Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Dongfang; Wang Xiliang; Chen Yitzai

2009-05-15

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrinmore » were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.« less

PYRETHROID INSECTICIDES: ISOFORM-DEPENDENT HYDROLYSIS, INDUCTION OF CYTOCHROME P450 3A4 AND EVIDENCE ON THE INVOLVEMENT OF THE PREGNANE X RECEPTOR

PubMed Central

Yang, Dongfang; Wang, Xiliang; Chen, Yi-tzai; Deng, Ruitang; Yan, Bingfang

2009-01-01

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity. PMID:19249324

Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

PubMed

Yang, Dongfang; Wang, Xiliang; Chen, Yi-Tzai; Deng, Ruitang; Yan, Bingfang

2009-05-15

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran

PubMed Central

Duysen, Ellen G.; Cashman, John R.; Schopfer, Lawrence M.; Nachon, Florian; Masson, Patrick; Lockridge, Oksana

2012-01-01

Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal’s well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1−/− mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5 mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100 mg/kg and chlorpyrifos oxon at 14 mg/kg was lethal to wild-type but not to ES1−/− mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase. PMID:22209767

The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin.

PubMed

Lin, Ni-Ni; Chen, Jia; Xu, Bin; Wei, Xia; Guo, Lei; Xie, Jian-Wei

2015-01-01

T-2 toxin poses a great threat to human health because it has the highest toxicity of the currently known trichothecene mycotoxins. To understand the in vivo toxicity and transformation mechanism of T-2 toxin, we investigated the role of one kind of principal phase I drug-metabolizing enzymes (cytochrome P450 [CYP450] enzymes) on the metabolism of T-2 toxin, which are crucial to the metabolism of endogenous substances and xenobiotics. We also investigated carboxylesterase, which also plays an important role in the metabolism of toxic substances. A chemical inhibition method and a recombinant method were employed to investigate the metabolism of the T-2 toxin by the CYP450 enzymes, and a chemical inhibition method was used to study carboxylesterase metabolism. Samples incubated with human liver microsomes were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC- QqQ MS) after a simple pretreatment. In the presence of a carboxylesterase inhibitor, only 20 % T-2 toxin was metabolized. When CYP enzyme inhibitors and a carboxylesterase inhibitor were both present, only 3 % of the T-2 toxin was metabolized. The contributions of the CYP450 enzyme family to T-2 toxin metabolism followed the descending order CYP3A4, CYP2E1, CYP1A2, CYP2B6 or CYP2D6 or CYP2C19. Carboxylesterase and CYP450 enzymes are of great importance in T-2 toxin metabolism, in which carboxylesterase is predominant and CYP450 has a subordinate role. CYP3A4 is the principal member of the CYP450 enzyme family responsible for T-2 toxin metabolism. The primary metabolite produced by carboxylesterase is HT-2, and the main metabolite produced by CYP 3A4 is 3'-OH T-2. The different metabolites show different toxicities. Our results will provide useful data concerning the toxic mechanism, the safety evaluation, and the health risk assessment of T-2 toxin.

In Vitro Metabolism of the Brominated Flame Retardants 2-Ethylhexyl-2,3,4,5-Tetrabromobenzoate (TBB) and Bis(2-Ethylhexyl) 2,3,4,5-Tetrabromophthalate (TBPH) in Human and Rat Tissues

PubMed Central

Roberts, Simon C.; Macaulay, Laura J.; Stapleton, Heather M.

2012-01-01

Due to the phaseout of polybrominated diphenyl ether (PBDE) flame retardants, new chemicals, such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) and bis(2-ethylhexyl) 2,3,4,5-tetrabromophthalate (TBPH), have been used as replacements in some commercial flame retardant mixtures. Both chemicals have been detected in indoor dust at concentrations approaching the concentrations of PBDEs; however, little is known about their fate, metabolism, or toxicity. The goal of this study was to investigate the potential metabolism of these two brominated flame retardants in human and rat tissues by conducting in vitro experiments with liver and intestinal subcellular fractions. In all the experiments, TBB was consistently metabolized to 2,3,4,5-tetrabromobenzoic acid (TBBA) via cleavage of the 2-ethylhexyl chain without requiring any added cofactors. TBBA was also formed in purified porcine carboxylesterase, but at a much faster rate of 6.29 ± 0.58 nmol min-1 mg protein-1. The estimated Km and Vmax values for TBB metabolism in human microsomes were 11.1 ± 3.9 μM and 0.644 ± 0.144 nmol min-1 mg protein-1, respectively. A similar Km of 9.3 ± 2.2 μM was calculated for porcine carboxylesterase, indicating similar enzyme specificity. While the rapid formation of TBBA may reduce the bioaccumulation potential of TBB in mammals and may be useful as a biomarker of TBB exposure, the toxicity of this brominated benzoic acid is unknown and may be a concern based on its structural similarity to other toxic pollutants. In contrast to TBB, no metabolites of TBPH were detected in human or rat subcellular fractions. However, a metabolic product of TBPH, mono(2-ethylhexyl) tetrabromophthalate (TBMEHP), was formed in purified porcine carboxylesterase at an approximate rate of 1.08 pmol min-1 mg protein-1. No Phase II metabolites of TBBA or TBMEHP were observed. More research is needed to understand the in vivo toxicokinetics and health effects of these compounds given their current ubiquitous presence in most US households and the resulting probability of chronic exposure, particularly to young children. PMID:22575079

Nuclear receptor-mediated regulation of carboxylesterase expression and activity.

PubMed

Staudinger, Jeff L; Xu, Chenshu; Cui, Yue J; Klaassen, Curtis D

2010-03-01

Emerging evidence demonstrates that several nuclear receptor (NR) family members regulate drug-inducible expression and activity of several important carboxylesterase (CES) enzymes in mammalian liver and intestine. Numerous clinically prescribed anticancer prodrugs, carbamate and pyrethroid insecticides, environmental toxicants and procarcinogens are substrates for CES enzymes. Moreover, a key strategy used in rational drug design frequently utilizes an ester linkage methodology to selectively target a prodrug, or to improve the water solubility of a novel compound. This review summarizes the current state of knowledge regarding NR-mediated regulation of CES enzymes in mammals and highlights their importance in drug metabolism, drug-drug interactions and toxicology. New knowledge regarding the transcriptional regulation of CES enzymes by NR proteins pregnane x receptor (NR1I2) and constitutive androstane receptor (NR1I3) has recently come to light through the use of knockout and transgenic mouse models. Novel insights regarding the species-specific cross-regulation of glucocorticoid receptor (NR3C1) and PPAR-alpha (NR1C1) signaling and CES gene expression are discussed. Elucidation of the role of NR-mediated regulation of CES enzymes in liver and intestine will have a significant impact on rational drug design and the development of novel prodrugs, especially for patients on combination therapy.

Carboxylesterase inhibitors

PubMed Central

Hatfield, M. Jason; Potter, Philip M.

2011-01-01

Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

Activation of a camptothecin prodrug by specific carboxylesterases as predicted by quantitative structure-activity relationship and molecular docking studies.

PubMed

Yoon, Kyoung Jin P; Krull, Erik J; Morton, Christopher L; Bornmann, William G; Lee, Richard E; Potter, Philip M; Danks, Mary K

2003-11-01

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (irinotecan, CPT-11) is a camptothecin prodrug that is metabolized by carboxylesterases (CE) to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase I inhibitor. CPT-11 has shown encouraging antitumor activity against a broad spectrum of tumor types in early clinical trials, but hematopoietic and gastrointestinal toxicity limit its administration. To increase the therapeutic index of CPT-11 and to develop other prodrug analogues for enzyme/prodrug gene therapy applications, our laboratories propose to develop camptothecin prodrugs that will be activated by specific CEs. Specific analogues might then be predicted to be activated, for example, predominantly by human liver CE(hCE1), by human intestinal CE (hiCE), or in gene therapy approaches using a rabbit liver CE (rCE). This study describes a molecular modeling approach to relate the structure of rCE-activated camptothecin prodrugs with their biological activation. Comparative molecular field analysis, comparative molecular similarity index analysis, and docking studies were used to predict the biological activity of a 4-benzylpiperazine derivative of CPT-11 [7-ethyl-10-[4-(1-benzyl)-1-piperazino]carbonyloxycamptothecin (BP-CPT)] in U373MG glioma cell lines transfected with plasmids encoding rCE or hiCE. BP-CPT has been reported to be activated more efficiently than CPT-11 by a rat serum esterase activity; however, three-dimensional quantitative structure-activity relationship studies predicted that rCE would activate BP-CPT less efficiently than CPT-11. This was confirmed by both growth inhibition experiments and kinetic studies. The method is being used to design camptothecin prodrugs predicted to be activated by specific CEs.

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication.

PubMed

Dunn, Emily N; Ferrara-Bowens, Teresa M; Chachich, Mark E; Honnold, Cary L; Rothwell, Cristin C; Hoard-Fruchey, Heidi M; Lesyna, Catherine A; Johnson, Erik A; Cerasoli, Douglas M; McDonough, John H; Cadieux, C Linn

2018-06-07

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Demonstration of carboxylesterase in cytology samples of human nasal respiratory epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodgers, D.A.; Nikula, K.J.; Avila, K.

1995-12-01

The epithelial lining of the nasal airways is a target for responses induced by a variety of toxicant exposures. The high metabolic capacity of this tissue has been suggested to play a role in both protection of the airways through detoxication of certain toxicants, as well as in activation of other compounds to more toxic metabolites. Specifically, nasal carboxylesterase (CE) has been shown to mediate the toxicity of inhaled esters and acrylates by converting them to more toxic acid and alcohol metabolites which can be cytotoxic and/or carcinogenic to the nasal mucosa. Due to difficulties in extrapolating rodent models tomore » human, new paradigms using human cells and tissues are essential to understanding and evaluating the metabolic processes in human nasal epithelium.« less

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH)

PubMed Central

Wheelock, Craig E.; Nishi, Kosuke; Ying, Andy; Jones, Paul D.; Colvin, Michael E.; Olmstead, Marilyn M.; Hammock, Bruce D.

2009-01-01

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (ki). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (~5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process. PMID:18023188

Carboxylesterase-mediated insecticide resistance: Quantitative increase induces broader metabolic resistance than qualitative change.

PubMed

Cui, Feng; Li, Mei-Xia; Chang, Hai-Jing; Mao, Yun; Zhang, Han-Ying; Lu, Li-Xia; Yan, Shuai-Guo; Lang, Ming-Lin; Liu, Li; Qiao, Chuan-Ling

2015-06-01

Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change. Copyright © 2014 Elsevier Inc. All rights reserved.

In Vivo and In Vitro Metabolites from the Main Diester and Monoester Diterpenoid Alkaloids in a Traditional Chinese Herb, the Aconitum Species

PubMed Central

Zhang, Min; Peng, Chong-sheng; Li, Xiao-bo

2015-01-01

Diester diterpenoid alkaloids (DDAs), such as aconitine (AC), mesaconitine (MA), and hypaconitine (HA), are both pharmacologically active compounds and toxic ingredients in a traditional Chinese herb, the Aconitum species. Many DDA metabolism studies have been performed to explore mechanisms for reducing toxicity in these compounds and in Aconitum species extracts for safe clinical administration. In this review, we summarize recent progress on the metabolism of toxic AC, MA, and HA and corresponding monoester diterpenoid alkaloids (MDAs) in the gastrointestinal tract and liver in different animal species and humans in vivo and/or in vitro, where these alkaloids are primarily metabolized by cytochrome P450 enzymes, carboxylesterases, and intestinal bacteria, which produces phase I metabolites, ester hydrolysed products, and lipoalkaloids. Furthermore, we classify metabolites detected in the blood and urine, where the aforementioned metabolites are absorbed and excreted. Less toxic MDAs and nontoxic alcohol amines are the primary DDA metabolites detected in the blood. Most other DDAs metabolites produced in the intestine and liver detected in the urine have not been reported in the blood. We propose an explanation for this nonconformity. Finally, taking AC, for instance, we generalize a process of toxicity reduction in the body after oral AC administration for the first time. PMID:25705235

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

PubMed

Holmes, Roger S; Wright, Matthew W; Laulederkind, Stanley J F; Cox, Laura A; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J; Potter, Phillip M; Redinbo, Matthew R; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J

2010-10-01

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

PubMed

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Assessment of the inhibitory effects of pyrethroids against human carboxylesterases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lei, Wei

