Impact and consequences of intensive chemotherapy on intestinal barrier and microbiota in acute myeloid leukemia: the role of mucosal strengthening.

Induction chemotherapy (7 + 3 regimen) remains the gold standard for patients with acute myeloid leukemia (AML) but is responsible for gut damage leading to several complications such as bloodstream infection (BSI). We aimed to investigate the impact of induction chemotherapy on the intestinal barrier of patients with AML and in wild-type mice. Next, we assessed the potential benefit of strengthening the mucosal barrier in transgenic mice releasing a recombinant protein able to reinforce the mucus layer (Tg222). In patients, we observed a decrease of plasma citrulline, which is a marker of the functional enterocyte mass, of short-chain fatty acids and of fecal bacterial load, except for Escherichia coli and Enterococcus spp., which became dominant. Both the α and ß-diversities of fecal microbiota decreased. In wild-type mice, citrulline levels decreased under chemotherapy along with an increase of E. coli and Enterococcus spp load associated with concomitant histologic impairment. By comparison with wild-type mice, Tg222 mice, 3 days after completing chemotherapy, had higher citrulline levels, a faster healing epithelium, and preserved α-diversity of their intestinal microbiota. This was associated with reduced bacterial translocations. Our results highlight the intestinal damage and the dysbiosis induced by the 7 + 3 regimen. As a proof of concept, our transgenic model suggests that strengthening the intestinal barrier is a promising approach to limit BSI and improve AML patients' outcome.

Functional and phylogenetic alterations in gut microbiome are linked to graft-versus-host disease severity.

Acute graft-versus-host disease (aGVHD) is the main complication of hematopoietic stem cell transplantation (HSCT). Changes in gut microbiota composition have been associated with subsequent aGVHD, and reconstitution of healthy microbiota is currently being explored as a therapeutic approach. However, the specific actors in the intestinal ecosystem involved in the pathologic process at the time of aGVHD onset are not yet fully known. We prospectively collected stool samples from patients who underwent allogeneic HSCT. Patients sampled at aGVHD onset were compared with non-GVHD patients. To identify phylogenetic and functional signatures of the disease process, we determined fecal short-chain fatty acid (SFCA) profiles and used high-throughput DNA sequencing and real-time quantitative polymerase chain reaction to assess the microbiota composition. Microbiota alterations were highly specific of gastrointestinal (GI) aGVHD severity. Bacterial biomass and α-diversity were lower in severe aGVHD. We identified several bacterial signatures associated with severe aGVHD at disease onset; a negative correlation was observed with anaerobic bacteria of the Lachnospiraceae, especially the Blautia genus, and Ruminococcaceae families. In parallel, in severe aGVHD patients, we showed a dramatic decrease in the levels of the main SFCAs: acetate (75.8%), propionate (95.8%), and butyrate (94.6%). Mild aGVHD patients were characterized by conserved levels of propionate and Blautia propionate producers. Butyrate was significantly decreased in all GI aGVHD stages, representing a potential diagnostic marker of the disease. Specific microbiota and metabolic alterations were thus associated with aGVHD severity and may be useful for diagnostic and pathophysiologic purposes.

Microbiota Is Involved in Post-resection Adaptation in Humans with Short Bowel Syndrome.

Short bowel syndrome (SBS) is characterized by severe intestinal malabsorption following restrictive surgery. The objective of this study was to determine the functional contribution of SBS-microbiota after resection. It is well-known that SBS-microbiota displayed specific features with a prevalence of Lactobacillus, a low amount of some anaerobic microbes (Clostridium leptum) and an accumulation of fecal lactate in some patients. Patients with jejuno-colonic anastomosis were stratified according to the presence of lactate in their feces and, we observe that the lactate-producing bacteria were predominant in the sub-group of patients accumulating fecal lactate. One case of D-encephalopathy crisis occurred when the D-lactate isoform accumulated in the feces and plasma bicarbonate levels decreased. The fecal sample at the time of the encephalopathy was transferred to germ free rats (SBS-H rats). The SBS-H microbiota conserved some characteristics of the SBS donnor, predominated by lactate-producing bacteria (mainly Lactobacillus), a low level of lactate-consuming bacteria and undetectable C. leptum. However, lactate did not accumulate in feces of recipient rats and the D-encephalopathy was not reproduced in SBS-H rats. This suggests that the intact small bowel of the recipient rats protected them from lactate accumulation and that D-lactate encephalopathy can occur only in the absence of small intestine. After fecal transfer, we also show that gnotobiotic rats exhibited high levels of circulating GLP-1 and ghrelin, two hormones that are known to be induced in SBS patients. Therefore, the microbiota of SBS is a reservoir of biological signals involved in post-resection adaptation.

