088 (HK-LJ88) and also that of some other lactobacilli inhibited the growth of H. pylori in vitro. Furthermore, the number of H. pylori in the infected stomach of germ-free mice was significantly decreased by the repeated oral administration of HK-LJ88. Observation by scanning electron microscopy revealed that no co-aggregation had occurred between H. pylori and HK-LJ88; instead, deformations of H. pylori (e.g. disappearance of spiral, bending of cell body, coccoid formation, degradations, etc.) appeared after incubation for 24 h with HK-LJ88. These results suggest that HK-LJ88 inhibited H. pylori activity probably not by co-aggregation but by some unknown mechanism involving HK-LJ88's cell surface molecules and that even non-living lactobacilli are possibly useful to support H. pylori eradication therapy.

Colonic Absorption of Low-Molecular-Weight Metabolites Influenced by the Intestinal Microbiome: A Pilot Study.

Low-molecular-weight metabolites produced by the intestinal microbiome play a direct role in health and disease. However, little is known about the ability of the colon to absorb these metabolites. It is also unclear whether these metabolites are bioavailable. Here, metabolomics techniques (capillary electrophoresis with time-of-flight mass spectrometry, CE-TOFMS), germ-free (GF) mice, and colonized (Ex-GF) mice were used to identify the colonic luminal metabolites transported to colonic tissue and/or blood. We focused on the differences in each metabolite between GF and Ex-GF mice to determine the identities of metabolites that are transported to the colon and/or blood. CE-TOFMS identified 170, 246, 166, and 193 metabolites in the colonic feces, colonic tissue, portal plasma, and cardiac plasma, respectively. We classified the metabolites according to the following influencing factors: (i) the membrane transport system of the colonocytes, (ii) metabolism during transcellular transport, and (iii) hepatic metabolism based on the similarity in the ratio of each metabolite between GF and Ex-GF mice and found 62 and 22 metabolites that appeared to be absorbed from the colonic lumen to colonocytes and blood, respectively. For example, 11 basic amino acids were transported to the systemic circulation from the colonic lumen. Furthermore, many low-molecular-weight metabolites influenced by the intestinal microbiome are bioavailable. The present study is the first to report the transportation of metabolites from the colonic lumen to colonocytes and somatic blood in vivo, and the present findings are critical for clarifying host-intestinal bacterial interactions.

Losartan attenuates the coronary perivasculitis through its local and systemic anti-inflammatory properties in a murine model of Kawasaki disease.

BACKGROUND: Kawasaki disease is a common systemic vasculitis that leads to coronary artery lesions. Besides its antihypertensive effects, losartan can modulate inflammation in cardiovascular disease. We examined whether losartan can attenuate coronary inflammation in a murine model of Kawasaki disease. METHODS AND RESULTS: Five-wk-old C57/BL6J male mice were intraperitoneally injected with Lactobacillus casei cell wall extract to induce coronary inflammation and divided into four groups: placebo, intravenous immunoglobulin (IVIG), losartan, and IVIG+losartan. After 2 wk, mice were harvested. The coronary perivasculitis was significantly attenuated by losartan but not by IVIG alone, and further dramatic attenuation by IVIG+losartan was observed. The frequency of Lactobacillus casei cell wall extract-induced myocarditis (80%) was markedly lowered by losartan (22%) and IVIG+losartan (0%). Furthermore, interleukin (IL)-6 mRNA was markedly attenuated by IVIG+losartan. Serum levels of IL-6, TNF-α, MCP-1, and IL-10 after Lactobacillus casei cell wall extract injection were slightly decreased by IVIG or losartan. Moreover, IL-1ß, IL-10, and MCP-1 levels were significantly decreased by IVIG+losartan. CONCLUSION: The addition of losartan to IVIG strongly attenuated the severity of coronary perivasculitis and the incidence of myocarditis, along with suppressing systemic/local cytokines as well as the activated macrophage infiltration. Therefore, losartan may be a potentially useful additive drug for the acute phase of Kawasaki disease to minimize coronary artery lesions.

A highly acid-resistant novel strain of Lactobacillus johnsonii No. 1088 has antibacterial activity, including that against Helicobacter pylori, and inhibits gastrin-mediated acid production in mice.

A novel strain of Lactobacillus johnsonii No. 1088 was isolated from the gastric juice of a healthy Japanese male volunteer, and characterized for its effectiveness in the stomach environment. Lactobacillus johnsonii No. 1088 was found to have the strongest acid resistance among several lactobacilli examined (>10% of cells survived at pH 1.0 after 2 h), and such a high acid resistance property was a specific characteristic of this strain of L. johnsonii. When cultured with various virulent bacteria, L. johnsonii No. 1088 inhibited the growth of Helicobacter pylori, Escherichia coli O-157, Salmonella Typhimurium, and Clostridium difficile, in which case its effectiveness was more potent than that of a type strain of L. johnsonii, JCM2012. In addition to its effect in vitro, L. johnsonii No. 1088 inhibited the growth of H. pylori in human intestinal microbiota-associated mice in both its live and lyophilized forms. Moreover, L. johnsonii No. 1088 suppressed gastric acid secretion in mice via decreasing the number of gastrin-positive cells in the stomach. These results taken together suggest that L. johnsonii No. 1088 is a unique lactobacillus having properties beneficial for supporting H. pylori eradication by triple therapy including the use of a proton pump inhibitor (PPI) and also for prophylaxis of gastroesophageal reflux disease possibly caused after H. pylori eradication as a side effect of PPI.

