Changes in bile acids, FGF-19 and sterol absorption in response to bile salt hydrolase active L. reuteri NCIMB 30242.

The size and composition of the circulating bile acid (BA) pool are important factors in regulating the human gut microbiota. Disrupted regulation of BA metabolism is implicated in several chronic diseases. Bile salt hydrolase (BSH)-active Lactobacillus reuteri NCIMB 30242, previously shown to decrease LDL-cholesterol and increase circulating BA, was investigated for its dose response effect on BA profile in a pilot clinical study. Ten otherwise healthy hypercholesterolemic adults, recruited from a clinical trial site in London, ON, were randomized to consume delayed release or standard release capsules containing L. reuteri NCIMB 30242 in escalating dose over 4 weeks. In another aspect, 4 healthy normocholesterolemic subjects with LDL-C below 3.4 mmol/l received delayed release L. reuteri NCIMB 30242 at a constant dose over 4 weeks. The primary outcome measure was the change in plasma BA profile over the intervention period. Additional outcomes included circulating fibroblast growth factor (FGF)-19, plant sterols and LDL-cholesterol as well as fecal microbiota and bsh gene presence. After one week of intervention subjects receiving delayed release L. reuteri NCIMB 30242 increased total BA by 1.13 ± 0.67 µmol/l (P = 0.02), conjugated BA by 0.67 ± 0.39 µmol/l (P = 0.02) and unconjugated BA by 0.46 ± 0.43 µmol/l (P = 0.07), which represented a greater than 2-fold change relative to baseline. Increases in BA were largely maintained post-week 1 and were generally correlated with FGF-19 and inversely correlated with plant sterols. This is the first clinical support showing that a BSH-active probiotic can significantly and rapidly influence BA metabolism and may prove useful in chronic diseases beyond hypercholesterolemia.

Bacterial bile metabolising gene abundance in Crohn's, ulcerative colitis and type 2 diabetes metagenomes.

We performed an analysis to determine the importance of bile acid modification genes in the gut microbiome of inflammatory bowel disease and type 2 diabetic patients. We used publicly available metagenomic datasets from the Human Microbiome Project and the MetaHIT consortium, and determined the abundance of bile salt hydrolase gene (bsh), 7 alpha-dehydroxylase gene (adh) and 7-alpha hydroxysteroid dehydrogenase gene (hsdh) in fecal bacteria in diseased populations of Crohn's disease (CD), Ulcerative Colitis (UC) and Type 2 diabetes mellitus (T2DM). Phylum level abundance analysis showed a significant reduction in Firmicute-derived bsh in UC and T2DM patients but not in CD patients, relative to healthy controls. Reduction of adh and hsdh genes was also seen in UC and T2DM patients, while an increase was observed in the CD population as compared to healthy controls. A further analysis of the bsh genes showed significant differences in the correlations of certain Firmicutes families with disease or healthy populations. From this observation we proceeded to analyse BSH protein sequences and identified BSH proteins clusters representing the most abundant strains in our analysis of Firmicute bsh genes. The abundance of the bsh genes corresponding to one of these protein clusters was significantly reduced in all disease states relative to healthy controls. This cluster includes bsh genes derived from Lachospiraceae, Clostridiaceae, Erysipelotrichaceae and Ruminococcaceae families. This metagenomic analysis provides evidence of the importance of bile acid modifying enzymes in health and disease. It further highlights the importance of identifying gene and protein clusters, as the same gene may be associated with health or disease, depending on the strains expressing the enzyme, and differences in the enzymes themselves.

Emerging science of the human microbiome.

The human gastrointestinal tract hosts a large number of microbial cells which exceed their mammalian counterparts by approximately 3-fold. The genes expressed by these microorganisms constitute the gut microbiome and may participate in diverse functions that are essential to the host, including digestion, regulation of energy metabolism, and modulation of inflammation and immunity. The gut microbiome can be modulated by dietary changes, antibiotic use, or disease. Different ailments have distinct associated microbiomes in which certain species or genes are present in different relative quantities. Thus, identifying specific disease-associated signatures in the microbiome as well as the factors that alter microbial populations and gene expression will lead to the development of new products such as prebiotics, probiotics, antimicrobials, live biotherapeutic products, or more traditional drugs to treat these disorders. Gained knowledge on the microbiome may result in molecular lab tests that may serve as personalized tools to guide the use of the aforementioned products and monitor interventional progress.

