Early intervention with Bifidobacterium lactis NCC2818 modulates the host-microbe interface independent of the sustained changes induced by the neonatal environment.

Inflammatory and metabolic diseases can originate during early-life and have been correlated with shifts in intestinal microbial ecology. Here we demonstrate that minor environmental fluctuations during the early neonatal period had sustained effects on the developing porcine microbiota and host-microbe interface. These inter-replicate effects appear to originate during the first day of life, and are likely to reflect very early microbiota acquisition from the environment. We statistically link early systemic inflammation with later local increases in inflammatory cytokine (IL-17) production, which could have important enteric health implications. Immunity, intestinal barrier function, host metabolism and host-microbiota co-metabolism were further modified by Bifidobacterium lactis NCC2818 supplementation, although composition of the in situ microbiota remained unchanged. Finally, our robust model identified novel, strong correlations between urinary metabolites (eg malonate, phenylacetylglycine, alanine) and mucosal immunoglobulin (IgM) and cytokine (IL-10, IL-4) production, thus providing the possibility of the development of urinary 'dipstick' tests to assess non-accessible mucosal immune development and identify early precursors (biomarkers) of disease. These results have important implications for infants exposed to neonatal factors including caesarean delivery, antibiotic therapy and delayed discharge from hospital environments, which may predispose to the development of inflammatory and metabolic diseases in later life.

Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype.

The postnatal environment, including factors such as weaning and acquisition of the gut microbiota, has been causally linked to the development of later immunological diseases such as allergy and autoimmunity, and has also been associated with a predisposition to metabolic disorders. We show that the very early-life environment influences the development of both the gut microbiota and host metabolic phenotype in a porcine model of human infants. Farm piglets were nursed by their mothers for 1 day, before removal to highly controlled, individual isolators where they received formula milk until weaning at 21 days. The experiment was repeated, to create two batches, which differed only in minor environmental fluctuations during the first day. At day 1 after birth, metabolic profiling of serum by (1)H nuclear magnetic resonance spectroscopy demonstrated significant, systemic, inter-batch variation which persisted until weaning. However, the urinary metabolic profiles demonstrated that significant inter-batch effects on 3-hydroxyisovalerate, trimethylamine-N-oxide and mannitol persisted beyond weaning to at least 35 days. Batch effects were linked to significant differences in the composition of colonic microbiota at 35 days, determined by 16 S pyrosequencing. Different weaning diets modulated both the microbiota and metabolic phenotype independently of the persistent batch effects. We demonstrate that the environment during the first day of life influences development of the microbiota and metabolic phenotype and thus should be taken into account when interrogating experimental outcomes. In addition, we suggest that intervention at this early time could provide 'metabolic rescue' for at-risk infants who have undergone aberrant patterns of initial intestinal colonisation.

Dietary supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production in lymphoid-associated tissues but increases systemic antibodies in healthy neonates.

Weaning is associated with a major shift in the microbial community of the intestine, and this instability may make it more acquiescent than the adult microbiota to long-term changes. Modulation achieved through dietary interventions may have potentially beneficial effects on the developing immune system, which is driven primarily by the microbiota. The specific aim of the present study was to determine whether immune development could be modified by dietary supplementation with the human probiotic Bifidobacterium lactis NCC2818 in a tractable model of weaning in infants. Piglets were reared by their mothers before being weaned onto a solid diet supplemented with B. lactis NCC2818, while sibling controls did not receive supplementation. Probiotic supplementation resulted in a reduction in IgA (P<0·0005) and IgM (P<0·009) production by mucosal tissues but had no effect on IgG production (P>0·05). Probiotic-supplemented pigs had more mast cells than unsupplemented littermates (P<0·0001), although numbers in both groups were low. In addition, the supplemented piglets made stronger serum IgG responses to fed and injected antigens (P<0·05). The present findings are consistent with B. lactis NCC2818 reducing intestinal permeability induced by weaning, and suggest that the piglet is a valuable intermediate between rodent models and human infants. The results also strongly suggest that measures of the effect of probiotic supplementation on the immune system need to be interpreted carefully as proxy measures of health benefit. However, they are useful in developing an understanding of the mechanism of action of probiotic strains, an important factor in predicting favourable health outcomes of nutritional intervention.

Weaning diet induces sustained metabolic phenotype shift in the pig and influences host response to Bifidobacterium lactis NCC2818.

BACKGROUND: The process of weaning causes a major shift in intestinal microbiota and is a critical period for developing appropriate immune responses in young mammals. OBJECTIVE: To use a new systems approach to provide an overview of host metabolism and the developing immune system in response to nutritional intervention around the weaning period. DESIGN: Piglets (n=14) were weaned onto either an egg-based or soya-based diet at 3 weeks until 7 weeks, when all piglets were switched onto a fish-based diet. Half the animals on each weaning diet received Bifidobacterium lactis NCC2818 supplementation from weaning onwards. Immunoglobulin production from immunologically relevant intestinal sites was quantified and the urinary (1)H NMR metabolic profile was obtained from each animal at post mortem (11 weeks). RESULTS: Different weaning diets induced divergent and sustained shifts in the metabolic phenotype, which resulted in the alteration of urinary gut microbial co-metabolites, even after 4 weeks of dietary standardisation. B lactis NCC2818 supplementation affected the systemic metabolism of the different weaning diet groups over and above the effects of diet. Additionally, production of gut mucosa-associated IgA and IgM was found to depend upon the weaning diet and on B lactis NCC2818 supplementation. CONCLUSION: The correlation of urinary (1)H NMR metabolic profile with mucosal immunoglobulin production was demonstrated, thus confirming the value of this multi-platform approach in uncovering non-invasive biomarkers of immunity. This has clear potential for translation into human healthcare with the development of urine testing as a means of assessing mucosal immune status. This might lead to early diagnosis of intestinal dysbiosis and with subsequent intervention, arrest disease development. This system enhances our overall understanding of pathologies under supra-organismal control.