Pyrethroids are broad-spectrum insecticides that widely used in many countries, while humans may be exposed to these toxins by drinking or eating pesticide-contaminated foods. This study aimed to investigate the inhibitory effects of six commonly used pyrethroids against two major human carboxylesterases (CES) including CES1 and CES2. Three optical probe substrates for CES1 (DME, BMBT and DMCB) and a fluorescent probe substrate for CES2 (DDAB) were used to characterize the inhibitory effects of these pyrethroids. The results demonstrated that most of the tested pyrethroids showed moderate to weak inhibitory effects against both CES1 and CES2, but deltamethrin displayed strong inhibitionmore » towards CES1. The IC{sub 50} values of deltamethrin against CES1-mediated BMBT, DME, and DMCB hydrolysis were determined as 1.58 μM, 2.39 μM, and 3.3 μM, respectively. Moreover, deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in living cells. Further investigation revealed that deltamethrin inhibited CES1-mediated BMBT hydrolysis via competitive manner but noncompetitively inhibited DME or DMCB hydrolysis. The inhibition behaviors of deltamethrin against CES1 were also studied by molecular docking simulation. The results demonstrated that CES1 had at least two different ligand-binding sites, one was the DME site and another was the BMBT site which was identical to the binding site of deltamethrin. In summary, deltamethrin was a strong reversible inhibitor against CES1 and it could tightly bind on CES1 at the same ligand-binding site as BMBT. These findings are helpful for the deep understanding of the interactions between xenobiotics and CES1. - Highlights: • The inhibitory effects of six commonly used pyrethroids on human carboxylesterases were investigated. • Deltamethrin displayed strong inhibitory effects against human carboxylesterase 1 (CES1). • Deltamethrin was cell membrane permeable and could inhibit intracellular CES1 in living cells. • Both experimental and docking studies demonstrated that CES1 had at least two different ligand-binding sites.« less

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

PubMed Central

Crow, J. Allen; Herring, Katye L.; Xie, Shuqi; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2009-01-01

Summary Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC50=8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (Kiapp=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (Kiapp=1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo. PMID:19761868

Organophosphorus Flame Retardants Inhibit Specific Liver Carboxylesterases and Cause Serum Hypertriglyceridemia

PubMed Central

2015-01-01

Humans are prevalently exposed to organophosphorus flame retardants (OPFRs) contained in consumer products and electronics, though their toxicological effects and mechanisms remain poorly understood. We show here that OPFRs inhibit specific liver carboxylesterases (Ces) and cause altered hepatic lipid metabolism. Ablation of the OPFR target Ces1g has been previously linked to dyslipidemia in mice. Consistent with OPFR inhibition of Ces1g, we also observe OPFR-induced serum hypertriglyceridemia in mice. Our findings suggest novel toxicities that may arise from OPFR exposure and highlight the utility of chemoproteomic and metabolomic platforms in the toxicological characterization of environmental chemicals. PMID:24597639

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Determination of Human Hepatic CYP2C8 and CYP1A2 Age-Dependent Expression to Support Human Health Risk Assessment for Early Ages

EPA Science Inventory

Predicting age-specific metabolism is important for evaluating age-related drug and chemical sensitivity. Multiple cytochrome P450s and carboxylesterase enzymes are responsible for human pyrethroid metabolism. Complete ontogeny data for each enzyme are needed to support in vitro ...

Identification and characterization of a novel carboxylesterase (FpbH) that hydrolyzes aryloxyphenoxypropionate herbicides.

PubMed

Wang, Chenghong; Qiu, Jiguo; Yang, Youjian; Zheng, Jinwei; He, Jian; Li, Shunpeng

2017-04-01

To identify and characterize a novel aryloxyphenoxypropionate (AOPP) herbicide-hydrolyzing carboxylesterase from Aquamicrobium sp. FPB-1. A carboxylesterase gene, fpbH, was cloned from Aquamicrobium sp. FPB-1. The gene is 798 bp long and encodes a protein of 265 amino acids. FpbH is smaller than previously reported AOPP herbicide-hydrolyzing carboxylesterases and shares only 21-35% sequence identity with them. FpbH was expressed in Escherichia coli BL21(DE3) and the product was purified by Ni-NTA affinity chromatography. The purified FpbH hydrolyzed a wide range of AOPP herbicides with catalytic efficiency in the order: haloxyfop-P-methyl > diclofop-methyl > fenoxaprop-P-ethyl > quizalofop-P-ethyl > fluazifop-P-butyl > cyhalofop-butyl. The optimal temperature and pH for FpbH activity were 37 °C and 7, respectively. FpbH is a novel AOPP herbicide-hydrolyzing carboxylesterase; it is a good candidate for mechanistic study of AOPP herbicide-hydrolyzing carboxylesterases and for bioremediation of AOPP herbicide-contaminated environments.

Esterase detoxification of acetylcholinesterase inhibitors by human or rat liver in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered...

Esterase detoxification of acetylcholinesterase inhibitors using human liver samples in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are consider...

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

PubMed

Fu, Jing; Sadgrove, Matthew; Marson, Lesley; Jay, Michael

2016-08-01

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Isoflavones enhance pharmacokinetic exposure of active lovastatin acid via the upregulation of carboxylesterase in high-fat diet mice after oral administration of Xuezhikang capsules.

PubMed

Feng, Dong; Ge, Chun; Tan, Zhao-Yi; Sun, Jian-Guo; Xie, Yuan; Yao, Lan; Yan, Cai-Xia; Aa, Ji-Ye; Wang, Guang-Ji

2018-06-19

Xuezhikang capsule (XZK) is a traditional Chinese medicine that contains lovastatin (Lv) for hyperlipidemia treatment, although it has fewer side effects than Lv. However, the pharmacokinetic mechanisms contributing to its distinct efficacy and low side effects are unclear. Mice were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia. We first conducted the pharmacokinetic studies in HFD mice following oral administration of Lv (10 mg/kg, i.g.) and found that HFD remarkably decreased the active form of Lv (the lovastatin acid, LvA) exposure in the circulation system, especially in the targeting organ liver, with a declined conversion from Lv to LvA, whereas the Lv (responsible for myotoxicity) exposure in muscle markedly increased. Then we compared the pharmacokinetic profiles of Lv in HFD mice after the oral administration of XZK (1200 mg/kg, i.g.) or an equivalent dose of Lv (10 mg/kg, i.g.). A higher exposure of LvA and lower exposure of Lv were observed after XZK administration, suggesting a pharmacokinetic interaction of some ingredients in XZK. Further studies revealed that HFD promoted the inflammation and inhibited carboxylesterase (CES) activities in the intestine and the liver, thus contributing to the lower transformation of Lv into LvA. In contrast, XZK inhibited the inflammation and upregulated CES in the intestine and the liver. Finally, we evaluated the effects of monacolins and phytosterols, the fractional extracts of isoflavones, on inflammatory LS174T or HepG2 cells, which showed that isoflavones inhibited inflammation, upregulated CES, and markedly enhanced the conversion of Lv into LvA. For the first time, we provide evidence that isoflavones and Lv in XZK act in concert to enhance the efficacy and reduce the side effects of Lv.

Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off-On Probe.

PubMed

Li, Dongyu; Li, Zhao; Chen, Weihua; Yang, Xingbin

2017-05-24

A new near-infrared fluorescence off-on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off-on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10 -3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.

Carboxylesterases: General detoxifying enzymes

PubMed Central

Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

2016-01-01

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

PubMed

Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

2012-03-01

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion. Copyright © 2011 Wiley Periodicals, Inc.

Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleming, Christopher D.; Bencharit, Sompop; Edwards, Carol C.

2010-07-19

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition ofmore » hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.« less

Structural insights into drug processing by human carboxylesterase 1: tamoxifen, mevastatin, and inhibition by benzil.

PubMed

Fleming, Christopher D; Bencharit, Sompop; Edwards, Carol C; Hyatt, Janice L; Tsurkan, Lyudmila; Bai, Feng; Fraga, Charles; Morton, Christopher L; Howard-Williams, Escher L; Potter, Philip M; Redinbo, Matthew R

2005-09-09

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

PubMed

Hedges, Laura; Brown, Susan; MacLeod, A Kenneth; Vardy, Audrey; Doyle, Edward; Song, Gina; Moreau, Marjory; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-06-04

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL int ) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CL int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL int values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Slow-binding inhibition of carboxylesterase and other serine hydrolases by chlorodifluoroacetaldehyde.

PubMed

Yin, H; Jones, J P; Anders, M W

1993-01-01

The chlorofluorocarbon substitute 1,2-dichloro-1,1-difluoroethane (HCFC-132b) undergoes oxidative metabolism in rats to give a range of metabolites, including chlorodifluoroacetaldehyde [Harris and Anders (1991) Chem. Res. Toxicol. 4, 180]. The present experiments were undertaken after studies to characterize an unidentified metabolite of HCFC-132b revealed that chlorodifluoroacetaldehyde was toxic in vivo: rats given chlorodifluoroacetaldehyde died showing signs of cholinergic stimulation. Because some fluoroketones are known inhibitors of hydrolases, including acetylcholinesterase, the inhibitory effects of chlorodifluoroacetaldehyde on acetylcholinesterase (electric eel and human erythrocyte), on pseudocholinesterase (horse serum), on carboxylesterase (pig liver), and on alpha-chymotrypsin (bovine pancreas) were studied. In aqueous solution, the ratio chlorodifluoroacetaldehyde:chlorodifluroacetaldehyde hydrate, as determined by 1H nuclear magnetic resonance spectroscopy, was 1:157. Chlorodifluoroacetaldehyde was a slow-binding inhibitor of both acetylcholinesterases, of pseudocholinesterase, and of carboxylesterase; the Ki values, corrected for the aldehyde:hydrate ratio, were 150 nM, 1.7 nM, 3.7 nM, and 23 pM, respectively, as determined by final velocity of the progress curves; the kon values were 9.1 x 10(4), 1.1 x 10(5), 3.2 x 10(4), and 9.2 x 10(5) M-1 min-1, respectively. Chlorodifluoroacetaldehyde did not inhibit alpha-chymotrypsin. Acetaldehyde and trichloroacetaldehyde were classical competitive inhibitors of acetylcholinesterase. These results show that hydrochlorofluorocarbon metabolites may exert significant biological effects.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

PubMed

Bianco, Karina; Otero, Sofía; Oliver, Agustina Balazote; Nahabedian, Daniel; Kristoff, Gisela

2014-11-01

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of magnitude higher than cholinesterase. A greater efficiency (Vmax/Km) was obtained using acetylthiocholine and p-nitrophenyl butyrate. Acute exposure to azinphos-methyl did not cause inhibition of cholinesterase activity until 10 mg L(-1) used. Carboxylesterases towards p-nitrophenyl butyrate was inhibited by azinphos-methyl being the IC502.20±0.75 mg L(-1) of azinphos-methyl. Subchronic exposure to environmental concentrations of azinphos-methyl (0.02 and 0.2 mg L(-1)) produced a decrease in survival, protein content and carboxylesterases activity despite no inhibition of cholinesterase activity was observed. B. straminea cholinesterase is not a sensible biomarker. On the contrary, carboxylesterases activity was inhibited by azinphos-methyl. Carboxylesterases could be protecting cholinesterase activity and therefore, protecting the organism from neurotoxicity. This work confirms the advantages of measuring cholinesterases and carboxylesterases jointly in aquatic biomonitoring of pesticide contamination. This becomes relevant in order to find more sensitive biomarkers and new strategies to protect non-target aquatic organisms from pesticide contamination. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of carboxylesterase 1 is associated with cholesteryl ester retention in human THP-1 monocyte/macrophages

PubMed Central

Crow, J. Allen; Middleton, Brandy L.; Borazjani, Abdolsamad; Hatfield, M. Jason; Potter, Philip M.; Ross, Matthew K.

2008-01-01

Cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase (CEH) yielding free cholesterol for export from macrophages. Hence, CEH has an important regulatory role in macrophage reverse cholesterol transport (RCT). CEH and human carboxylesterase 1 (CES1) appear to be the same enzyme. CES1 is inhibited by oxons, the bioactive metabolites of organophosphate (OP) pesticides. Here, we show that CES1 protein is robustly expressed in human THP-1 monocytes/macrophages and its biochemical activity inhibited following treatment of cell lysates and intact cells with chlorpyrifos oxon, paraoxon, or methyl paraoxon (with nanomolar IC50 values) or after immunodepletion of CES1 protein. CES1 protein expression in cells is unaffected by 24-h paraoxon treatment, suggesting the reduced hydrolytic activity is due to covalent inhibition of CES1 by oxons and not down-regulation of expression. Most significantly, treatment of cholesterol-loaded macrophages with either paraoxon (a non-specific CES inhibitor) or benzil (a specific CES inhibitor) caused enhanced retention of intracellular cholesteryl esters and a “foamy” phenotype, consistent with reduced cholesteryl ester mobilization. Thus, exposure to OP pesticides, which results in the inhibition of CES1, may also inhibit macrophage RCT, an important process in the regression of atherosclerosis. PMID:18762277

Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip.

PubMed

Nomura, Taiji

2017-01-01

Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily.

Response of digestive enzymes and esterases of ecotoxicological concern in earthworms exposed to chlorpyrifos-treated soils.