Cerebral low-molecular metabolites influenced by intestinal microbiota: a pilot study.

Recent studies suggest that intestinal microbiota influences gut-brain communication. In this study, we aimed to clarify the influence of intestinal microbiota on cerebral metabolism. We analyzed the cerebral metabolome of germ-free (GF) mice and Ex-GF mice, which were inoculated with suspension of feces obtained from specific pathogen-free mice, using capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS). CE-TOFMS identified 196 metabolites from the cerebral metabolome in both GF and Ex-GF mice. The concentrations of 38 metabolites differed significantly (p < 0.05) between GF and Ex-GF mice. Approximately 10 of these metabolites are known to be involved in brain function, whilst the functions of the remainder are unclear. Furthermore, we observed a novel association between cerebral glycolytic metabolism and intestinal microbiota. Our work shows that cerebral metabolites are influenced by normal intestinal microbiota through the microbiota-gut-brain axis, and indicates that normal intestinal microbiota closely connected with brain health and disease, development, attenuation, learning, memory, and behavior.

Critical role of gut microbiota in the production of biologically active, free catecholamines in the gut lumen of mice.

There is increasing interest in the bidirectional communication between the mammalian host and prokaryotic cells. Catecholamines (CA), candidate molecules for such communication, are presumed to play an important role in the gut lumen; however, available evidence is limited because of the lack of actual data about luminal CA. This study evaluated luminal CA levels in the gastrointestinal tract and elucidated the involvement of gut microbiota in the generation of luminal CA by comparing the findings among specific pathogen-free mice (SPF-M), germ-free mice (GF-M), and gnotobiotic mice. Substantial levels of free dopamine and norepinephrine were identified in the gut lumen of SPF-M. The free CA levels in the gut lumen were lower in GF-M than in SPF-M. The majority of CA was a biologically active, free form in SPF-M, whereas it was a biologically inactive, conjugated form in GF-M. The association of GF-M with either Clostridium species or SPF fecal flora, both of which have abundant ß-glucuronidase activity, resulted in the drastic elevation of free CA. The inoculation of E. coli strain into GF-M induced a substantial amount of free CA, but the inoculation of its mutant strain deficient in the ß-glucuronidase gene did not. The intraluminal administration of DA increased colonic water absorption in an in vivo ligated loop model of SPF-M, thus suggesting that luminal DA plays a role as a proabsorptive modulator of water transport in the colon. These results indicate that gut microbiota play a critical role in the generation of free CA in the gut lumen.

Impact of intestinal microbiota on intestinal luminal metabolome.

Low-molecular-weight metabolites produced by intestinal microbiota play a direct role in health and disease. In this study, we analyzed the colonic luminal metabolome using capillary electrophoresis mass spectrometry with time-of-flight (CE-TOFMS) -a novel technique for analyzing and differentially displaying metabolic profiles- in order to clarify the metabolite profiles in the intestinal lumen. CE-TOFMS identified 179 metabolites from the colonic luminal metabolome and 48 metabolites were present in significantly higher concentrations and/or incidence in the germ-free (GF) mice than in the Ex-GF mice (p < 0.05), 77 metabolites were present in significantly lower concentrations and/or incidence in the GF mice than in the Ex-GF mice (p < 0.05), and 56 metabolites showed no differences in the concentration or incidence between GF and Ex-GF mice. These indicate that intestinal microbiota highly influenced the colonic luminal metabolome and a comprehensive understanding of intestinal luminal metabolome is critical for clarifying host-intestinal bacterial interactions.

Cd1d-dependent regulation of bacterial colonization in the intestine of mice.

The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen-free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I-like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with alpha-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d-/- mice in comparison to WT mice under specific pathogen-free conditions. Germ-free Cd1d-/- mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.

Movement and fixation of intestinal microbiota after administration of human feces to germfree mice.

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the Gammmaproteobacteria group and the family Coriobacteriaceae, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the Clostridium group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.

Dietary nucleic acid and intestinal microbiota synergistically promote a shift in the Th1/Th2 balance toward Th1-skewed immunity.

