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Every day, up to 1 g of cholesterol, composed of the unabsorbed dietary cholesterol, the biliary cholesterol secretion, and cholesterol of cells sloughed from the intestinal epithelium, enters the colon. All cholesterol arriving in the large intestine can be metabolized by the colonic bacteria. Cholesterol is mainly converted into coprostanol, a non-absorbable sterol that is excreted in the feces. Interestingly, cholesterol-to-coprostanol conversion in human populations is variable, with a majority of high converters and a minority of low or inefficient converters. Two major pathways have been proposed, one involving the direct stereospecific reduction of the Δ5 double bond direct while the indirect pathway involves the intermediate formation of 4-cholelesten-3-one and coprostanone. Despite the fact that intestinal cholesterol conversion was discovered more than a century ago, only a few cholesterol-to-coprostanol-converting bacterial strains have been isolated and characterized. Moreover, the responsible genes were mainly unknown until recently. Interestingly, cholesterol-to-coprostanol conversion is highly regulated by the diet. Finally, this gut bacterial metabolism has been linked to health and disease, and recent evidence suggests it could contribute to lower blood cholesterol and cardiovascular risks.

ObjectiveBased on animal models and human studies, there is now strong suspicion that host/microbiota mutualism in the context of gut microbial dysbiosis could influence immunity and multiple sclerosis (MS) evolution. Our goal was to seek evidence of deregulated microbiota-induced systemic immune responses in patients with MS.MethodsWe investigated gut and systemic commensal-specific antibody responses in healthy controls (n = 32), patients with relapsing-remitting MS (n = 30), and individuals with clinically isolated syndromes (CISs) (n = 15). Gut microbiota composition and diversity were compared between controls and patients by analysis of 16S ribosomal ribonucleic acid (rRNA) sequencing. Autologous microbiota and cultivable bacterial strains were used in bacterial flow cytometry assays to quantify autologous serum IgG and secretory IgA responses to microbiota. IgG-bound bacteria were sorted by flow cytometry and identified using 16S rRNA sequencing.ResultsWe show that commensal-specific gut IgA responses are drastically reduced in patients with severe MS, disease severity being correlated with the IgA-coated fecal microbiota fraction (r = −0.647, p < 0.0001). At the same time, IgA-unbound bacteria elicit qualitatively broad and quantitatively increased serum IgG responses in patients with MS and CIS compared with controls (4.1% and 2.5% vs 1.9%, respectively, p < 0.001).ConclusionsGut and systemic microbiota/immune homeostasis are perturbed in MS. Our results argue that defective IgA responses in MS are linked to a breakdown of systemic tolerance to gut microbiota leading to an enhanced triggering of systemic IgG immunity against gut commensals occurring early in MS.

Owing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMT) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24hours, fresh samples should not be exposed to temperatures above 20°C, and refrigeration at 4°C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufcient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a -80°C standard freezer and then rapidly thawed at 37°C, retained the best revivifcation potential as proven by 16S rRNA profles, metabolomic fngerprints, and fow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efcient in preserving viability of lyophilized transplants, either in their crude or purifed form, an option that can be attractive for fecal transplant biobanking and oral formulation. Te human intestinal microbiota ensures several essential functions that support maintenance of health and well-being. One of them is the prevention of colonization with allochtonous microbes including pathogens, also known as barrier efect or competitive exclusion. Tis is dependent on the maintenance of ecological homeostasis of the gut ecosystem1 . Another well-established function of the intestinal microbiota is its contribution to immune maturation and maintenance of a balanced immune tolerance and reactivity. It is mediated by a constant and intimate crosstalk between intestinal microbiota and the gut associated lymphoid tissues2 . Here again, a balanced microbiota-host crosstalk is dependent on the maintenance of ecological homeostasis of the gut ecosystem. In healthy adults, microbiota composition is individual specifc and for a given person, it remains fairly stable over time3 . Upon mild stress such as exposure to specifc nutrients or diverse conditions of modern daily life or clinical practices, including antibiotics, the intestinal microbiota can be temporarily altered but will eventually return to its original composition; it is resilient4 . Nonetheless, marked alterations of dietary habits, environmental exposures, drug therapy or surgical interventions will induce alterations of the intestinal microbiota that may exceed the resilience capacity5,6 and lead to onset of dysbiosis, i.e. distorted intestinal microbiota composition. Dysbiosis is now acknowledged as a common feature of numerous non-infectious diseases of modern societies, which have had a constant increase in incidence since the middle of the past century. Obesity is one of these