Changes in Composition and Function of Human Intestinal Microbiota Exposed to Chlorpyrifos in Oil as Assessed by the SHIME® Model.

The presence of pesticide residues in food is a public health problem. Exposure to these substances in daily life could have serious effects on the intestine-the first organ to come into contact with food contaminants. The present study investigated the impact of a low dose (1 mg/day in oil) of the pesticide chlorpyrifos (CPF) on the community structure, diversity and metabolic response of the human gut microbiota using the SHIME® model (six reactors, representing the different parts of the gastrointestinal tract). The last three reactors (representing the colon) were inoculated with a mixture of feces from human adults. Three time points were studied: immediately before the first dose of CPF, and then after 15 and 30 days of CPF-oil administration. By using conventional bacterial culture and molecular biology methods, we showed that CPF in oil can affect the gut microbiota. It had the greatest effects on counts of culturable bacteria (with an increase in Enterobacteria, Bacteroides spp. and clostridia counts, and a decrease in bifidobacterial counts) and fermentative activity, which were colon-segment-dependent. Our results suggest that: (i) CPF in oil treatment affects the gut microbiota (although there was some discordance between the culture-dependent and culture-independent analyses); (ii) the changes are "SHIME®-compartment" specific; and (iii) the changes are associated with minor alterations in the production of short-chain fatty acids and lactate.

Extensive Intestinal Resection Triggers Behavioral Adaptation, Intestinal Remodeling and Microbiota Transition in Short Bowel Syndrome.

Extensive resection of small bowel often leads to short bowel syndrome (SBS). SBS patients develop clinical mal-absorption and dehydration relative to the reduction of absorptive area, acceleration of gastrointestinal transit time and modifications of the gastrointestinal intra-luminal environment. As a consequence of severe mal-absorption, patients require parenteral nutrition (PN). In adults, the overall adaptation following intestinal resection includes spontaneous and complex compensatory processes such as hyperphagia, mucosal remodeling of the remaining part of the intestine and major modifications of the microbiota. SBS patients, with colon in continuity, harbor a specific fecal microbiota that we called "lactobiota" because it is enriched in the Lactobacillus/Leuconostoc group and depleted in anaerobic micro-organisms (especially Clostridium and Bacteroides). In some patients, the lactobiota-driven fermentative activities lead to an accumulation of fecal d/l-lactates and an increased risk of d-encephalopathy. Better knowledge of clinical parameters and lactobiota characteristics has made it possible to stratify patients and define group at risk for d-encephalopathy crises.

Enhanced Ghrelin Levels and Hypothalamic Orexigenic AgRP and NPY Neuropeptide Expression in Models of Jejuno-Colonic Short Bowel Syndrome.

Short bowel syndrome (SBS) patients developing hyperphagia have a better outcome. Gastrointestinal endocrine adaptations help to improve intestinal functions and food behaviour. We investigated neuroendocrine adaptations in SBS patients and rat models with jejuno-ileal (IR-JI) or jejuno-colonic (IR-JC) anastomosis with and without parenteral nutrition. Circulating levels of ghrelin, PYY, GLP-1, and GLP-2 were determined in SBS rat models and patients. Levels of mRNA for proglucagon, PYY and for hypothalamic neuropeptides were quantified by qRT-PCR in SBS rat models. Histology and immunostaining for Ki67, GLP-1 and PYY were performed in SBS rats. IR-JC rats, but not IR-JI, exhibited significantly higher crypt depths and number of Ki67-positive cells than sham. Fasting and/or postprandial plasma ghrelin and PYY concentrations were higher, or tend to be higher, in IR-JC rats and SBS-JC patients than in controls. Proglucagon and Pyy mRNA levels were significantly enhanced in IR-JC rats. Levels of mRNA coding hypothalamic orexigenic NPY and AgRP peptides were significantly higher in IR-JC than in sham rats. We demonstrate an increase of plasma ghrelin concentrations, major changes in hypothalamic neuropeptides levels and greater induction of PYY in SBS-JC rats and patients suggesting that jejuno-colonic continuity creates a peculiar environment promoting further gut-brain adaptations.

Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat.