PubMed

Sanchez-Hernandez, Juan C; Ríos, Juan Manuel; Attademo, Andrés M

2018-03-01

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity of digestive enzymes (phosphatase, β-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg -1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls), carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue, only the carboxylesterase and lipase activities were significantly depressed (42-67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterases in the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes.

Rhipicephalus (Boophilus) microplus strain Deutsch, 5 BAC clone sequencing, including two encoding Cytochrome P450s and one encoding CzEst9 carboxylesterase

USDA-ARS?s Scientific Manuscript database

The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. BAC clones give insight into the genome struct...

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun†,‡

PubMed Central

Fleming, Christopher D.; Edwards, Carol C.; Kirby, Stephen D.; Maxwell, Donald M.; Potter, Philip M.; Cerasoli, Douglas M.; Redinbo, Matthew R.

2008-01-01

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 104-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure. PMID:17407327

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

PubMed Central

Wheelock, Craig E.; Eder, Kai J.; Werner, Inge; Huang, Huazhang; Jones, Paul D.; Brammell, Benjamin F.; Elskus, Adria A.; Hammock, Bruce D.

2006-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish. PMID:16011852

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

USGS Publications Warehouse

Wheelock, C.E.; Eder, K.J.; Werner, I.; Huang, H.; Jones, P.D.; Brammell, B.F.; Elskus, A.A.; Hammock, B.D.

2005-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 ??g/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 ??g/l), but not a low dose (1.2 ??g/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ???30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish. ?? 2005 Elsevier B.V. All rights reserved.

The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and β-glucuronidase in human colorectal tumours

PubMed Central

Tobin, Peter; Clarke, Stephen; Seale, J Paul; Lee, Soon; Solomon, Michael; Aulds, Sally; Crawford, Michael; Gallagher, James; Eyers, Tony; Rivory, Laurent

2006-01-01

Aims Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by β-glucuronidase may represent a significant pathway of SN-38 formation. Methods The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 µm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). Results The rate of conversion of irinotecan (9.6 µM) was lower in tumour tissue than matched normal colon mucosa samples (0.30 ± 0.14 pmol min−1 mg−1 protein and 0.77 ± 0.59 pmol min−1 mg−1 protein, respectively; P < 0.005). In contrast, no significant difference was observed in β-glucuronidase activity between tumour and matched normal colon samples (4.56 ± 6.9 pmol min−1 mg−1 protein and 3.62 ± 2.95 pmol min−1 mg−1 protein, respectively, using 9.6 µM SN-38 glucuronide; P > 0.05). β-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2 > 0.23, P < 0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of β-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P < 0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. Conclusions Tumour β-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo. PMID:16842384

Juvenile Hormone Induction of Esterases: A Mechanism for the Regulation of Juvenile Hormone Titer

PubMed Central

Whitmore, Donald; Whitmore, Elaine; Gilbert, Lawrence I.

1972-01-01

Within a few hours after injection of juvenile hormone into Hyalophora gloveri pupae, several fast-migrating carboxylesterases (EC 3.1.1.1) that are sensitive to diisopropylfluorophosphate appear in the hemolymph. Treatment of the pupae with puromycin or actinomycin D prevents the appearance of these hemolymph enzymes, suggesting de novo synthesis of the carboxylesterases. Of the several other compounds investigated, only a potent mimic of the juvenile hormone is able to induce these enzymes. When the induced enzymes are incubated in vitro with 14C-labeled juvenile hormone, the hormone is rapidly and efficiently degraded. It is suggested that these induced carboxylesterases play an important role in the regulation of juvenile hormone titer. Images PMID:4504374

The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family.

PubMed

Johnson, Glynis; Moore, Samuel W

2013-09-01

Short linear motifs confer evolutionary flexibility on proteins as they can be added with relative ease allowing the acquisition of new functions. Such motifs may mediate a variety of signalling functions. The adhesion-mediating Leu-Arg-Glu (LRE) motif is enriched in laminin beta 2, and has been observed in other proteins, including members of the carboxylesterase/cholinesterase family. It acts as a stop signal for growing axons in the developing neuromuscular junction, binding to the voltage-gated calcium channel. In this bioinformatic analysis, we have investigated the presence of the motif in proteins of the neuromuscular junction, and have also examined its structural position and potential for ligand interaction, as well as phylogenetic conservation, in the carboxylesterase/cholinesterase family. The motif was observed to occur with a significantly higher frequency than expected in the UniProt/Swiss-Prot database, as well as in four individual species (human, mouse, Caenorhabditis elegans and Drosophila melanogaster). Examination of its presence in neuromuscular junction proteins showed it to be enriched in certain proteins of the synaptic basement membrane, including laminin, agrin, acetylcholinesterase and tenascin. A highly significant enrichment was observed in cytoskeletal proteins, particularly intermediate filament proteins and members of the spectrin family. In the carboxylesterase/cholinesterase family, the motif was observed in four conserved positions in the protein structure. It is present in the majority of mammalian acetylcholinesterases, as well as acetylcholinesterases from electric fish and a number of invertebrates. In insects, it is present in the ace-2, rather than in the synaptic ace-1, enzyme. It is also observed in the cholinesterase-like adhesion molecules (neuroligins, neurotactin and glutactin). It is never seen in butyrylcholinesterases, which do not mediate cell adhesion. In conclusion, the significant enrichment of the motif in certain classes of protein, as well as its conserved presence and structural positioning in one protein family, suggests that it has specific functions both in cell adhesion in the neuromuscular junction and in maintaining the structural integrity of the cytoskeleton. Copyright © 2013 Elsevier Inc. All rights reserved.

Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1.

PubMed

Yin, Kun; Lv, Min; Wang, Qiaoning; Wu, Yixuan; Liao, Chunyang; Zhang, Weiwei; Chen, Lingxin

2016-10-15

Mercury is a toxic heavy metal and presents significant threats to organisms and natural ecosystems. Recently, the mercury remediation as well as its detection by environmental-friendly biotechnology has received increasing attention. In this study, carboxylesterase E2 from mercury-resistant strain Pseudomonas aeruginosa PA1 has been successfully displayed on the outer membrane of Escherichia coli Top10 bacteria to simultaneously adsorb and detect mercury ion (Hg(2+)). The transmission electron microscopy analysis shows that Hg(2+) can be absorbed by carboxylesterase E2 and accumulated on the outer membrane of surface-displayed E. coli bacteria. The adsorption of Hg(2+) followed a physicochemical, equilibrated and saturatable mechanism, which well fits the traditional Langmuir adsorption model. The surface-displayed system can be regenerated through regulating pH values. As its activity can be inhibited by Hg(2+), carboxylesterase E2 has been used to detect the concentration of Hg(2+) in water samples. The developed surface display system will be of great potential in the simultaneous bioremediation and biodetection of environmental mercury pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

PubMed Central

Sobek, H; Görisch, H

1989-01-01

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported. PMID:2508625

Age-Dependent Human Hepatic Carboxylesterase 1 (Ces1) ...

EPA Pesticide Factsheets

Human hepatic carboxylesterase 1 and 2 (CES1 and CES2) are important for ester- and amide- bond containing pharmaceutical and environmental chemical disposition. Despite concern regarding juvenile sensitivity to such compounds, CES1 and CES2 ontogeny has not been well characterized. To define human hepatic microsomal and cytosolic CES1 and CES2 expression during early postnatal life, microsomal and cytosolic fractions were prepared using liver samples from subjects without liver disease [N=165, 1d-18 yrs]. Proteins were fractionated, detected and quantitated by western blotting. Median microsomal CES1 was lower among samples from subjects < 3 weeks of age (N=36) compared to the rest of the population (N=126; 6.27 vs 17.5 pmoles/mg microsomal protein, respectively; p<0.001; Kruskal Wallis test). Cytosolic CES1 increased sequentially with expression being lowest among samples from individuals between birth and 3 weeks of age (N=36), markedly greater among those from ages 3 weeks to 6 years (N=90), and then modestly greater still among those over 6 years of age (N=36; median values = 4.7, 15.8, and 16.6 pmoles/mg cytosolic protein, respectively; p values <0.001 and 0.05, respectively, Kruskal Wallis test). Microsomal CES2 also increased sequentially across the same three age groups with median values of 1.8, 2.9, and 4.2 pmoles/mg microsomal protein, respectively (p<0.001, both), whereas for cytosolic CES2, only the youngest age group differed from the two older g

Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens.

PubMed

Islam, Md Zaherul; Yun, Hae Keun

2016-08-01

Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines.

Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

PubMed Central

Islam, Md. Zaherul; Yun, Hae Keun

2016-01-01

Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines. PMID:27493610

Identification and characterization of novel benzil (diphenylethane-1,2-dione) analogues as inhibitors of mammalian carboxylesterases.

PubMed

Wadkins, Randy M; Hyatt, Janice L; Wei, Xin; Yoon, Kyoung Jin P; Wierdl, Monika; Edwards, Carol C; Morton, Christopher L; Obenauer, John C; Damodaran, Komath; Beroza, Paul; Danks, Mary K; Potter, Philip M

2005-04-21

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

Analysis of the Inhibition of Mammalian Carboxylesterases by Novel Fluorobenzoins and Fluorobenzils

PubMed Central

Hicks, Latorya D.; Hyatt, Janice L; Moak, Teri; Edwards, Carol C.; Tsurkan, Lyudmila; Wierdl, Monika; Ferreira, Antonio; Wadkins, Randy M.; Potter, Philip M.

2007-01-01

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived Ki values with the fluorobenzoins. PMID:17399985

Characterization and optimization of carboxylesterase-catalyzed esterification between capric acid and glycerol for the production of 1-monocaprin in reversed micellar system.

PubMed

Park, Kyung Min; Kwon, Oh Taek; Ahn, Seon Min; Lee, JaeHwan; Chang, Pahn-Shick

2010-02-28

Calotropis procera R. Br. carboxylesterase (EC 3.1.1.1) solubilized in reversed micellar glycerol droplets containing a very small amount of water (less than 5ppm) and stabilized by a surfactant effectively catalyzed the esterification between glycerol and capric acid to produce 1-monocaprin. Reaction variables including surfactant types, organic solvent media, reaction time, G-value ([glycerol]/[capric acid]), R-value ([water]/[surfactant]), pH, temperature, and types of metal ion inhibitors on the carboxylesterase-catalyzed esterification were characterized and optimized to efficiently produce 1-monocaprin. Bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and isooctane were the most effective surfactant and organic solvent medium, respectively, for 1-monocaprin formation in reversed micelles. The optimum G- and R-values were 3.0 and 0.05, respectively, and the optimum pH and temperature were determined to be 10.0 and 60 degrees C, respectively. K(m,app.) and V(max,app.) were calculated from a Hanes-Woolf plot, and the values were 9.64 mM and 2.45 microM/min mg protein, respectively. Among various metal ions, Cu(2+) and Fe(2+) severely inhibited carboxylesterase-catalyzed esterification activity (less than 6.0% of relative activity). Copyright 2009 Elsevier B.V. All rights reserved.

Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

PubMed

Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

2016-04-01

A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

Identification and Expression Profile of Multiple Genes in Response to Magnesium Exposure in Culex quinquefasciatus Larvae

DTIC Science & Technology

2010-11-01

Vol. 47, no. 6 Potter and Wadkins 2006; Cui et al. 2007a, 2007b; Cummins et al. 2007; Holmes et al. 2009). One study showed that the activities of...carboxylesterases in re- sponse to three insecticides (i.e.,malathion, propoxur, and permethrin) differed by mosquito species: high activities of...al. 2004). Chitotrio- sidase secreted by activated human macrophages is involved in the defense against chitin-containing pathogens such as fungi

Role of geraniol against lead acetate-mediated hepatic damage and their interaction with liver carboxylesterase activity in rats.

PubMed

Ozkaya, Ahmet; Sahin, Zafer; Kuzu, Muslum; Saglam, Yavuz Selim; Ozkaraca, Mustafa; Uckun, Mirac; Yologlu, Ertan; Comakli, Veysel; Demirdag, Ramazan; Yologlu, Semra

2018-02-01

In this study, the effect of geraniol (50 mg/kg for 30 d), a natural antioxidant and repellent/antifeedant monoterpene, in a rat model of lead acetate-induced (500 ppm for 30 d) liver damage was evaluated. Hepatic malondialdehyde increased in the lead acetate group. Reduced glutathione unchanged, but glutathione S-transferase, glutathione reductase, as well as carboxylesterase activities decreased in geraniol, lead acetate and geraniol + lead acetate groups. 8-OhDG immunoreactivity, mononuclear cell infiltrations and hepatic lead concentration were lower in the geraniol + lead acetate group than the lead acetate group. Serum aspartate aminotransferase and alanine aminotransferase activities increased in the Pb acetate group. In conclusion, lead acetate causes oxidative and toxic damage in the liver and this effect can reduce with geraniol treatment. However, we first observed that lead acetate, as well as geraniol, can affect liver carboxylesterase activity.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anand, Sathanandam S.; Bruckner, James V.; Haines, Wendy T.