Background: The mechanisms responsible for calorie restriction-induced improvement in insulin sensitivity have not been fully elucidated. Greater insight can be achieved through deep biological phenotyping of subjects undergoing calorie restriction, and integration of big data.\ud \ud Materials and Methods: An integrative approach was applied to investigate associations between change in insulin sensitivity and factors from host, microbiota and lifestyle after a 6-week calorie restriction period in 27 overweight or obese adults (ClinicalTrials.gov: NCT01314690). Partial least squares regression was used to determine associations of change (week 6 – baseline) between insulin sensitivity markers and lifestyle factors (diet and physical activity), subcutaneous adipose tissue (sAT) gene expression, metabolomics in serum, urine and feces, and gut microbiota composition. ScaleNet, a network learning approach based on spectral consensus strategy (SCS, developed by us) was used for reconstruction of biological networks.\ud \ud Results: A spectrum of variables from lifestyle factors (10 nutrients), gut microbiota (10 metagenomics species) and host multi-omics (metabolic features: 84 from serum, 73 from urine, and 131 from feces; and 257 subcutaneous adipose tissue gene probes) most associated with insulin sensitivity were identified. Biological network reconstruction using SCS, highlighted links between changes in insulin sensitivity, serum branched chain amino acids, sAT genes involved in endoplasmic reticulum stress and ubiquitination, and gut metagenomic species. Linear regression analysis to model how changes of select variables over the calorie restriction period contribute to changes in insulin sensitivity, showed greatest contributions from gut metagenomic species and fiber intake.\ud \ud Conclusions: This work has enhanced previous knowledge on links between host glucose homeostasis, lifestyle factors and microbiota, and has identified potential biomarkers that may be used in future studies to predict and improve individual response to weight-loss interventions. Furthermore, this is the first study showing integration of the wide range of data presented herein, identifying 115 variables of interest with respect to insulin sensitivity from the initial input, consisting of 9,986 variables.

International audience; BACKGROUND: Commensals induce local IgA responses essential to the induction of tolerance to gut microbiota, but it remains unclear whether antimicrobiota responses remain confined to the gut.OBJECTIVE: The aim of this study was to investigate systemic and intestinal responses against the whole microbiota under homeostatic conditions and in the absence of IgA.METHODS: We analyzed blood and feces from healthy donors, patients with selective IgA deficiency (SIgAd), and patients with common variable immunodeficiency (CVID). Immunoglobulin-coated bacterial repertoires were analyzed by using combined bacterial fluorescence-activated cell sorting and 16S rRNA sequencing. Bacterial lysates were probed by using Western blot analysis with healthy donor sera.RESULTS: Although absent from the healthy gut, serum antimicrobiota IgG are present in healthy subjects and increased in patients with SIgAd. IgG converges with nonoverlapping secretory IgA specificities to target the same bacteria. Each individual subject targets a diverse microbiota repertoire with a proportion that correlates inversely with systemic inflammation. Finally, intravenous immunoglobulin preparations target CVID gut microbiota much less efficiently than healthy microbiota.CONCLUSION: Secretory IgA and systemic IgG converge to target gut microbiota at the cellular level. SIgAd-associated inflammation is inversely correlated with systemic anticommensal IgG responses, which might serve as a second line of defense. We speculate that patients with SIgAd could benefit from oral IgA supplementation. Our data also suggest that intravenous immunoglobulin preparations can be supplemented with IgG from IgA-deficient patient pools to offer better protection against gut bacterial translocations in patients with CVID.