Previous studies have suggested that intestinal microbiota modulates colonic epithelium renewal. The objective of our work was to study the effects of microbiota on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic crypts and amount of cell cycle-related proteins were compared between germ-free (GF), conventional (CV), and conventionalized rats by histochemistry and Western blot. Ki67 and proliferating cell nuclear antigen (PCNA) were used as surrogates for proliferative cells; p21(cip1) and p27(kip1) were markers of cell cycle arrest; anti- and proapoptotic proteins, Bcl2 and Bax, respectively, were also studied. We observed 40% increase of the crypt proliferative area 2 days after inoculation of GF rats with a complex microbiota. This recruitment of proliferative cells may account for the 30% increase of crypt depth observed between CV and GF rats. The hyperproliferative boost induced by microbiota was compensated by a fourfold increase of p21(cip1) and p27(kip1) involved in cell cycle arrest and a 30% drop of antiapoptotic Bcl2 protein while Bax was unchanged. Inductions of p21(cip1), p27(kip1), and PCNA protein were not paralleled by an increase of the corresponding mRNA. We also showed that p21(cip1) induction by microbiota was partially restored by Bacteroides thetaiotaomicron, Ruminococcus gnavus, and Clostridium paraputrificum. Colonization of the colon by a complex microbiota increases the crypt depth of colon epithelium. This event takes place in conjunction with a multistep process: a hyperproliferative boost accompanied by compensatory events as induction of p21(cip1) and p27(kip1) and decrease of Bcl2.

Drastic changes in fecal and mucosa-associated microbiota in adult patients with short bowel syndrome.

Short bowel syndrome (SBS) is observed in Humans after a large resection of gut. Since the remnant colon and its associated microbiota play a major role in the outcome of patients with SBS, we studied the overall qualitative and quantitative microbiota composition of SBS adult patients compared to controls. The population was composed of 11 SBS type II patients (with a jejuno-colonic anastomosis) and 8 controls without intestinal pathology. SBS patients had 38 +/- 30 cm remnant small bowel length and 66 +/- 19% of residual colon. The repartition of proteins, lipids, carbohydrates and fibres was expressed as % of total oral intake in patients and controls. The microbiota was profiled from stool and biopsy samples with temporal temperature gradient gel electrophoresis and quantitative PCR. We show here that microbiota of SBS patients is unbalanced with a high prevalence of Lactobacillus along with a sub-dominant presence and poor diversity of Clostridium leptum, Clostridium coccoides and Bacteroidetes. In addition, Lactobacillus mucosae was detected within the fecal and mucosa-associated microbiota of SBS patients, whereas it remained undetectable in controls. Thus, in SBS the microbial composition was deeply altered in fecal and mucosal samples, with a shift between dominant and sub-dominant microbial groups and the prevalence of L. mucosae.

Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome.

In short bowel syndrome (SBS), although a remaining colon improves patient outcome, there is no direct evidence of a mucosal colonic adaptation in humans. This prospective study evaluates morphology, proliferation status, and transporter expression level in the epithelium of the remaining colon of adult patients compared with controls. The targeted transporters were Na+/H+ exchangers (NHE2 and 3) and oligopeptide transporter (PepT1). Twelve adult patients with a jejuno-colonic anastomosis were studied at least 2 yr after the last surgery and compared with 11 healthy controls. The depth of crypts and number of epithelial cells per crypt were quantified. The proliferating and apoptotic cell contents were evaluated by revealing Ki67, PCNA, and caspase-3. NHE2, NHE3, PepT1 mRNAs, and PepT1 protein were quantified by quantitative RT-PCR and Western blot, respectively. In patients with SBS compared with controls, 1) hyperphagia and severe malabsorption were documented, 2) crypt depth and number of cells per crypt were 35% and 22% higher, respectively (P < 0.005), whereas the proliferation and apoptotic levels per crypt were unchanged, and 3) NHE2 mRNA was unmodified; NHE3 mRNA was downregulated near the anastomosis and unmodified distally, and PepT1 mRNA and protein were unmodified. We concluded that, in hyperphagic patients with SBS with severe malabsorption, adaptive colonic changes include an increased absorptive surface with an unchanged proliferative/apoptotic ratio and well-preserved absorptive NHE2, NHE3, and PepT1 transporters. This is the first study showing a controlled nonpharmacological hyperplasia in the colon of patients with SBS.

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle.

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.

Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle.

Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of L-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.

Diallyl disulfide (DADS) increases histone acetylation and p21(waf1/cip1) expression in human colon tumor cell lines.