2006-04-15

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 {mu}M) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450more » metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 {mu}M of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax = 325.3 {+-} 53.4 nmol/h/ml and Km = 165.4 {+-} 41.9 {mu}M. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax = 1981.8 {+-} 132.3 nmol/h/g liver and Km = 172.5 {+-} 22.5 {mu}M. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax = 2611.3 {+-} 134.1 nmol/h/g liver) and higher affinity (Km = 74.9 {+-} 5.9 {mu}M) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4)« less

Gene cloning, expression, and characterization of a new carboxylesterase from Serratia sp. SES-01: comparison with Escherichia coli BioHe enzyme.

PubMed

Kwon, Min-A; Kim, Hyun Suk; Oh, Joon Young; Song, Bong Keun; Song, Jae Kwang

2009-02-01

The carboxylesterase-encoding gene (bioHs) of a newly isolated strain, Serratia sp. SES-01, was cloned from the genomic DNA library by detecting formation of transparent halo around the colony on LB-tributyrin agar plates. The amino acid sequence of BioHs was highly similar to the members of the BioH enzyme family involved in the biotin biosynthetic pathway; it showed the highest similarity (91%) with that of Serratia proteamaculans. To compare BioHs with other BioH enzymes, the relatively well-known bioHe gene of E. coli was cloned with PCR. After we achieved high-level expression of soluble BioHs and BioHe through the exploration of different culture conditions, the purified BioHs and BioHe enzymes were characterized in terms of specificity, activity, and stability. BioHe was generally more robust to a change in temperature and pH and an addition of organic solvents than BioHs. The two enzymes exhibited a strong preference for carboxylesterase rather than for thioesterase and were optimal at relatively low temperatures (20-40 degrees ) and alkaline pHs (7.5-9.0). The results in this study strongly suggested that both the BioHs and BioHe enzymes would be potential candidates for use as a carboxylesterase in many industrial applications.

Prilocaine- and lidocaine-induced methemoglobinemia is caused by human carboxylesterase-, CYP2E1-, and CYP3A4-mediated metabolic activation.

PubMed

Higuchi, Ryota; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

2013-06-01

Prilocaine and lidocaine are classified as amide-type local anesthetics for which serious adverse effects include methemoglobinemia. Although the hydrolyzed metabolites of prilocaine (o-toluidine) and lidocaine (2,6-xylidine) have been suspected to induce methemoglobinemia, the metabolic enzymes that are involved remain uncharacterized. In the present study, we aimed to identify the human enzymes that are responsible for prilocaine- and lidocaine-induced methemoglobinemia. Our experiments revealed that prilocaine was hydrolyzed by recombinant human carboxylesterase (CES) 1A and CES2, whereas lidocaine was hydrolyzed by only human CES1A. When the parent compounds (prilocaine and lidocaine) were incubated with human liver microsomes (HLM), methemoglobin (Met-Hb) formation was lower than when the hydrolyzed metabolites were incubated with HLM. In addition, Met-Hb formation when prilocaine and o-toluidine were incubated with HLM was higher than that when lidocaine and 2,6-xylidine were incubated with HLM. Incubation with diisopropyl fluorophosphate and bis-(4-nitrophenyl) phosphate, which are general inhibitors of CES, significantly decreased Met-Hb formation when prilocaine and lidocaine were incubated with HLM. An anti-CYP3A4 antibody further decreased the residual formation of Met-Hb. Met-Hb formation after the incubation of o-toluidine and 2,6-xylidine with HLM was only markedly decreased by incubation with an anti-CYP2E1 antibody. o-Toluidine and 2,6-xylidine were further metabolized by CYP2E1 to 4- and 6-hydroxy-o-toluidine and 4-hydroxy-2,6-xylidine, respectively, and these metabolites were shown to more efficiently induce Met-Hb formation than the parent compounds. Collectively, we found that the metabolites produced by human CES-, CYP2E1-, and CYP3A4-mediated metabolism were involved in prilocaine- and lidocaine-induced methemoglobinemia.

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE PAGES

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; ...

2017-03-08

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Molecular characterization of the amplified carboxylesterase gene associated with organophosphorus insecticide resistance in the brown planthopper, Nilaparvata lugens.

PubMed

Small, G J; Hemingway, J

2000-12-01

Widespread resistance to organophosphorus insecticides (OPs) in Nilaparvata lugens is associated with elevation of carboxylesterase activity. A cDNA encoding a carboxylesterase, Nl-EST1, has been isolated from an OP-resistant Sri Lankan strain of N. lugens. The full-length cDNA codes for a 547-amino acid protein with high homology to other esterases/lipases. Nl-EST1 has an N-terminal hydrophobic signal peptide sequence of 24 amino acids which suggests that the mature protein is secreted from cells expressing it. The nucleotide sequence of the homologue of Nl-EST1 in an OP-susceptible, low esterase Sri Lankan strain of N. lugens is identical to Nl-EST1. Southern analysis of genomic DNA from the Sri Lankan OP-resistant and susceptible strains suggests that Nl-EST1 is amplified in the resistant strain. Therefore, resistance to OPs in the Sri Lankan strain is through amplification of a gene identical to that found in the susceptible strain.

Characteristics of carboxylesterase genes and their expression-level between acaricide-susceptible and resistant Tetranychus cinnabarinus (Boisduval).

PubMed

Wei, Peng; Shi, Li; Shen, Guangmao; Xu, Zhifeng; Liu, Jialu; Pan, Yu; He, Lin

2016-07-01

Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung.

PubMed

Gabriele, Morena; Puccini, Paola; Lucchi, Marco; Vizziello, Anna; Gervasi, Pier Giovanni; Longo, Vincenzo

2018-04-01

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (V max ) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the V max of pNPA, as CES1 marker, were much greater (30-50-fold) than those of FD, as a specific CES2 marker. Conversely, the K m values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5-5-fold), FD or 4-MUA (8-15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ. Copyright © 2018 Elsevier Inc. All rights reserved.

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

PubMed Central

Abass, Kasim Sakran

2014-01-01

Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k cat/K m), and the K m and k cat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (k cat/K m), and the K m and k cat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester. PMID:24864243

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

PubMed

Denton, P W; Nochi, T; Lim, A; Krisko, J F; Martinez-Torres, F; Choudhary, S K; Wahl, A; Olesen, R; Zou, W; Di Santo, J P; Margolis, D M; Garcia, J V

2012-09-01

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides.

PubMed

Gong, Y-H; Ai, G-M; Li, M; Shi, X-Y; Diao, Q-Y; Gao, X-W

2017-12-01

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain. © 2017 The Royal Entomological Society.

Association of a carboxylesterase 1 polymorphism with appetite reduction in children and adolescents with attention-deficit/hyperactivity disorder treated with methylphenidate.

PubMed

Bruxel, E M; Salatino-Oliveira, A; Genro, J P; Zeni, C P; Polanczyk, G V; Chazan, R; Rohde, L A; Hutz, M H

2013-10-01

Carboxylesterase 1 is the enzyme involved in methylphenidate (MPH) metabolism. The aim of this study was to evaluate the association between a -75 T>G polymorphism and appetite reduction in children with attention-deficit/hyperactivity disorder (ADHD). A sample of 213 children with ADHD was investigated. The primary outcome was appetite reduction measured by the Barkley Stimulant Side Effect Rating Scale applied at baseline, at 1 and 3 months of treatment. MPH doses were augmented until no further clinical improvement or significant adverse events occurred. The G allele presented a trend for association with appetite reduction scores (P=0.05). A significant interaction between the G allele and treatment over time for appetite reduction scores was also observed (P=0.03). The G allele carriers presented a higher risk for appetite reduction worsening when compared with T allele homozygotes (odds ratio=3.47, P=0.01). The present results suggest an influence of carboxylesterase 1 -75 T>G polymorphism on the worsening of appetite reduction with MPH treatment in youths with ADHD.

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

NASA Astrophysics Data System (ADS)

Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

2014-07-01

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

Shengjiang Xiexin Decoction Alters Pharmacokinetics of Irinotecan by Regulating Metabolic Enzymes and Transporters: A Multi-Target Therapy for Alleviating the Gastrointestinal Toxicity.

PubMed

Guan, Huan-Yu; Li, Peng-Fei; Wang, Xiao-Ming; Yue, Jia-Jing; He, Yang; Luo, Xiao-Mei; Su, Mei-Feng; Liao, Shang-Gao; Shi, Yue

2017-01-01

Shengjiang Xiexin decoction (SXD), a classic traditional Chinese medical formula chronicled in Shang Han Lun , is used in modern clinical practice to decrease gastrointestinal toxicity induced by the chemotherapeutic drug irinotecan (CPT-11). In this study, the effect of SXD on the pharmacokinetics of CPT-11 and its active metabolites (SN-38 and SN-38G), and the underlying mechanisms were further examined. An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of CPT-11, SN-38, and SN-38G in the plasma, bile, liver, intestine, and intestinal contents of control and SXD-pre-treated rats after intravenous administration of CPT-11. SXD pretreatment increased the area under the curve (AUC) and the initial plasma concentration (C 0 ) of CPT-11 but decreased the plasma clearance (CL). The AUC and the maximum plasma concentration (C max ) of SN-38 decreased, whereas the C max of SN-38G increased. Compared with that of the control group, the biliary excretion of CPT-11, SN-38, and SN-38G was inhibited. The CPT-11, SN-38, and SN-38G concentrations in the liver, intestine, and intestinal contents were different between the two groups. Furthermore, the hepatic expression of multidrug resistance-associated protein-2 (Mrp-2), P-glycoprotein (P-gp), and carboxylesterase 2 (CES2) was significantly down-regulated by SXD, while the hepatic and jejunal uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) expression was elevated. The hydrolysis of CPT-11 to SN-38 by CES and the glucuronidation of SN-38 to SN-38G by UGT were affected by liver and jejunum S9 fractions from rats pre-treated with SXD. Therefore, this study demonstrated for the first time that SXD could alter the pharmacokinetics of CPT-11 and its metabolites to alleviate CPT-11-induced diarrhea. And the underlying mechanism of drug interaction between CPT-11 and SXD involves decreasing hepatic Mrp-2 and P-gp expression and altering the activities of CES and UGT.

Multifunctional Bioreactor System for Human Intestine Tissues

PubMed Central

2017-01-01

The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments. PMID:29333491

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

PubMed

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota.

PubMed

von Klitzing, Eliane; Ekmekciu, Ira; Kühl, Anja A; Bereswill, Stefan; Heimesaat, Markus M

2017-01-01

Within seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction. Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum. With respect to the intestinal microbiota composition "humanized" mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation.

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

PubMed

Dorywalska, Magdalena; Dushin, Russell; Moine, Ludivine; Farias, Santiago E; Zhou, Dahui; Navaratnam, Thayalan; Lui, Victor; Hasa-Moreno, Adela; Casas, Meritxell Galindo; Tran, Thomas-Toan; Delaria, Kathy; Liu, Shu-Hui; Foletti, Davide; O'Donnell, Christopher J; Pons, Jaume; Shelton, David L; Rajpal, Arvind; Strop, Pavel

2016-05-01

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR. ©2016 American Association for Cancer Research.

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota

PubMed Central

von Klitzing, Eliane; Ekmekciu, Ira; Kühl, Anja A.; Bereswill, Stefan

2017-01-01

Background Within seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction. Methodology/Principal findings Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum. Conclusion/Significance With respect to the intestinal microbiota composition “humanized” mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation. PMID:28414794

Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin

PubMed Central

Grigoraki, Linda; Pipini, Dimitra; Labbé, Pierrick; Chaskopoulou, Alexandra; Weill, Mylene; Vontas, John

2017-01-01

Background Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. Principal findings Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. Significance The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies. PMID:28394886

Human Carboxylesterase 1 Stereoselectively Binds the Nerve Agent Cyclosarin and Spontaneously Hydrolyzes the Nerve Agent Sarin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hemmert, Andrew C.; Otto, Tamara C.; Wierdl, Monika

Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex withmore » the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P{sub R} enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P{sub S} isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P{sub S} isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.« less

Homology modeling and metabolism prediction of human carboxylesterase-2 using docking analyses by GriDock: a parallelized tool based on AutoDock 4.0

NASA Astrophysics Data System (ADS)

Vistoli, Giulio; Pedretti, Alessandro; Mazzolari, Angelica; Testa, Bernard

2010-09-01

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted to developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas less has been done to predict the activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES2. The study involved first a homology modeling of the hCES2 protein based on the model of hCES1 since the two proteins share a high degree of homology (≅73%). A set of 40 known substrates of hCES2 was taken from the literature; the ligands were docked in both their neutral and ionized forms using GriDock, a parallel tool based on the AutoDock4.0 engine which can perform efficient and easy virtual screening analyses of large molecular databases exploiting multi-core architectures. Useful statistical models (e.g., r 2 = 0.91 for substrates in their unprotonated state) were calculated by correlating experimental pKm values with distance between the carbon atom of the substrate's ester group and the hydroxy function of Ser228. Additional parameters in the equations accounted for hydrophobic and electrostatic interactions between substrates and contributing residues. The negatively charged residues in the hCES2 cavity explained the preference of the enzyme for neutral substrates and, more generally, suggested that ligands which interact too strongly by ionic bonds (e.g., ACE inhibitors) cannot be good CES2 substrates because they are trapped in the cavity in unproductive modes and behave as inhibitors. The effects of protonation on substrate recognition and the contrasting behavior of substrates and products were finally investigated by MD simulations of some CES2 complexes.

Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans.

PubMed

Miyake, Masateru; Koga, Toshihisa; Kondo, Satoshi; Yoda, Noriaki; Emoto, Chie; Mukai, Tadashi; Toguchi, Hajime

2017-01-01

An adequate evaluation system for drug intestinal absorption is essential in the pharmaceutical industry. Previously, we established a novel prediction system of drug intestinal absorption in humans, using the mini-Ussing chamber equipped with human intestinal tissues. In this system, the TI value was defined as the sum of drug amounts transported to the basal-side component (X corr ) and drug amounts accumulated in the tissue (T corr ), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. In order to apply this system to the screening assay, it is important to understand the differences between animal and human tissues in the intestinal absorption of drugs. In this study, the transport index (TI) values of three drugs, with different levels of membrane permeability, were determined to evaluate the rank order of drug absorbability in intestinal tissues from rats, dogs, and monkeys. The TI values in small intestinal tissues in rats and dogs showed a good correlation with those in humans. On the other hand, the correlation of TI values in monkeys was lower compared to rats and dogs. The rank order of the correlation coefficient between human and investigated animal tissues was as follows: dog (r 2 =0.978), rat (r 2 =0.955), and monkey (r 2 =0.620). TI values in large intestinal tissues from rats (r 2 =0.929) and dogs (r 2 =0.808) also showed a good correlation. The obtained TI values in small intestinal tissues in rats and dogs were well correlated with the fraction of drug absorbed (F a ) in humans. From these results, the mini-Ussing chamber, equipped with intestinal tissues in rats and dogs, would be useful as a screening tool in the drug discovery stage. In addition, the obtained TI values can be used for the prediction of the F a in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

PubMed Central

Ganguli, Kriston; Collado, Maria Carmen; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W. Allan; Rautava, Samuli

2015-01-01

Background Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Methods TNF-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Results Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. Conclusion The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human intestinal epithelial cells and directly modulates intestinal epithelial cell innate immune gene expression. PMID:25580735

Elevated carboxylesterase activity contributes to the lambda-cyhalothrin insensitivity in quercetin fed Helicoverpa armigera (Hübner).

PubMed

Chen, Chengyu; Liu, Ying; Shi, Xueyan; Desneux, Nicolas; Han, Peng; Gao, Xiwu

2017-01-01

Quercetin as one of the key plant secondary metabolite flavonol is ubiquitous in terrestrial plants. In this study, the decrease in sensitivity to lambda-cyhalothrin was observed in quercetin-fed Helicoverpa armigera larvae. In order to figure out the mechanisms underlying the decreased sensitivity of H. armigera larvae to lambda-cyhalothrin by quercetin induction, the changes in carboxylesterase activity and in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin were examined. The LC50 value of quercetin-fed H. armigera larvae to lambda-cyhalothrin showed 2.41-fold higher than that of the control. S, S, S-Tributyl phosphorotrithioate (DEF) treatment showed a synergism effect on lambda-cyhalothrin toxicity to quercetin-fed H. armigera. Moreover, the activity of carboxylesterase was significantly higher in quercetin-fed H. armigera larvae after fed on quercetin for 48 h. The in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera larvae midgut was 289.82 nmol 3-PBA/mg protein/min, which is significant higher than that in the control group (149.60 nmol 3-PBA/mg protein/min). The elevated CarE enzyme activity and corresponding increased hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera contributed to the enhanced tolerance to lambda-cyhalothrin.

Elevated carboxylesterase activity contributes to the lambda-cyhalothrin insensitivity in quercetin fed Helicoverpa armigera (Hübner)

PubMed Central

Chen, Chengyu; Liu, Ying; Desneux, Nicolas; Han, Peng; Gao, Xiwu

2017-01-01

Quercetin as one of the key plant secondary metabolite flavonol is ubiquitous in terrestrial plants. In this study, the decrease in sensitivity to lambda-cyhalothrin was observed in quercetin-fed Helicoverpa armigera larvae. In order to figure out the mechanisms underlying the decreased sensitivity of H. armigera larvae to lambda-cyhalothrin by quercetin induction, the changes in carboxylesterase activity and in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin were examined. The LC50 value of quercetin-fed H. armigera larvae to lambda-cyhalothrin showed 2.41-fold higher than that of the control. S, S, S-Tributyl phosphorotrithioate (DEF) treatment showed a synergism effect on lambda-cyhalothrin toxicity to quercetin-fed H. armigera. Moreover, the activity of carboxylesterase was significantly higher in quercetin-fed H. armigera larvae after fed on quercetin for 48 h. The in-vitro hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera larvae midgut was 289.82 nmol 3-PBA/mg protein/min, which is significant higher than that in the control group (149.60 nmol 3-PBA/mg protein/min). The elevated CarE enzyme activity and corresponding increased hydrolytic metabolic capacity to lambda-cyhalothrin in quercetin-fed H. armigera contributed to the enhanced tolerance to lambda-cyhalothrin. PMID:28817718

Influence of polychlorinated aromatic compounds on the biotransformation and toxicity of organophosphorus pesticides (OP) to the Daphnia magna

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tonkopii, V.; Zagrebin, A.; Sherstneva, L.

1995-12-31

The effect of different polychlorinated aromatics (DDT, Aroclor 1254, certain polychlorinated biphenyls and dibenzofurans) on the toxicity of OP (DDVP paraoxon, malaoxon) to Daphnia magna was studied. Pretreatment of daphnids with chlorinated compounds during 72 hours in nontoxic concentrations (1/5--1/20 CL{sub 50}) has been shown to reduce the toxicity of OP for hydrobionts. For study of influence of chlorinated compounds on biotransformation of OP the activity of enzymes which are hydrolyzing the OP was investigated in Daphnia`s homogenates or microsomes. The activity of carboxylesterase (tributyrinase, aliesterase) and arylesterase (phosphorylphosphatase) with usage as substrates accordingly {alpha}-naphthylacetate and paraoxon was measured. Besidesmore » that the activity of cholinesterase with application of propionylthiocholine as substrate was determined. After polychlorinated aromatic compounds treatment of daphnids activities of both aryl-and carboxylesterase increased markedly. It decreased the inhibition of Daphnia`s cholinesterase caused by incubation with OP in concentrations 0.5--1.0 CL{sub 50}. Thus the induction by chlorinate aromatics of OP metabolizing enzymes seems to play the important role in reduction of OP toxicity to Daphnia magna. Perhaps the aryl- and carboxylesterase of Daphnia can be used as biomarkers of pollution by polychlorinated aromatics in water.« less

Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.

PubMed

Nomura, Taiji; Murase, Tatsunori; Ogita, Shinjiro; Kato, Yasuo

2015-07-01

6-Tuliposides A (PosA) and B (PosB), which are the major secondary metabolites in tulip (Tulipa gesneriana), are enzymatically converted to the antimicrobial lactonized aglycons, tulipalins A (PaA) and B (PaB), respectively. We recently identified a PosA-converting enzyme (TCEA) as the first reported member of the lactone-forming carboxylesterases. Herein, we describe the identification of another lactone-forming carboxylesterase, PosB-converting enzyme (TCEB), which preferentially reacts with PosB to give PaB. This enzyme was isolated from tulip pollen, which showed high PosB-converting activity. Purified TCEB exhibited greater activity towards PosB than PosA, which was contrary to that of the TCEA. Novel cDNA (TgTCEB1) encoding the TCEB was isolated from tulip pollen. TgTCEB1 belonged to the carboxylesterase family and was approximately 50% identical to the TgTCEA polypeptides. Functional characterization of the recombinant enzyme verified that TgTCEB1 catalyzed the conversion of PosB to PaB with an activity comparable with the native TCEB. RT-qPCR analysis of each part of plant revealed that TgTCEB1 transcripts were limited almost exclusively to the pollen. Furthermore, the immunostaining of the anther cross-section using anti-TgTCEB1 polyclonal antibody verified that TgTCEB1 was specifically expressed in the pollen grains, but not in the anther cells. N-terminal transit peptide of TgTCEB1 was shown to function as plastid-targeted signal. Taken together, these results indicate that mature TgTCEB1 is specifically localized in plastids of pollen grains. Interestingly, PosB, the substrate of TgTCEB1, accumulated on the pollen surface, but not in the intracellular spaces of pollen grains. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

PubMed Central

Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

2013-01-01

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development.

PubMed

Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H; Nagy, Melinda S; Chin, Alana M; Thomson, Matthew; Klein, Ophir D; Spence, Jason R

2017-03-15

The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research. © 2017. Published by The Company of Biologists Ltd.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation.

PubMed

Lanik, Wyatt E; Xu, Lily; Luke, Cliff J; Hu, Elise Z; Agrawal, Pranjal; Liu, Victoria S; Kumar, Rajesh; Bolock, Alexa M; Ma, Congrong; Good, Misty

2018-02-15

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

New catalytic roles for serine esterases: a 19F-NMR study of the interaction of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone with chicken liver carboxylesterase.

PubMed

Bowles, M R; King, G J; Berndt, M C; Zerner, B

1996-12-05

The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.

Understanding How Commensal Obligate Anaerobic Bacteria Regulate Immune Functions in the Large Intestine

PubMed Central

Maier, Eva; Anderson, Rachel C.; Roy, Nicole C.

2014-01-01

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases. PMID:25545102

Understanding how commensal obligate anaerobic bacteria regulate immune functions in the large intestine.

PubMed

Maier, Eva; Anderson, Rachel C; Roy, Nicole C

2014-12-24

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases.

The intestine is a blender

NASA Astrophysics Data System (ADS)

Yang, Patricia; Lamarca, Morgan; Kravets, Victoria; Hu, David

According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines Contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

The intestine is a blender

NASA Astrophysics Data System (ADS)

Yang, Patricia; Lamarca, Morgan; Hu, David

2015-11-01

According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crow, J. Allen; Bittles, Victoria; Herring, Katye L.

2012-01-01

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL,more » EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals. -- Highlights: ► IC{sub 50} values and bimolecular rate constants (k{sub inact}/K{sub I}) of human recombinant CES1, CES2, and MGL proteins and chlorpyrifos oxon, paraoxon and methyl paraoxon were determined. ► The IC{sub 50} values for the oxons with CES1, CES2, and MGL followed the rank order: chlorpyrifos oxon > paraoxon > methyl paraoxon. ► The order of reactivity for the oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. ► Chlorpyrifos oxon was less reactive with MGL than with either CES1 or CES2.« less

Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

USDA-ARS?s Scientific Manuscript database

Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

Screening of Exopolysaccharide-Producing Lactobacillus and Bifidobacterium Strains Isolated from the Human Intestinal Microbiota▿

PubMed Central

Ruas-Madiedo, Patricia; Moreno, José Antonio; Salazar, Nuria; Delgado, Susana; Mayo, Baltasar; Margolles, Abelardo; de los Reyes-Gavilán, Clara G.

2007-01-01

Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains. PMID:17483284

Xanthine oxido-reductase activity in ischemic human and rat intestine.

PubMed

Bianciardi, Paola; Scorza, Roberto; Ghilardi, Giorgio; Samaja, Michele

2004-09-01

We measured time course and extent of xanthine dehydrogenase (XD) to xanthine oxidase (XO) conversion in ischemic human and rat intestine. To model normothermic no-flow ischemia, we incubated fresh biopsies for 0, 2, 4, 8 and 16h. At t = 0h, XO was less in humans than in rats (P < 0.0004), while XD was essentially the same (P = NS). After 16h incubation at 37 degrees C, there was no appreciable XD-to-XO conversion and no change in neither XO nor XD activity in human intestine. In contrast, the rat intestine had XO/(XO + XD) ratio doubled in the first 2h and then maintained that value until t = 16 h. In conclusion, no XO-to-XD conversion was appreciable after 16 h no-flow normothermic ischemia in human intestine; in contrast, XO activity in rats increased sharply after the onset of ischemia. An immunohistochemical labelling study shows that, whereas XO + XD expression in liver tissue is localised in both hepatocytes and endothelial cells, in the intestine that expression is mostly localised in epithelial cells. We conclude that XO may be considered as a major source of reactive oxygen species in rats but not in humans.