The human microbiome and bile acid metabolism: dysbiosis, dysmetabolism, disease and intervention.

INTRODUCTION: Recent evidence indicates that the human gut microbiome plays a significant role in health and disease. Dysbiosis, defined as a pathological imbalance in a microbial community, is becoming increasingly appreciated as a 'central environmental factor' that is both associated with complex phenotypes and affected by host genetics, diet and antibiotic use. More recently, a link has been established between the dysmetabolism of bile acids (BAs) in the gut to dysbiosis. AREAS COVERED: BAs, which are transformed by the gut microbiota, have been shown to regulate intestinal homeostasis and are recognized as signaling molecules in a wide range of metabolic processes. This review will examine the connection between BA metabolism as it relates to the gut microbiome and its implication in health and disease. EXPERT OPINION: A disrupted gut microbiome, including a reduction of bile salt hydrolase (BSH)-active bacteria, can significantly impair the metabolism of BAs and may result in an inability to maintain glucose homeostasis as well as normal cholesterol breakdown and excretion. To better understand the link between dysbiosis, BA dysmetabolism and chronic degenerative disease, large-scale metagenomic sequencing studies, metatranscriptomics, metaproteomics and metabolomics should continue to catalog functional diversity in the gastrointestinal tract of both healthy and diseased populations. Further, BSH-active probiotics should continue to be explored as treatment options to help restore metabolic levels.

Improvement of gastrointestinal health status in subjects consuming Lactobacillus reuteri NCIMB 30242 capsules: a post-hoc analysis of a randomized controlled trial.

OBJECTIVE: Gastrointestinal (GI) symptoms are conditions that are frequently observed in clinical practice. A post-hoc analysis has been undertaken to evaluate the effect of bile salt hydrolase-active L. reuteri NCIMB 30242 on GI health status based on Rome III questionnaire response in otherwise healthy hypercholesterolemic subjects. RESEARCH DESIGN/METHODS: A total of 127 subjects received either L. reuteri NCIMB 30242 or placebo capsules over a 9-week intervention in a randomized, double-blind, placebo-controlled, parallel-arm, multicenter study. Subjects were asked to complete the Rome III diagnostic GI questionnaire prior to the baseline and end point visits of the clinical study. MAIN OUTCOME MEASURE: GI health status was evaluated, per questionnaire, by assessing all questions with 5- or 7-point response scales for symptoms of the stomach and intestines. RESULTS: Subjects receiving L. reuteri NCIMB 30242 reported significant improvements in general GI health status (p = 0.029) and in symptoms related to diarrhea (p = 0.018) as compared to placebo over the intervention period. Further, a greater proportion of L. reuteri-treated subjects showed improved general GI health status (p = 0.042) and improved diarrhea symptoms (p = 0.03). CONCLUSIONS: L. reuteri NCIMB 30242 capsules appear to be well tolerated and potentially beneficial for GI health status. Further clinical investigation is warranted for the treatment of functional GI disorders.

Novel nitric oxide producing probiotic wound healing patch: preparation and in vivo analysis in a New Zealand white rabbit model of ischaemic and infected wounds.

The treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we report design and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing. Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has been designed and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand white rabbit model for ischaemic wound healing. Kaplan-Meier survival curves showed increased wound closure with gNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113). Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likely to close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355), and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This study may provide an effective, safe and less costly alternative for treating chronic wounds.

Surface-attached amphipathic peptides reduce hemorrhage in vivo.