International audience; Paradoxically, loss of immunoglobulin A (IgA), one of the most abundant antibodies, does not irrevocably lead to severe infections in humans but rather is associated with relatively mild respiratory infections, atopy, and autoimmunity. IgA might therefore also play covert roles, not uniquely associated with control of pathogens. We show that human IgA deficiency is not associated with massive quantitative perturbations of gut microbial ecology. Metagenomic analysis highlights an expected pathobiont expansion but a less expected depletion in some typically beneficial symbionts. Gut colonization by species usually present in the oropharynx is also reminiscent of spatial microbiota disorganization. IgM only partially rescues IgA deficiency because not all typical IgA targets are efficiently bound by IgM in the intestinal lumen. Together, IgA appears to play a nonredundant role at the forefront of the immune/microbial interface, away from the intestinal barrier, ranging from pathobiont control and regulation of systemic inflammation to preservation of commensal diversity and community networks.

SummarySegmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer’s patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer’s patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer’s patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

We explored the possibility that the biliary protein fraction may support part of the variation in the nucleating activity previously measured in gallbladder biles of pigs. Eighteen gallbladder aspirates freshly obtained from three dietary groups (0, 5, or 10% beta-cyclodextrin) of six pigs were chromatographed to purify their total protein fraction. Proteins were quantified, and analysed through electrophoresis and immunoblotting or enzyme-linked immunosorbent assay for albumin, and five putative effectors of cholesterol crystallisation, mucins, immunoglobulin A, 130 kDa, apolipoprotein A-I, and anionic polypeptide fraction. Each total protein fraction was also assayed for its ability to influence cholesterol precipitation, when added to supersaturated model bile. The current data provided evidence that the cholesterol crystallisation-promoting activity of biliary proteins in model biles increased with the beta-cyclodextrin dietary content. This occurred without any significant change in the total biliary protein content, but was associated with a significant decrease in the concentration of albumin and apolipoprotein A-I, resulting in changes in the overall balance of proteins in bile. Comparison of these results with the crystallisation figures previously obtained from the corresponding native biles led us to conclude that biliary proteins might influence the outcome of the crystallisation process, namely the final crystal concentration at equilibrium, but would not systematically represent a major driving force for determining the velocity of crystal formation in native bile of pigs.

In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile.Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0105% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0510% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet.We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.

The Staphylococcus hyicus lip gene was cloned in Lactococcus lactis . Pancreatic insufficiency was induced by ligation of the pancreatic duct in pigs. In pigs who had undergone pancreatic ligation, the coefficient of fat absorption was higher after consumption of lipase-expressing L. lactis (91.9% ± 3.7%) than that after consumption of the inactive control strain (78.4% ± 2.4%).

Ursodeoxycholic acid (UDCA)-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. UDCA is the 7β-OH epimer of the primary (i.e. synthesized by the liver) bile acid chenodeoxycholic acid (CDCA). Epimerization proceeds in two subsequent and reversible steps, catalysed by a 7α- and a 7β-hydroxysteroid dehydrogenase (7α- and 7β- HSDH), with 7oxo-lithocholic acid (7oxo-LCA) as the intermediate product. The aim of this study was to test the 7α- and 7β-HSDH activities of anaerobic whole cell cultures of a number of lactic acid bacteria and human intestinal isolates, using CDCA, UDCA and 7oxo- LCA as the substrates. Among 140 strains tested, 21 exhibited at least one of both 7-HSDH activities. 7α-HSDH activity was detected in six strains, 7β-HSDH in nine strains, and both activities in six other strains. All active strains were isolated from normal human and infant faeces. They belonged to the genera Clostridium , Eubacterium and Ruminococcus, whereas no strain of Lactobacillus, Bifidobacterium or Streptococcus was found to be active under our study conditions. The present study therefore revealed, for the first time, a number of normal human intestinal isolates supporting the epimerization of CDCA to UDCA, and further extended our knowledge of those intestinal bacteria which are responsible for 7α- or 7β-HSDH activity.Key words: screening, bile acids, epimerization, intestinal microflora, lactic acid bacteria, probiotics.