Diallyl disulfide (DADS) is a naturally occurring organosulfur compound, from garlic, which exerts pleiotropic biological effects. In rodents, DADS inhibits colon chemically induced carcinogenesis. DADS anti-promoting effect may partly result from its ability to inhibit tumoral cell proliferation in vivo and in vitro. As far as DADS may modulate the expression of a subset of genes, we investigated DADS effect on histone acetylation, in two human colon tumor cell lines. Our study demonstrates that in Caco-2 and HT-29 cells treated for 6 h, 200 microM DADS increases histone H3 acetylation (x2 and x1.4, respectively). In Caco-2 cells, we also observed histone H4 hyperacetylation, preferentially at the lysine residues 12 and 16. We explored the effects of DADS and one of its metabolites, allyl mercaptan (AM), on histone deacetylase (HDAC) activity: using nuclear extracts of Caco-2 cells, 200 microM DADS decreased HDAC activity by 29% and AM at the same concentration was more efficient (92% inhibition). We also observed that DADS induced an increase in p21(waf1/cip1) expression, at mRNA and protein levels, in both cell lines. This effect was associated with an accumulation of cells in the G2 phase of the cell cycle. Our results suggest that in Caco-2 and HT-29 cells, DADS could inhibit cell proliferation through the inhibition of HDAC activity, histone hyperacetylation and increase in p21(waf1/cip1) expression. The present study provides evidence for cellular and molecular responses triggered by DADS that could be linked to its effect on histone acetylation and play a role in its protective properties on colon carcinogenesis.

Repetitive treatments of colon HT-29 cells with diallyl disulfide induce a prolonged hyperacetylation of histone H3 K14.

Diallyl disulfide (DADS) is a sulfur compound derived from garlic. Several studies carried out in rodents have revealed protective effects of DADS against colon carcinogenesis. The antipromoting effects of DADS may be partly related to its ability to inhibit tumoral cell proliferation. In a previous study, we have shown that in two human colon tumor cell lines (HT-29 and Caco-2) seeded at a low density (0.2 x 10(6) cells/100-mm petri dish), DADS antiproliferative effects were associated with a transient increase of histone H3 K14 acetylation. Moreover, DADS could inhibit nuclear histone deacetylase activity. Therefore, in the present study, we examined the possible effects of different experimental conditions (HT-29 cells at high density, repetitive treatments with DADS) on the pattern of DADS-induced histone hyperacetylation. Using HT-29 cells seeded at a higher density (5 x 10(6) cells/100-mm petri dish), we found that DADS induced histone H3 K14 hyperacetylation rapidly (3 h). When administrated as single treatments, the DADS effect on histone H3 K14 remained transient. In contrast, repetitive treatment with DADS resulted in a prolonged hyperacetylation of histone H3 K14. Whatever the cell culture conditions were, DADS had no effect on histone H4 acetylation. Thus, in vitro, the cell density and pattern of DADS treatment influenced the HT-29 nuclear response to DADS. DADS belongs to food-borne molecules that may play a role in chromatin remodeling and contribute to the nutritional modulation of gene expression.

Inhibition of human colon carcinoma cell growth by ammonia: a non-cytotoxic process associated with polyamine synthesis reduction.

Ammonia, produced by bacterial degradation of unabsorbed and endogenous nitrogenous compounds, is found to be present at millimolar concentrations in the colon lumen. From in vivo animal experiments, this metabolite has been shown to alter colonic epithelial cell morphology and to increase compensatory cell proliferation when present in excess. In this in vitro study, using the human colon adenocarcinoma HT-29 Glc(-/+) cell line treated with increasing doses of NH(4)Cl, we found that 20 mM NH(4)Cl, a concentration close to that found in the large intestine lumen, was able to increase the volume of vacuolar lysosomes and to repress HT-29 Glc(-/+) cell proliferation. This growth-inhibitory effect was not correlated with decrease of cell viability, with modification of cell differentiation and change of the cell distribution in the different cell cycle phases, thus indicating a proportional slowdown in all cell cycle phases. In contrast to what is found in healthy colonocytes, ammonia was not metabolized by HT-29 cells into carbamoyl-phosphate (carbamoyl-P) and citrulline, indicating that ammonia was likely acting on cells by itself. This agent was shown to markedly reduce cellular ornithine decarboxylase (ODC) activity resulting in a threefold decrease in the capacity of HT-29 cells to synthetize polyamines, these latter metabolites being strictly necessary for cell growth. The unexpected finding that ammonia is acting as an antimitotic agent against tumoral HT-29 colonic cells may be related to the inability of these cells to metabolize this compound.