Nigella sativa (black cumin) seed extract alleviates symptoms of allergic diarrhea in mice, involving opioid receptors.

The incidence of food hypersensitivity and food allergies is on the rise and new treatment approaches are needed. We investigated whether N. sativa, one of its components, thymoquinone, or synthetic opioid receptor (OR)-agonists can alleviate food allergy. Hence, ovalbumin (OVA)-sensitized BALB/c-mice were pre-treated either with a hexanic N. sativa seed extract, thymoquinone, kappa-(U50'4889) or mu-OR-agonists (DAMGO) and subsequently challenged intra-gastrically with OVA. All 4 treatments significantly decreased clinical scores of OVA-induced diarrhea. N. sativa seed extract, thymoquinone, and U50'488 also decreased intestinal mast cell numbers and plasma mouse mast cell protease-1 (MMCP-1). DAMGO, in contrast, had no effect on mast cell parameters but decreased IFNγ, IL-4, IL-5, and IL-10 concentration after ex vivo re-stimulation of mesenteric lymphocytes. The effects on allergy symptoms were reversible by OR-antagonist pre-treatment, whereas most of the effects on immunological parameter were not. We demonstrate that N. sativa seed extract significantly improves symptoms and immune parameters in murine OVA-induced allergic diarrhea; this effect is at least partially mediated by thymoquinone. ORs may also be involved and could be a new target for intestinal allergy symptom alleviation. N. sativa seed extract seems to be a promising candidate for nutritional interventions in humans with food allergy.

Probiotic Lactobacillus reuteri alleviates the response to gastric distension in rats.

Probiotic lactic acid bacteria have been reported to alleviate symptoms in patients with irritable bowel syndrome. However, they have not been tested for use in functional gastric disease. We therefore investigated if strains previously shown to protect from response to colorectal distension (CRD) in rats also modulate response to gastric distension (GD). Healthy, male Sprague-Dawley rats were treated with viable, heat-killed, gamma-irradiated Lactobacillus reuteri or viable Lactobacillus plantarum wild type (WT), L. plantarum Dlt¯mutant, conditioned medium or medium control (9 d), and subjected to GD under anesthesia using an i.g. Teflon catheter. Effects were measured by heart rate (HR) changes during noxious distension (60 mm Hg) compared to baseline HR values. We also investigated the localization of viable, green fluorescent protein-transfected bacteria in the stomach mucosa. Viable L. reuteri decreased the bradycardia induced by noxious GD compared to placebo controls (P < 0.001). Heat-killed or gamma-irradiated L. reuteri and conditioned medium did not have a protective effect in GD. Viable L. plantarum WT and Dlt¯mutant, previously shown to be effective antinociceptive agents in CRD, showed no protective effect in GD. All viable bacteria were associated with the pars glandularis of the rat stomach. Thus, we conclude that the antinociceptive mechanisms of action of probiotic bacteria differ between the stomach and the colon. Symptom alleviation cannot be attributed to the localization of the bacteria in the stomach. Information derived from effects of CRD cannot be extrapolated to effects in the stomach, which are likely to be strain and organ specific.

A metabolic system-wide characterisation of the pig: a model for human physiology.

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major inter-species differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.

Effect of oral lactulose on clinical and immunohistochemical parameters in patients with inflammatory bowel disease: a pilot study.

BACKGROUND: The prebiotic potential of lactulose is well established and preclinical studies demonstrated a protective effect of lactulose in murine models of colitis. The aim of the present study was to investigate the clinical and histological efficacy of lactulose in patients with inflammatory bowel disease (IBD), for which probiotic therapy yielded promising results. METHODS: Patients were treated with standard medication alone or combined with 10 g lactulose daily as adjuvant therapy for 4 months. Clinical efficacy of treatment was assessed using clinical activity indices, a quality of life index (IBDQ), endoscopic scores, defecation frequency and monitoring corticosteroid medication. Orsomucoid, alpha1-antitrypsin and other laboratory parameters were determined. In addition, in some participants colonic biopsies were analyzed with haematoxylin-eosin staining or with antibodies against HLA-DR, CD68, IgA and CD3, and evaluated systematically. All measurements were performed both at enrollment and at the end of the trial. RESULTS: 14 patients presenting ulcerative colitis (UC) and 17 patients presenting Crohn's disease (CD), most of them in a clinically active state, were enrolled in this pilot study. After 4 month no significant improvement of clinical activity index, endoscopic score or immunohistochemical parameters was observed in CD or UC patients receiving lactulose in comparison to the control group. However, significant improvement of quality of life was observed in UC patients receiving lactulose compared to the control group (p = 0.04). CONCLUSION: The findings of the present pilot study indicate that oral lactulose has no beneficial effects in IBD patients in particular with regard to clinical activity, endoscopic score or immunohistochemical parameters. The importance of the beneficial effect of lactulose in UC patients regarding the quality of life needs further evaluation in larger controlled clinical trials.

Effect of orally administered probiotic E. coli strain Nissle 1917 on intestinal mucosal immune cells of healthy young pigs.

Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients; however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered orally to young pigs at two concentrations (10(9) and 10(11)CFU/d for 21 days) on the gut-associated lymphatic tissue. This probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin, prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (10(11) CFU/d) showing an increase in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple modulation of immune cell distribution.