The objective of the present study was to evaluate the consequence of Camembert consumption on the composition and metabolism of human intestinal microbiota. Camembert cheese was compared with milk fermented by yoghurt starters andLactobacillus caseias a probiotic reference. The experimental model was the human microbiota-associated (HM) rat. HM rats were fed a basal diet (HMB group), a diet containing Camembert made from pasteurised milk (HMCp group) or a diet containing fermented milk (HMfm group). The level of micro-organisms from dairy products was measured in faeces using cultures on a specific medium and PCR–temporal temperature gradient gel electrophoresis. The metabolic characteristics of the caecal microbiota were also studied: SCFA, NH3, glycosidase and reductase activities, and bile acid degradations. The results showed that micro-organisms from cheese comprised 105–108bacteria/g faecal sample in the HMCp group.Lactobacillusspecies from fermented milk were detected in HMfm rats. Consumption of cheese and fermented milk led to similar changes in bacterial metabolism: a decrease in azoreductase activity and NH3concentration and an increase in mucolytic activities. However, specific changes were observed: in HMCp rats, the proportion of ursodeoxycholic resulting from chenodeoxycholic epimerisation was higher; in HMfm rats, α and β-galactosidases were higher than in other groups and both azoreductases and nitrate reductases were lower. The results show that, as for fermented milk, Camembert consumption did not greatly modify the microbiota profile or its major metabolic activities. Ingested micro-organisms were able to survive in part during intestinal transit. These dairy products exert a potentially beneficial influence on intestinal metabolism.

This study was performed to determine whether erythrocyte and digestive lumenal polyamine concentrations are affected by a soy protein diet when compared to a casein diet. We also determined the effects of these diets on colonic cell proliferation. Sixteen pigs received either a 16% soy protein or casein diet for 25 days. The erythrocyte putrescine was higher in pigs fed the soy protein diet. Significant levels of polyamines were observed in the digestive lumen on both diets. Lumenal putrescine and cadaverine were higher in the proximal colon in the casein group. Lumenal spermidine was higher in the caecum and colon in the soy protein group. No significant differences in the ornithine decarboxylase activity nor in the proliferative cell nuclear antigen labelling index were observed in the colonic mucosa regardless of the regimen. These results indicate that the dietary source of protein induces significant changes in lumenal polyamines in the colon. The physiological effects of these changes need to be further investigated.

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A technique for permanent fistulation of the bile duct, associated with a permanent fistulation of the duodenum allowing continuous reintroduction of the collected bile, was used to investigate the level and daily kinetics of biliary excretion in the conscious pig. Seven Large-White pigs of 45 kg live weight were fistulated. Volumes and total excreted bile salts were measured in hourly samples. Results show that within 24 hrs.the volume of the collected bile was about 2100 ml and the total amount of bile salts about 37 g. The bile excretion (volume and bile salts) was minimum before the morning meal (9:00 a. m.) and the total amount of the excreted bile salts immediately increased after diet intake. Within 2 hrs. after the morning and afternoon meals, values were about 300 p. 100 higher than those determined at 9:00 a. m. This increase was essentially the result of the excretion of a highly concentrated bile (200 to 300 p. 100 more than the values at 9:00 a. m.). On the other hand, the bile volumes were only affected 2 to 3 hrs. after diet intake.

The aim of this work was to investigate the effects on bile secretion of flow rate and site of reinfusion of the collected bile to the animal. Thirty-two pigs weighing 50 +/- 3 kg at the beginning of the experiment were fitted with a reentrant fistula in the lower common bile duct and in the upper duodenum. Bile collected from the bile duct was reinfused in four different ways (four groups of 8 animals each): into the duodenum or the lower common bile duct at a constant flow rate using a peristaltic pump, or into the duodenum or the lower common bile duct at a rate mimicking the flow rate of the secretion using an automatic apparatus. Reinfusing the bile into the lower common bile duct at a rate mimicking the secretion rate provided a daily bile acid production about 21% higher than the level recorded with the other three methods. This was mainly due to a higher bile acid concentration since the bile flow was only slightly affected by the treatment.