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy

PubMed Central

2013-01-01

Introduction Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury. Methods Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility. Results There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups. Conclusions Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis. PMID:24423414

In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses.

PubMed

Chen, Ying; Zhou, Wenda; Roh, Terrence; Estes, Mary K; Kaplan, David L

2017-01-01

There is a need for functional in vitro 3D human intestine systems that can bridge the gap between conventional cell culture studies and human trials. The successful engineering in vitro of human intestinal tissues relies on the use of the appropriate cell sources, biomimetic scaffolds, and 3D culture conditions to support vital organ functions. We previously established a compartmentalized scaffold consisting of a hollow space within a porous bulk matrix, in which a functional and physiologically relevant intestinal epithelium system was generated using intestinal cell lines. In this study, we adopt the 3D scaffold system for the cultivation of stem cell-derived human small intestinal enteriods (HIEs) to engineer an in vitro 3D model of a nonstransformed human small intestinal epithelium. Characterization of tissue properties revealed a mature HIE-derived epithelium displaying four major terminally differentiated epithelial cell types (enterocytes, Goblet cells, Paneth cells, enteroendocrine cells), with tight junction formation, microvilli polarization, digestive enzyme secretion, and low oxygen tension in the lumen. Moreover, the tissue model demonstrates significant antibacterial responses to E. coli infection, as evidenced by the significant upregulation of genes involved in the innate immune response. Importantly, many of these genes are activated in human patients with inflammatory bowel disease (IBD), implicating the potential application of the 3D stem-cell derived epithelium for the in vitro study of host-microbe-pathogen interplay and IBD pathogenesis.

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kacham, R.; Karanth, S.; Baireddy, P.

2006-01-15

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequentialmore » dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less capable than adults at detoxifying many organophosphorus insecticides including chlorpyrifos and parathion, toxicant-selective differences in detoxification play a role in sequence-dependent toxicity in both neonatal and adult rats with these two insecticides.« less

Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

PubMed Central

Van den Bogert, Bartholomeus; Boekhorst, Jos; Herrmann, Ruth; Smid, Eddy J.; Zoetendal, Erwin G.; Kleerebezem, Michiel

2013-01-01

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine. PMID:24386196

The Intestinal Microbiome and Health

PubMed Central

Tuddenham, Susan; Sears, Cynthia L.

2015-01-01

Purpose of Review A diverse array of microbes colonizes the human intestine. In this review we seek to outline the current state of knowledge on what characterizes a “healthy” or “normal” intestinal microbiome, what factors modify the intestinal microbiome in the healthy state and how the intestinal microbiome affects normal host physiology Recent Findings What constitutes a “normal” or “healthy” intestinal microbiome is an area of active research, but key characteristics may include diversity, richness and a microbial community’s resilience and ability to resist change. A number of factors, including age, the host immune system, host genetics, diet and antibiotic use appear to modify the intestinal microbiome in the normal state. New research shows that the microbiome likely plays a critical role in the healthy human immune system and metabolism. Summary It is clear that there is a complicated bi-directional relationship between the intestinal microbiota and host which is vital to health. An enhanced understanding of this relationship will be critical not only to maximize and maintain human health but also to shape our understanding of disease and to foster new therapeutic approaches. PMID:26237547

Clostridium perfringens Sialidases: Potential Contributors to Intestinal Pathogenesis and Therapeutic Targets

PubMed Central

Li, Jihong; Uzal, Francisco A.; McClane, Bruce A.

2016-01-01

Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene (clostridial myonecrosis), remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections. PMID:27869757

Clostridium perfringens Sialidases: Potential Contributors to Intestinal Pathogenesis and Therapeutic Targets.

PubMed

Li, Jihong; Uzal, Francisco A; McClane, Bruce A

2016-11-19

Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene (clostridial myonecrosis), remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections.

Notum deacylates Wnts to suppress signalling activity

PubMed Central

Howell, Steve; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O’Reilly, Nicola; Snijders, Ambrosius P.; Jones, E. Yvonne; Vincent, Jean-Paul

2015-01-01

Signalling by Wnts is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnts from the cell surface. However, this view fails to explain specificity since glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which likely help Notum colocalise with Wnts. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnts and thus constitutes the first known extracellular protein deacylase. PMID:25731175

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Pathogenicity of Shigella in chickens.

PubMed

Shi, Run; Yang, Xia; Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

2014-01-01

Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance.

Pathogenicity of Shigella in Chickens

PubMed Central

Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

2014-01-01

Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance. PMID:24949637

Population Pharmacokinetics of Methylphenidate in Healthy Adults Emphasizing Novel and Known Effects of Several Carboxylesterase 1 (CES1) Variants

PubMed Central

Stage, C; Bergmann, TK; Ferrero‐Milliani, L; Bjerre, D; Thomsen, R; Dalhoff, KP; Rasmussen, HB; Jürgens, G

2016-01-01

The aim of this study was to identify demographic and genetic factors that significantly affect methylphenidate (MPH) pharmacokinetics (PK), and may help explain interindividual variability and further increase the safety of MPH. d‐MPH plasma concentrations, demographic covariates, and carboxylesterase 1 (CES1) genotypes were gathered from 122 healthy adults and analyzed using nonlinear mixed effects modeling. The structural model that best described the data was a two‐compartment disposition model with absorption transit compartments. Novel effects of rs115629050 and CES1 diplotypes, as well as previously reported effects of rs71647871 and body weight, were included in the final model. Assessment of the independent and combined effect of CES1 covariates identified several specific risk factors that may result in severely increased d‐MPH plasma exposure. PMID:27754602

Gene expression analysis and enzyme assay reveal a potential role of the carboxylesterase gene CpCE-1 from Cydia pomonella in detoxification of insecticides.

PubMed

Yang, Xue-Qing

2016-05-01

Carboxylesterases (CarEs) are responsible for metabolism of xenobiotics including insecticides in insects. Understanding the expression patterns of a such detoxifying gene and effect of insecticides on its enzyme activity are important to clarify the function of this gene relevant to insecticides-detoxifying process, but little information is available in the codling moth Cydia pomonella (L.). In this study, we investigated the expression profiles of CarE gene CpCE-1 at different developmental stages and in different tissues of C. pomonella, as well as the larvae exposed to chlorpyrifos-ethyl and lambda-cyhalothrin by using absolute real-time quantitative PCR (absolute RT-qPCR). Results indicated that CpCE-1 expression was significantly altered during C. pomonella development stages, and this expression differed between sexes, with a higher transcript in females than males. Meanwhile, CpCE-1 is overexpressed in cuticle, midgut and head than silk gland, fat body and Malpighian tubules. Exposure of third instar larvae to a non-lethal dosage of chlorpyrifos-ethyl and lambda-cyhalothrin resulted in induction of CpCE-1 transcript. The total carboxylesterase enzyme activity was inhibited by chlorpyrifos-ethyl in vivo; in contrast, the activity of Escherichia coli produced recombinant CpCE-1 was significantly inhibited by both lambda-cyhalothrin and chlorpyrifos-ethyl in vitro. These results suggested that CpCE-1 in C. pomonella is potentially involved in the development and in detoxification of chlorpyrifos-ethyl and lambda-cyhalothrin.

Correlation between Pesticide Resistance and Enzyme Activity in the Diamondback Moth, Plutella xylostella

PubMed Central

Gong, Ya-Jun; Wang, Ze-Hua; Shi, Bao-Cai; Kang, Zong-Jiang; Zhu, Liang; Jin, Gui-Hua; Weig, Shu-Jun

2013-01-01

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most important pests that has developed high pesticide resistance. The resistances of five Chinese populations of this moth, four resistant strains (from Beijing, Henan, Fujian, and Guangdong) and one susceptible strain, to five pesticides were determined, and the activities of carboxylesterase, glutathione S-transferase, and acetylcholine esterase were tested in all five populations. The correlations between pesticide resistance and enzyme activity were analyzed. The results showed that the resistance status to the five pesticides was different among the five populations. The resistance ratios of the Beijing and Henan populations to spinosad were 5.84 and 8.22, respectively, and those to beta-cypermethrin were 4.91 and 4.98, respectively. These ratios were higher than those for the Fujian and Guangdong populations. The Fujian population was more sensitive to abamectin and chlorpyrifos than the susceptible population (the resistance ratios were 0.14 and 0.91, respectively); in fact, the median lethal concentration for P. xylostella was significantly higher for chlorpyrifos than that for any of the other four pesticides. The carboxylesterase activity in P. xylostella showed positive correlations with the resistance to spinosad, beta-cypermethrin, chlorpyrifos, and abamectin, but no correlation was observed between the carboxylesterase activity and resistance to emamectin benzoate, between glutathione S-transferase activity and resistance to any of the five pesticides tested, or between acetylcholine esterase activity and any of the pesticides except for emamectin benzoate. PMID:24766444

Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

PubMed Central

Ohki, Taku; Shibata, Naoki; Higuchi, Yoshiki; Kawashima, Yasuyuki; Takeo, Masahiro; Kato, Dai-ichiro; Negoro, Seiji

2009-01-01

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s−1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-Oɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold. PMID:19521995

Rifaximin Exerts Beneficial Effects Independent of its Ability to Alter Microbiota Composition.

PubMed

Kang, Dae J; Kakiyama, Genta; Betrapally, Naga S; Herzog, Jeremy; Nittono, Hiroshi; Hylemon, Phillip B; Zhou, Huiping; Carroll, Ian; Yang, Jing; Gillevet, Patrick M; Jiao, Chunhua; Takei, Hajime; Pandak, William M; Iida, Takashi; Heuman, Douglas M; Fan, Sili; Fiehn, Oliver; Kurosawa, Takao; Sikaroodi, Masoumeh; Sartor, R B; Bajaj, Jasmohan S

2016-08-25

Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin. Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100 mg/kg from days 16-30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1β, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1β, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation). All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice. Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.

[Breast milk: its nutritional composition and functional properties].

PubMed

Tackoen, M

2012-09-01

Human milk is a complex biological fluid with thousands of components. The milk composition in the mammalian species is specific and adapted to the needs of the offspring. It contains macronutrients (proteins, lipids and carbohydrates), micronutrients (minerals and vitamins) and numerous biologically active substrates. Human milk not only covers the nutritional needs of the newborn but protects the baby against infection, inflammation and oxidative stress. It has immunomodulation properties and confers trophical protection to the intestinal mucosa. The newborn infant is particularly immature: innate immunity, adaptive immunity and intestinal immaturity. Human milk will offer this exogenous protective and immunomodulating source. The development of the composition of the intestinal microflora of the neonate will be impacted by pre- and probiotic components of human milk. Current scientific knowledge of human milk properties highlights interdependency of the different components, ontogeny of the intestinal function, development of the mucosal intestinal immune system, colonization by the intestinal microbiota and protection against pathogens. Quality of these interactions influences the newborn's short and long-term health status. The promotion of breastfeeding with the support of the Baby Friendly Hospital Initiative (BFHI) program and labeling has been shown to have positive impact in public health.

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner.

PubMed

Drummond, Coyne G; Bolock, Alexa M; Ma, Congrong; Luke, Cliff J; Good, Misty; Coyne, Carolyn B

2017-02-14

Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.

Canine intestinal contents vs. simulated media for the assessment of solubility of two weak bases in the human small intestinal contents.

PubMed

Kalantzi, Lida; Persson, Eva; Polentarutti, Britta; Abrahamsson, Bertil; Goumas, Konstantinos; Dressman, Jennifer B; Reppas, Christos

2006-06-01

This study was conducted to assess the relative usefulness of canine intestinal contents and simulated media in the prediction of solubility of two weak bases (dipyridamole and ketoconazole) in fasted and fed human intestinal aspirates that were collected under conditions simulating those in bioavailability/bioequivalence studies. After administration of 250 mL of water or 500 mL of Ensure plus [both containing 10 mg/mL polyethylene glycol (PEG) 4000 as nonabsorbable marker], intestinal aspirates were collected from the fourth part of the duodenum of 12 healthy adults and from the mid-jejunum of four Labradors. Pooled samples were analyzed for PEG, pH, buffer capacity, osmolality, surface tension, pepsin, total carbohydrates, total protein content, bile salts, phospholipids, and neutral lipids. The shake-flask method was used to measure the solubility of dipyridamole and ketoconazole in pooled human and canine intestinal contents and in fasted-state-simulating intestinal fluid (FaSSIF) and fed-state-simulating intestinal fluid (FeSSIF) containing various bile salts and pH-buffering agents. For both compounds, solubility in canine contents may be predictive of human intralumenal solubility in the fasting state but not in the fed state. The poor agreement of results in canine and human aspirates can be attributed to the higher bile salt content in canine bile. Solubility in FaSSIF containing a mixture of bile salts from crude bile predicted satisfactorily the intralumenal solubility of both drugs in the fasted state in humans. Solubility in FeSSIF, regardless of the identity of bile salts or of the buffering species, deviated from intralumenal values in the fed human aspirates by up to 40%. This was attributed to the lack of lipolytic products in FeSSIF, the higher bile salt content of FeSSIF, and the lower pH of FeSSIF. FaSSIF containing a mixture of bile salts from crude bile, and FeSSIF containing lipolytic products and, perhaps, having lower bile salt content but slightly higher pH, should be more useful than canine intestinal aspirates for predicting intralumenal solubilities in humans.