BACKGROUND: The leading causes of death for trauma patients in civilian and combat settings are traumatic brain injury and uncontrolled hemorrhage, respectively. This study examines the hemostatic activity of an ideal amphipathic peptide (IAP) attached to a biocompatible surface. METHODS: Procoagulant properties of IAP attached to a surface were first tested, in vitro, using factor Xa and thrombin generation assays and thromboelastography. Rabbits and swine were used for in vivo studies. Injuries were performed using scalpel blade 11, and free bleeding was allowed for five seconds. While bleeding, IAP coupled to a hydrogel or QuikClot were applied to the wound and the time was recorded until bleeding stopped. RESULTS: Results show that when IAP is attached to a surface, both factor IXa and factor Xa activities are promoted. Thromboelastography shows that surface-attached IAP results in earlier onset and stronger clot formation. In rabbits, the incorporation of IAP onto a biocompatible hydrogel reduces bleeding times by 40% (p < 0.03). In pigs, bleeding times are reduced 30% to 50% (p < 0.02) by surface-coupled IAP. Finally, using a rabbit liver laceration model, the properties of surface-coupled IAP are less damaging when compared with QuikClot, a currently used material in external hemorrhagic injuries. CONCLUSIONS: This study provides a relevant proof of concept for the development of IAP coupled to a biocompatible surface as a hemostatic agent, that is potentially safer than the commercially available QuikClot.

A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis.

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.

Characterization of an ideal amphipathic peptide as a procoagulant agent.

On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000-12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys(7)Leu(15) composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (gamma-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.

Focal arterial inflammation is augmented in mice with a deficiency of the protein C gene.

Increased risk of thrombosis, with propitious conditions for fibrin deposition, along with upregulation of inflammation, are important factors that enhance plaque formation in atherosclerosis. Evidence supporting the role of anticoagulant protein C (PC) as an inflammatory agent has emerged, supplementing its well-known function as an anticoagulant. Thus, we sought to examine whether a PC deficiency would lead to an enhanced response to an acute arterial hyperplasic challenge. The presentation of early arterial inflammation was studied using a copper/silicone arterial cuff model of accelerated focal neointimal remodeling in mice with a heterozygous total deficiency of PC (PC+/-). Increased inflammation, cell proliferation, cell migration, fibrin elevation, and tissue necrosis were observed in the treated arteries of PC+/- mice, as compared to arteries of equally challenged age- and gender-matched WT mice. These results indicate that PC+/- mice subjected to this challenge displayed enhanced focal arterial inflammation and thrombosis, leading to larger neointimas and subsequent localized occlusion, as compared to their WT counterparts.

A protein C deficiency exacerbates inflammatory and hypotensive responses in mice during polymicrobial sepsis in a cecal ligation and puncture model.

During the systemic inflammatory state induced by sepsis, the potential for coagulopathy exists because of up-regulation of natural procoagulants and anti-fibrinolytics, and down-regulation of natural anti-coagulants, with protein C (PC) being a critical example of the latter case. PC functions as an anti-coagulant, profibrinolytic, and anti-inflammatory agent, and, thus, its administration or deficiency may affect the course and outcome of sepsis in patients. In this study, a cecal ligation and puncture model of septic peritonitis was applied to wild-type mice and littermates with a targeted heterozygous deficiency of PC (PC(+/-)) to characterize the importance of a PC-deficiency on polymicrobial sepsis. An enhanced mortality rate was found to accompany a PC deficiency. Plasma cytokines, as well as organ-specific expression of cytokine transcripts, were elevated in PC(+/-) mice. No signs of severe disseminated intravascular coagulation (DIC) were observed in wild-type or PC(+/-) mice, as indicated by an increase in fibrinogen levels and the invariability of platelet counts after cecal ligation and puncture. Consumption of coagulation factors was similar in both genotypes and a decrease in the PC mRNA and protein levels was more prominent in PC(+/-) mice. Renal and organ muscle damage was enhanced in PC(+/-) mice, as shown by increases in plasma blood urea nitrogen, creatinine, and creatinine kinase. Hypotension and bradycardia were more enhanced in PC(+/-) mice than in wild-type mice, thus provoking a more severe septic shock response. Thus, the hemodynamic role of PC during sepsis is of critical importance to the outcome of the disease.