Bile flow, biliary lipids and bile acid pool have been studied in growing pigs adapted to a semipurified diet of 2, 10 or 20% lard. After a 5-day period of apparent digestibility measurement, bile secretion was studied in previously fistulated pigs. The apparent digestibility of lard was maintained at a very high level, whatever quantity of lard was ingested. Daily bile acid output was 49% higher with the 10% lard diet than with the 2% lard diet, whereas no further increase was observed with the 20% lard diet. There was a direct relationship between bile acid output and pool size. Biliary phospholipid output increased by 29.0% between 2 and 10% dietary lard and by 69.5% between 10 and 20% lard. The corresponding rise in biliary cholesterol output was +33% between 2 and 10 or 10 and 20% lard in the diet. A study of biliary kinetics showed that this adaptation of biliary lipid to dietary fat was observed whatever time of the light/dark cycle was considered. These results, together with a recent demonstration of the implication of endogenous phospholipids in fat digestion, suggested that, in the course of biliary lipid adaptation to dietary fat content, biliary phospholipid could act in synergy with bile salt, resulting in the maintenance of excellent dietary fat digestion.

A study of the relationship between bile secretion and nutrition in the pig requires a complete and continuous collection of the bile and its reinfusion to the animal. In most of the studies performed in different species, bile has been directly reinfused into the duodenum, leading to the exclusion of the sphincter of Oddi from the biliary pathway. It has been postulated that such an exclusion could inhibit gallbladder emptying. The aim of the present work was to study postprandial gallbladder emptying in the pig, depending on the site of bile reinfusion, i.e. the duodenum or the lower bile duct. The gallbladder bile was coloured with indocyanine green (ICG) and marker secretion was recorded after a test-meal. The results showed that after meal intake, the gallbladder emptied over a similar period of time and according to similar kinetics, whatever the site of bile reinfusion.

The digestive physiology of the germ-free animal has a number of characteristics (cecal hypertrophy, slower small intestine cell renewal, slower gastric emptying and intestinal transit) which distinguish it from that of the conventional animal. If the germ-free model is to be used to determine the role of gastrointestinal microflora in the nutrition of the conventional animal, it is essential to complete the study of these characteristics by data on digestive enzymes in the germ-free. The present paper analyzes these data. There is little information on salivary amylase and none on gastric proteolytic enzymes and intestinal peptidases. More complete data on exocrine pancreas enzymes and intestinal disaccharidases show that the digestive equipment is similar in germ-free and conventional animals. Bile salts, not considered as digestive enzymes, are qualitatively and quantitatively different, depending on the digestive tract bacterial environment. In general, the germ-free animal has some characteristics which should permit better utilization of the diet ingested. Measurements of apparent digestibility do not confirm this hypothesis since results obtained in germ-free and conventional animals of the same species are contradictory.

Bile salt output and digestive utilization of a high-lipid diet were studied in pig bile collected from the common bile duct and infused into the duodenum then into the distal ileum of the animal. Five Large-White pigs of 50 kg body-weight were fitted with three permanent fistulae, one in the common bile duct for complete collection of bile secretion, and the other two in the duodenum and the ileum (at about 80 cm from the ileo-caecal valve) for returning the secretion to the animal. The diversion of the bile into the distal ileum led to a significant 82 p. 100 decrease in daily bile acid output and a 36 p. 100 decrease in apparent digestibility of dietary energy when compared with values obtained when bile was infused into the duodenum. The latter decrease was mainly due to a marked diminution ( - 60.4 p. 100) of the digestive utilization of the dietary lipids. The results show the role of the pig small intestine in maintaining an efficient enterohepatic circulation of bile acid as a result of optimal utilization of the dietary lipids.