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application.

PubMed

Fujikawa, Yuuta; Nampo, Taiki; Mori, Masaya; Kikkawa, Manami; Inoue, Hideshi

2018-03-01

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

PubMed Central

Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

2013-01-01

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

PubMed

Jung, Kwang Bo; Lee, Hana; Son, Ye Seul; Lee, Ji Hye; Cho, Hyun-Soo; Lee, Mi-Ok; Oh, Jung-Hwa; Lee, Jaemin; Kim, Seokho; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

2018-01-01

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5 mCherry ) and intestine-specific homeobox enhanced green fluorescence protein (ISX eGFP ). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. © FASEB.

A Revised Model for Dosimetry in the Human Small Intestine

DOE Office of Scientific and Technical Information (OSTI.GOV)

John Poston; Nasir U. Bhuiyan; R. Alex Redd

2005-02-28

A new model for an adult human gastrointestinal tract (GIT) has been developed for use in internal dose estimations to the wall of the GIT and to the other organs and tissues of the body from radionuclides deposited in the lumenal contents of the five sections of the GIT. These sections were the esophasgus, stomach, small intestine, upper large intestine, and the lower large intestine. The wall of each section was separated from its lumenal contents.

The Role of Intestinal Alkaline Phosphatase in Inflammatory Disorders of Gastrointestinal Tract.

PubMed

Bilski, Jan; Mazur-Bialy, Agnieszka; Wojcik, Dagmara; Zahradnik-Bilska, Janina; Brzozowski, Bartosz; Magierowski, Marcin; Mach, Tomasz; Magierowska, Katarzyna; Brzozowski, Tomasz

2017-01-01

Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients.

The Role of Intestinal Alkaline Phosphatase in Inflammatory Disorders of Gastrointestinal Tract

PubMed Central

Wojcik, Dagmara; Zahradnik-Bilska, Janina; Mach, Tomasz

2017-01-01

Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients. PMID:28316376

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models

PubMed Central

Govers, Coen; van der Meulen, Jan; van Hoef, Angeline; Stoopen, Geert; Hamers, Astrid; Hoekman, Arjan; de Vos, Ric; Bovee, Toine F. H.; Smits, Mari; Mes, Jurriaan J.

2016-01-01

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies. PMID:27631494

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

PubMed

de Wit, Nicole J W; Hulst, Marcel; Govers, Coen; van der Meulen, Jan; van Hoef, Angeline; Stoopen, Geert; Hamers, Astrid; Hoekman, Arjan; de Vos, Ric; Bovee, Toine F H; Smits, Mari; Mes, Jurriaan J; Hendriksen, Peter J M

2016-01-01

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

Metabolism and Fitness of Urinary Tract Pathogens.

PubMed

Alteri, Christopher J; Mobley, Harry L T

2015-06-01

Among common infections, urinary tract infections (UTI) are the most frequently diagnosed urologic disease. The majority of UTIs are caused by uropathogenic Escherichia coli. The primary niche occupied by E. coli is the lower intestinal tract of mammals, where it resides as a beneficial component of the commensal microbiota. Although it is well-known that E. coli resides in the human intestine as a harmless commensal, specific strains or pathotypes have the potential to cause a wide spectrum of intestinal and diarrheal diseases. In contrast, extraintestinal E. coli pathotypes reside harmlessly in the human intestinal microenvironment but, upon access to sites outside of the intestine, become a major cause of human morbidity and mortality as a consequence of invasive UTI (pyelonephritis, bacteremia, or septicemia). Thus, extraintestinal pathotypes like uropathogenic E. coli (UPEC) possess an enhanced ability to cause infection outside of the intestinal tract and colonize the urinary tract, the bloodstream, or cerebrospinal fluid of human hosts. Due to the requirement for these E. coli to replicate in and colonize both the intestine and extraintestinal environments, we posit that physiology and metabolism of UPEC strains is paramount. Here we discuss that the ability to survive in the urinary tract depends as much on bacterial physiology and metabolism as it does on the well-considered virulence determinants.

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids.

PubMed

Hill, David R; Huang, Sha; Tsai, Yu-Hwai; Spence, Jason R; Young, Vincent B

2017-12-18

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

Effects of Probiotics, Prebiotics, and Synbiotics on Human Health

PubMed Central

Markowiak, Paulina; Śliżewska, Katarzyna

2017-01-01

The human gastrointestinal tract is colonised by a complex ecosystem of microorganisms. Intestinal bacteria are not only commensal, but they also undergo a synbiotic co-evolution along with their host. Beneficial intestinal bacteria have numerous and important functions, e.g., they produce various nutrients for their host, prevent infections caused by intestinal pathogens, and modulate a normal immunological response. Therefore, modification of the intestinal microbiota in order to achieve, restore, and maintain favourable balance in the ecosystem, and the activity of microorganisms present in the gastrointestinal tract is necessary for the improved health condition of the host. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota. They may be consumed in the form of raw vegetables and fruit, fermented pickles, or dairy products. Another source may be pharmaceutical formulas and functional food. This paper provides a review of available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health. The mechanism of beneficial action of those substances is discussed, and verified study results proving their efficacy in human nutrition are presented. PMID:28914794

Effects of Probiotics, Prebiotics, and Synbiotics on Human Health.

PubMed

Markowiak, Paulina; Śliżewska, Katarzyna

2017-09-15

The human gastrointestinal tract is colonised by a complex ecosystem of microorganisms. Intestinal bacteria are not only commensal, but they also undergo a synbiotic co-evolution along with their host. Beneficial intestinal bacteria have numerous and important functions, e.g., they produce various nutrients for their host, prevent infections caused by intestinal pathogens, and modulate a normal immunological response. Therefore, modification of the intestinal microbiota in order to achieve, restore, and maintain favourable balance in the ecosystem, and the activity of microorganisms present in the gastrointestinal tract is necessary for the improved health condition of the host. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota. They may be consumed in the form of raw vegetables and fruit, fermented pickles, or dairy products. Another source may be pharmaceutical formulas and functional food. This paper provides a review of available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health. The mechanism of beneficial action of those substances is discussed, and verified study results proving their efficacy in human nutrition are presented.

Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

PubMed

Hanioka, Nobumitsu; Isobe, Takashi; Kinashi, Yu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

2016-07-01

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

PubMed

Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

2015-11-02

Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

Molecular Epidemiology of Human Intestinal Amoebas in Iran

PubMed Central

Hooshyar, H; Rostamkhani, P; Rezaian, M

2012-01-01

Many microscopic-based epidemiological surveys on the prevalence of human intestinal pathogenic and non-pathogenic protozoa including intestinal amoeba performed in Iran show a high prevalence of human intestinal amoeba in different parts of Iran. Such epidemiological studies on amoebiasis are confusing, mainly due to recently appreciated distinction between the Entamoeba histolytica, E. dispar and E. moshkovskii. Differential diagnosis can be done by some methods such as PCR-based methods, monoclonal antibodies and the analysis of isoenzyme typing, however the molecular study of these protozoa in Iran is low. Based on molecular studies, it seems that E. dispar is predominant species especially in the central and northern areas of Iran and amoebiasis due to E. histolytica is a rare infection in the country. It is suggested that infection with E. moshkovskii may be common among Iranians. Considering the importance of molecular epidemiology of amoeba in Iran and also the current data, the present study reviews the data currently available on the molecular distribution of intestinal human amoeba in Iran. PMID:23193500

Intestinal organoids: A model of intestinal fibrosis for evaluating anti-fibrotic drugs

PubMed Central

Rodansky, Eva S.; Johnson, Laura A.; Huang, Sha; Spence, Jason R.; Higgins, Peter D. R.

2016-01-01

Background & Aims Intestinal fibrosis is a critical complication of Crohn’s disease (CD). Current in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, while in vivo rodent models do not fully recapitulate human disease and have limited utility for large-scale screening. Here, we exploit recent advances in stem cell derived human intestinal organoids (HIOs) as a new human model of fibrosis in CD. Methods Human pluripotent stem cells were differentiated into HIOs. We identified myofibroblasts, the key effector cells of fibrosis, by immunofluorescence staining for alpha-smooth muscle actin (αSMA), vimentin, and desmin. We examined the fibrogenic response of HIOs by treatment with transforming growth factor beta (TGFβ) in the presence or absence of the anti-fibrotic drug spironolactone. Fibrotic response was assayed by expression of fibrogenic genes (COL1A1 (collagen, type I, alpha 1), ACTA2 (alpha smooth muscle actin), FN1 (fibronectin 1), MYLK (myosin light chain kinase), and MKL1 (megakaryoblastic leukemia (translocation) 1)) and proteins (αSMA). Results Immunofluorescent staining of organoids identified a population of myofibroblasts within the HIO mesenchyme. TGFβ stimulation of HIOs produced a dose-dependent pro-fibrotic response. Spironolactone treatment blocked the fibrogenic response of HIOs to TGFβ. Conclusions HIOs contain myofibroblasts and respond to a pro-fibrotic stimulus in a manner that is consistent with isolated human myofibroblasts. HIOs are a promising model system that might bridge the gap between current in vitro and in vivo models of intestinal fibrosis in IBD. PMID:25828392

The metabolic profile of acteoside produced by human or rat intestinal bacteria or intestinal enzyme in vitro employed UPLC-Q-TOF-MS.

PubMed

Cui, Qingling; Pan, Yingni; Xu, Xiaotong; Zhang, Wenjie; Wu, Xiao; Qu, Shouhe; Liu, Xiaoqiu

2016-03-01

Acteoside, the main and representative phenylethanoid glycosides of Herba Cistanches, possesses wide bioactivities but low oral bioavailability. It may serve as the prodrug and be converted into the active forms in gastrointestinal tract, which mainly occurred in intestinal tract composed of intestinal bacteria and intestinal enzyme. Intestinal bacteria, a new drug target, take a significant role on exerting pharmacological effects of drugs by oral administration. In this paper, acteoside was incubated with human or rat intestinal bacteria or rat intestinal enzyme for 36 h to seek metabolites responsible for pharmacodynamics. The samples were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Besides the parent compound, 14 metabolites were detected and identified based on their retention times and fragmentation patterns in their MS spectra including 8 degradation metabolites, 2 isomers in intestinal bacteria and intestinal enzyme samples and 4 parent metabolites only found in intestinal enzymes. The metabolic pathway of acteoside was thus proposed. Identification of these metabolites of acteoside by the intestinal bacteria or intestinal enzyme gave an insight to clarify pharmacological mechanism of traditional Chinese medicines and identify the real active molecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Systematic review: microbial dysbiosis and nonalcoholic fatty liver disease.

PubMed

Wieland, A; Frank, D N; Harnke, B; Bambha, K

2015-11-01

The human intestinal microbiota is a key regulator of host metabolic and immune functions and alterations in the microbiome ('dysbiosis') have been implicated in several human diseases. Because of the anatomical links between the intestines and the liver, dysbiosis may also disrupt hepatic function and thereby contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). To perform a comprehensive review of the medical literature investigating associations between intestinal dysbiosis and NAFLD, with a particular emphasis on studies that characterise the microbiome in NAFLD. We conducted a search of PubMed, Embase, and Web of Science using multiple search terms including: 'NAFLD, NASH, fatty liver, steatohepatitis' combined with 'metagenome, microbiom*, microbiota*, fecal flora, intestinal flora, gut bacteria'. Results were manually reviewed and studies selected based on relevance to intestinal microbiota and NAFLD. We also included studies that addressed potential mechanistic models of pathways linking the dysbiosis to NAFLD. Nine studies (five human and four animal models) were identified in our search that assessed associations between specific intestinal microbiota composition and NAFLD. We reviewed and summarised the results of additional investigations that more broadly addressed the mechanisms by which the microbiome may impact NAFLD pathogenesis. Investigations in humans and animals demonstrate associations between intestinal dysbiosis and NAFLD; however, causality has not been proven and mechanistic links require further delineation. As the field of microbiome research matures in techniques and study design, more detailed insights into NAFLD pathogenesis and its associations with the intestinal microbiota will be elucidated. © 2015 John Wiley & Sons Ltd.

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

PubMed

Liévin-Le Moal, Vanessa

2013-06-01

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier. © 2013 John Wiley & Sons Ltd.

Magnetic-Fe/Fe(3)O(4)-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages.