BACKGROUND: Intestinal microbiota are known to play an important role in the establishment of oral tolerance, thereby protecting the organism from food allergies. Dietary intake of nucleic acid (NA) is also reported to have such an anti-allergic effect; however, one unsolved question is whether or not dietary NA would act through a process of toll-like receptor 9 signaling activated by DNA containing a CpG motif, a well-known sequence leading to immunostimulatory activity. In this study, we focused on the question of whether the addition of dietary NA lacking CpG motifs would allow continued modulation of the Th1/Th2 balance. METHODS: Germ free (GF) and Bifidobacterium-infantis-monoassociated BALB/c mice were maintained on either an NA-free casein diet or on an NA-supplemented casein diet for 4 weeks. Thereafter, both the in vivo anti-casein antibody levels and in vitro splenocyte cytokine secretion pattern were evaluated. RESULTS: Feeding with a casein diet elicited a substantial increase in the serum anti-casein-specific IgG1, IgG2a, and IgE levels of GF mice fed the NA free-diet. The in vitro cytokine production profile showed that enhanced IL-4 production in the GF mice fed the NA free-diet was markedly reduced by the supplementation with dietary NA in both the GF and B.-infantis-monoassociated mice. In addition, IFN-gamma secretion increased in the B.-infantis-reconstituted mice fed the diet containing NA. CONCLUSIONS: These results suggest that dietary intake of NA devoid of CpG motifs may prevent the development of allergies via acceleration of Th1-dominant immunity.

Allergic symptoms and microflora in schoolchildren.

We studied 867 junior high school children and administered a questionnaire documenting allergic symptoms and environmental variables, and measured Immunoglobulin E serum levels and the immunoglobulin G titers of serum antibody to microflora. A total of 716 subjects were ultimately used for statistics; those with at least two of the following allergic symptoms: asthma, rhinitis, eczema, or food allergy, showed significantly higher IgG titers to Bactroides vulgatus than other groups. This finding suggests that a species of the Bacteroides genus of the intestinal microflora tends to affect the gut issues, but further studies are needed to clarify this.

Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice.

Indigenous microbiota have several beneficial effects on host physiological functions; however, little is known about whether or not postnatal microbial colonization can affect the development of brain plasticity and a subsequent physiological system response. To test the idea that such microbes may affect the development of neural systems that govern the endocrine response to stress, we investigated hypothalamic-pituitary-adrenal (HPA) reaction to stress by comparing germfree (GF), specific pathogen free (SPF) and gnotobiotic mice. Plasma ACTH and corticosterone elevation in response to restraint stress was substantially higher in GF mice than in SPF mice, but not in response to stimulation with ether. Moreover, GF mice also exhibited reduced brain-derived neurotrophic factor expression levels in the cortex and hippocampus relative to SPF mice. The exaggerated HPA stress response by GF mice was reversed by reconstitution with Bifidobacterium infantis. In contrast, monoassociation with enteropathogenic Escherichia coli, but not with its mutant strain devoid of the translocated intimin receptor gene, enhanced the response to stress. Importantly, the enhanced HPA response of GF mice was partly corrected by reconstitution with SPF faeces at an early stage, but not by any reconstitution exerted at a later stage, which therefore indicates that exposure to microbes at an early developmental stage is required for the HPA system to become fully susceptible to inhibitory neural regulation. These results suggest that commensal microbiota can affect the postnatal development of the HPA stress response in mice.

The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease.

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti-kappa+ phenotype, which represents plasma cell-like B cells. Moreover, treatment of those B. vulgatus-implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti-kappa+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.

Lactobacillus gasseri OLL2716 as a probiotic in clarithromycin-resistant Helicobacter pylori infection.

BACKGROUNDS AND AIM: Clarithromycin (CAM)-resistant Helicobacter pylori sometimes offers serious problems with eradication by antibiotics. The aim of this study was to determine whether a probiotic can be an alternative therapy in CAM-resistant Hp infection. METHODS: The effects of Lactobacillus gasseri (strain OLL2716) on the growth of CAM-susceptible and CAM-resistant H. pylori and interleukin (IL)-8 production provoked by these strains were examined by in vitro experiments. Moreover, mice were infected with these CAM-susceptible or CAM-resistant H. pylori, and were treated with CAM or L. gasseri. RESULTS: In vitro experiments demonstrated that L. gasseri inhibited the growth of H. pylori and suppressed H. pylori-associated IL-8 production. Such effects were noted in CAM-resistant and CAM-susceptible H. pylori. Similarly, in an in vivo model of H. pylori infection, H. pylori colonization was significantly decreased by L. gasseri. CONCLUSION: Therefore, L. gasseri was found to act as a probiotic in CAM-resistant H. pylori infection.

Bacteroides ovatus as the predominant commensal intestinal microbe causing a systemic antibody response in inflammatory bowel disease.

To clarify what bacterial species of commensal intestinal microbes are recognized as the antigens that induce a serum antibody response in patients with inflammatory bowel disease (IBD), 72 subjects consisting of 12 Crohn's disease patients, 30 ulcerative colitis patients, and 30 healthy volunteers were examined for their titers of serum antibody to these intestinal bacteria. In IBD patients, as a result, significant elevations of both the immunoglobulin G (IgG) and IgA titers to Bacteroides ovatus were found. Immunoblotting showed that a definite 19.5-kDa band of B. ovatus was bound to the serum antibody raised in IBD patients. It was thus concluded that B. ovatus causes serum antibody responses in IBD patients, and a 19.5-kDa molecule of this bacterium appears to be the responsible antigen, although the role of this event in pathogenesis remains unclear.

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model.

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.