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.

D-lactate is normally present in the blood of mammals at nanomolar concentrations due to methylglyoxal metabolism; millimolar d-lactate concentrations can arise due to excess gastrointestinal microbial production. Grain overload in ruminants, short-bowel syndrome in humans, and diarrhea in calves can all result in profound D-lactic acidemia, with remarkably similar neurological manifestations. In the past, D-lactate was thought to be excreted mainly in the urine, and metabolized slowly by the enzyme d-alpha-hydroxy acid dehydrogenase. More recent studies reported that mammals have a relatively high capacity for D-lactate metabolism and identified a putative mammalian D-lactate dehydrogenase. A growing body of literature is also emerging describing subclinical elevation of D-lactate as an indicator of sepsis and trauma. This article describes advances in the understanding of D-lactate metabolism, D-lactic acidosis in ruminants and humans, and subclinical elevation of d-lactate.

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

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.

Colonic epithelial cells are constantly exposed to high levels of bacterial DNA in the intestinal lumen and must recognize and respond appropriately to pathogens, while they maintain a tolerance to nonpathogenic commensal bacterial strains. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9). The aim of this study was to investigate TLR9 expression and localization in colonic epithelial cells under basal conditions and in response to bacterial DNA. HT-29 cells were exposed to DNA from various strains of commensal and pathogenic microbes. Recognition of microbial components and discrimination of harmful pathogens from commensal bacteria and from self are fundamental elements of the innate immune system. Tolllike receptors (TLRs) are responsible for recognizing various pathogen-associated molecular patterns, including lipoproteins (Toll-like receptor 2 [TLR2]), lipopolysaccharides (TLR4), and flagellin (TLR5). Bacterial DNA contains unmethylated 2Ј-deoxyribo(cytidine-phosphate-guanine) (CpG) dinucleotides flanked by specific sequences that activate the vertebrate innate immune system through TLR9. Bacterial DNA differs from mammalian DNA by its 20-fold-greater frequency of unmethylated CpG sequences (32). These sequences activate a signaling cascade via transcription factors NF-B and AP-1 and stimulate the proliferation of B cells and the secretion of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and tumor necrosis factor alpha) required to eliminate an invading pathogen (15,33). Intestinal epithelial cells are constantly exposed to high levels of bacterial DNA and must recognize and respond appropriately to the presence of pathogenic bacteria. These cells interact with microbes in the lumen, and TLR signaling is a key component of communication between intes-