Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.

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A breakdown in intestinal barrier function and increased bacterial translocation are key events in the pathogenesis of sepsis and liver disease. Altering gut microflora with noninvasive and immunomodulatory probiotic organisms has been proposed as an adjunctive therapy to reduce the level of bacterial translocation and prevent the onset of sepsis. The purpose of this study was to determine the efficacy of a probiotic compound in attenuating hepatic and intestinal injury in a mouse model of sepsis. Wild-type and interleukin-10 (IL-10) gene-deficient 129 Sv/Ev mice were fed the probiotic compound VSL#3 for 7 days. To induce sepsis, the mice were injected with lipopolysaccharide (LPS) and D-galactosamine (GalN) in the presence and absence of the peroxisome proliferator-activated receptor gamma (PPAR␥) inhibitor GW9662. The mice were killed after 6 hours, and their colons were removed for the measurement of the cytokine production and epithelial function. The functional permeability was assessed by the mannitol movement and cyclic adenosine monophosphate-dependent chloride secretion in tissue mounted in Ussing chambers. The livers were analyzed for bacterial translocation, cytokine production, histological injury, and PPAR␥ levels. The tissue levels of tumor necrosis factor alpha, interferon gamma, IL-6, and IL-12p35 ribonucleic acid were measured by semiquantitative reverse transcription polymerase chain reaction. L iver dysfunction and failure contribute to the high mortality rates seen in patients with Gram-negative sepsis. The presence of lipopolysaccharide (LPS) from Gram-negative bacteria in the systemic circulation results in the activation of the innate immune system and the secretion of high levels of proinflammatory cytokines. In animal models, LPS challenge can induce a systemic reaction resulting in a sepsis-like condition characterized by fever, hypotension, and widespread tissue damage. D-Galactosamine (GalN) increases the susceptibility of mice to LPS-induced shock by impairing liver metabolism. 1 Challenging mice with low doses of LPS in conjunction with GalN results in massive liver apoptosis and increased mortality.Tumor necrosis factor alpha (TNF-␣) plays a central role in the overwhelming systemic inflammatory response to LPS. 2 However, the complete blockade of TNF-␣ production does not improve survival in animals or humans 3 The activation of nuclear factor kappa B (NF-B) has been shown to play a key role in the pathogenesis of sepsis and is a pivotal step in the regulation of several immune and proinflammatory genes, including TNF-␣. 4 The modulation of NF-B activity has been proposed as a strategy for reducing the mortality associated with sepsis. Peroxisome proliferator-activated receptor gamma (PPAR␥) is a nuclear hormone receptor and transcription

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase-PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.

Probiotics have been shown to reduce the incidence of colon cancer in animal models. The mechanisms responsible for this activity are poorly defined. Conjugated linoleic acids (CLA) are a group of isomers of linoleic acid (LA) possessing anti-inflammatory and anticarcinogenic properties, which can be produced from LA by certain bacterial strains. In this study, the ability of probiotic bacteria to exert anticarcinogenic effects through the production of CLA was assessed. Incubation of probiotic bacteria (VSL3, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Bifidobacterium breve, B. infantis, B. longum, and Streptococcus thermophilus) in the presence of LA yielded CLA production as measured by gas chromatography. Conditioned medium, containing probiotic-produced CLA, reduced viability and induced apoptosis of HT-29 and Caco-2 cells, as assessed by MTT assay and DNA laddering, respectively. Western blotting demonstrated an increased expression of PPARgamma in cells treated with conditioned medium compared with LA alone. Incubation of murine feces with LA after administering VSL3 yielded 100-fold more CLA than feces collected prior to VSL3 feeding. This study supports a role for supplemental probiotics as a strategy both for attenuating inflammation and for preventing colon cancer.