PubMed

Wang, Hongwang; Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor; Troyer, Deryl L; Bossmann, Stefan H

2012-01-01

The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

PubMed

Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

2017-10-01

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D 3 , there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Intestinal Parasitic Infections in Human Immunodeficiency Virus-Infected and Noninfected Persons in a High Human Immunodeficiency Virus Prevalence Region of Cameroon.

PubMed

Nkenfou, Céline Nguefeu; Tchameni, Sandrine Mboula; Nkenfou, Carine Nguefeu; Djataou, Patrice; Simo, Ulrich Florian; Nkoum, Alexandre Benjamin; Estrin, William

2017-09-01

The problem of intestinal parasitic infection in human immunodeficiency virus (HIV)-infected people requires careful consideration in the developing world where poor nutrition is associated with poor hygiene and several coinfecting diseases. Studies have addressed this issue in Cameroon, especially in the low HIV prevalence area. The current study was conducted to determine the prevalence of intestinal parasitosis in people living with HIV (PLHIV) in Adamaoua and to identify associated risk factors. Stool and blood specimens from study participants were screened for intestinal parasites and anti-HIV antibodies, respectively. Of 235 participants, 68 (28.9%) were HIV positive, 38 of them on antiretroviral treatment (ART). The overall prevalence of intestinal parasites was 32.3%. Of 68 PLHIV, 32.3% (22/68) were infected with intestinal parasites, compared with 32.3% (54/167) of the HIV-negative patients. Univariate analysis showed no difference between the prevalence of intestinal parasites among PLHIV and HIV-negative patients ( P = 0.69). ART was not associated with the prevalence of intestinal parasites. Multivariate analysis showed that the quality of water and the personal hygiene were the major risk factors associated to intestinal parasitosis. The level of education was associated with HIV serostatus: the higher the level of education, the lower the risk of being infected with HIV ( P = 0.00). PLHIV and the general population should be screened routinely for intestinal parasites and treated if infected.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo12

PubMed Central

Ko, Jae Sung; Leone, Serena; Nanthakumar, N Nanda

2016-01-01

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α– and IL-1β–induced expression of IL-8, MIP-3α, and MCP-1 to 48–51% and pathogen-induced IL-8 and MCP-1 to 26–30% of positive controls (P < 0.001). GOS reduced TNF-α– and IL-1β–induced inflammatory responses to 25–26% and pathogen-induced IL-8 and MCP-1 to 36–39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01). Conclusion: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation. PMID:26701795

Three-Dimensional Coculture Of Human Small-Intestine Cells

NASA Technical Reports Server (NTRS)

Wolf, David; Spaulding, Glen; Goodwin, Thomas J.; Prewett, Tracy

1994-01-01

Complex three-dimensional masses of normal human epithelial and mesenchymal small-intestine cells cocultured in process involving specially designed bioreactors. Useful as tissued models for studies of growth, regulatory, and differentiation processes in normal intestinal tissues; diseases of small intestine; and interactions between cells of small intestine and viruses causing disease both in small intestine and elsewhere in body. Process used to produce other tissue models, leading to advances in understanding of growth and differentiation in developing organisms, of renewal of tissue, and of treatment of myriad of clinical conditions. Prior articles describing design and use of rotating-wall culture vessels include "Growing And Assembling Cells Into Tissues" (MSC-21559), "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), and "In Vitro, Matrix-Free Formation Of Solid Tumor Spheroids" (MSC-21843).

An update discussion on the current assessment of the safety of veterinary antimicrobial drug residues in food with regard to their impact on the human intestinal microbiome.

PubMed

Cerniglia, Carl E; Pineiro, Silvia A; Kotarski, Susan F

2016-05-01

The human gastrointestinal tract ecosystem consists of complex and diverse microbial communities that have now been collectively termed the intestinal microbiome. Recent scientific breakthroughs and research endeavours have increased our understanding of the important role the intestinal microbiome plays in human health and disease. The use of antimicrobial new animal drugs in food-producing animals may result in the presence of low levels of drug residues in edible foodstuffs. There is concern that antimicrobial new animal drugs in or on animal-derived food products at residue-level concentrations could disrupt the colonization barrier and/or modify the antimicrobial resistance profile of human intestinal bacteria. Therapeutic doses of antimicrobial drugs have been shown to promote shifts in the intestinal microbiome, and these disruptions promote the emergence of antimicrobial-resistant bacteria. To assess the effects of antimicrobial new animal drug residues in food on human intestinal bacteria, many national regulatory agencies and international committees follow a harmonized process, VICH GL36(R), which was issued by a trilateral organization of the European Union, the USA, and Japan called the International Cooperation on Harmonization of Technical Requirements for Veterinary Medicinal Products (VICH). The guidance describes a general approach currently used by national regulatory agencies and international committees to assess the effects of antimicrobial new animal drug residues in animal-derived food on human intestinal bacteria. The purpose of this review is to provide an overview of this current approach as part of the antimicrobial new animal drug approval process in participating countries, give insights on the microbiological endpoints used in this safety evaluation, and discuss the availability of new information. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing.

PubMed

Gao, Shuai; Yan, Liying; Wang, Rui; Li, Jingyun; Yong, Jun; Zhou, Xin; Wei, Yuan; Wu, Xinglong; Wang, Xiaoye; Fan, Xiaoying; Yan, Jie; Zhi, Xu; Gao, Yun; Guo, Hongshan; Jin, Xiao; Wang, Wendong; Mao, Yunuo; Wang, Fengchao; Wen, Lu; Fu, Wei; Ge, Hao; Qiao, Jie; Tang, Fuchou

2018-06-01

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution.

Intestinal Stem Cell Dynamics: A Story of Mice and Humans.

PubMed

Hodder, Michael C; Flanagan, Dustin J; Sansom, Owen J

2018-06-01

Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitation of small intestinal permeability during normal human drug absorption

PubMed Central

2013-01-01

Background Understanding the quantitative relationship between a drug’s physical chemical properties and its rate of intestinal absorption (QSAR) is critical for selecting candidate drugs. Because of limited experimental human small intestinal permeability data, approximate surrogates such as the fraction absorbed or Caco-2 permeability are used, both of which have limitations. Methods Given the blood concentration following an oral and intravenous dose, the time course of intestinal absorption in humans was determined by deconvolution and related to the intestinal permeability by the use of a new 3 parameter model function (“Averaged Model” (AM)). The theoretical validity of this AM model was evaluated by comparing it to the standard diffusion-convection model (DC). This analysis was applied to 90 drugs using previously published data. Only drugs that were administered in oral solution form to fasting subjects were considered so that the rate of gastric emptying was approximately known. All the calculations are carried out using the freely available routine PKQuest Java (http://www.pkquest.com) which has an easy to use, simple interface. Results Theoretically, the AM permeability provides an accurate estimate of the intestinal DC permeability for solutes whose absorption ranges from 1% to 99%. The experimental human AM permeabilities determined by deconvolution are similar to those determined by direct human jejunal perfusion. The small intestinal pH varies with position and the results are interpreted in terms of the pH dependent octanol partition. The permeability versus partition relations are presented separately for the uncharged, basic, acidic and charged solutes. The small uncharged solutes caffeine, acetaminophen and antipyrine have very high permeabilities (about 20 x 10-4 cm/sec) corresponding to an unstirred layer of only 45 μm. The weak acid aspirin also has a large AM permeability despite its low octanol partition at pH 7.4, suggesting that it is nearly completely absorbed in the first part of the intestine where the pH is about 5.4. Conclusions The AM deconvolution method provides an accurate estimate of the human intestinal permeability. The results for these 90 drugs should provide a useful benchmark for evaluating QSAR models. PMID:23800230

Glutamine: commercially essential or conditionally essential? A critical appraisal of the human data.

PubMed

Buchman, A L

2001-07-01

Glutamine is a nonessential amino acid that can be synthesized from glutamate and glutamic acid by glutamate-ammonia ligase. Glutamine is an important fuel source for the small intestine. It was proposed that glutamine is necessary for the maintenance of normal intestinal morphology and function in the absence of luminal nutrients. However, intestinal morphologic and functional changes related to enteral fasting and parenteral nutrition are less significant in humans than in animal models and may not be clinically significant. Therefore, it is unclear whether glutamine is necessary for the preservation of normal intestinal morphology and function in humans during parenteral nutrition. It was suggested that both glutamine-supplemented parenteral nutrition and enteral diets may pre-vent bacterial translocation via the preservation and augmentation of small bowel villus morphology, intestinal permeability, and intestinal immune function. However, it is unclear whether clinically relevant bacterial translocation even occurs in humans, much less whether there is any value in the prevention of such occurrences. Results of the therapeutic use of glutamine in humans at nonphysiologic doses indicate limited efficacy. Although glutamine is generally recognized to be safe on the basis of relatively small studies, side effects in patients receiving home parenteral nutrition and in those with liver-function abnormalities have been described. Therefore, on the basis of currently available clinical data, it is inappropriate to recommend glutamine for therapeutic use in any condition.

Activation of Intestinal Human Pregnane X Receptor Protects against Azoxymethane/Dextran Sulfate Sodium–Induced Colon Cancer

PubMed Central

Cheng, Jie; Fang, Zhong-Ze; Nagaoka, Kenjiro; Okamoto, Minoru; Qu, Aijuan; Tanaka, Naoki; Kimura, Shioko

2014-01-01

The role of intestinal human pregnane X receptor (PXR) in colon cancer was determined through investigation of the chemopreventive role of rifaximin, a specific agonist of intestinal human PXR, toward azoxymethane (AOM)/dextran sulfate sodium (DSS)–induced colon cancer. Rifaximin treatment significantly decreased the number of colon tumors induced by AOM/DSS treatment in PXR-humanized mice, but not wild-type or Pxr-null mice. Additionally, rifaximin treatment markedly increased the survival rate of PXR-humanized mice, but not wild-type or Pxr-null mice. These data indicated a human PXR–dependent therapeutic chemoprevention of rifaximin toward AOM/DSS-induced colon cancer. Nuclear factor κ-light-chain-enhancer of activated B cells–mediated inflammatory signaling was upregulated in AOM/DSS-treated mice, and inhibited by rifaximin in PXR-humanized mice. Cell proliferation and apoptosis were also modulated by rifaximin treatment in the AOM/DSS model. In vitro cell-based assays further revealed that rifaximin regulated cell apoptosis and cell cycle in a human PXR-dependent manner. These results suggested that specific activation of intestinal human PXR exhibited a chemopreventive role toward AOM/DSS-induced colon cancer by mediating anti-inflammation, antiproliferation, and proapoptotic events. PMID:25277138

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection.

PubMed

Saxena, Kapil; Simon, Lukas M; Zeng, Xi-Lei; Blutt, Sarah E; Crawford, Sue E; Sastri, Narayan P; Karandikar, Umesh C; Ajami, Nadim J; Zachos, Nicholas C; Kovbasnjuk, Olga; Donowitz, Mark; Conner, Margaret E; Shaw, Chad A; Estes, Mary K

2017-01-24

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

PubMed Central

Saxena, Kapil; Simon, Lukas M.; Zeng, Xi-Lei; Blutt, Sarah E.; Crawford, Sue E.; Sastri, Narayan P.; Karandikar, Umesh C.; Ajami, Nadim J.; Zachos, Nicholas C.; Kovbasnjuk, Olga; Donowitz, Mark; Conner, Margaret E.; Shaw, Chad A.; Estes, Mary K.

2017-01-01

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine. PMID:28069942

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones

PubMed Central

Parkinson, Elizabeth I.; Hatfield, M. Jason; Tsurkan, Lyudmila; Hyatt, Janice L.; Edwards, Carol C.; Hicks, Latorya D.; Yan, Bing; Potter, Philip M.

2011-01-01

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: One containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X = CH2, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X =N), or conversion of the compounds into substrates for the enzymes (when X = S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH3 to C8H17] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH3 to C6H13], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group. PMID:21733699

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones.

PubMed

Parkinson, Elizabeth I; Jason Hatfield, M; Tsurkan, Lyudmila; Hyatt, Janice L; Edwards, Carol C; Hicks, Latorya D; Yan, Bing; Potter, Philip M

2011-08-01

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: one containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X=CH₂, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X=N), or conversion of the compounds into substrates for the enzymes (when X=S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH₃ to C₈H₁₇] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH₃ to C₆H₁₃], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group. Copyright © 2011 Elsevier Ltd. All rights reserved.

Possible mechanism for inhibition of morphine formation from 6-acetylmorphine after intake of street heroin.

PubMed

Andersson, Maria; Björkhem-Bergman, Linda; Beck, Olof

2015-07-01

Heroin is de-acetylated in the body to morphine in two steps. The intermediate 6-acetylmorphine (6-AM) is formed rapidly and is considered important for the pharmacological effect of heroin. In urine drug testing, an atypical pattern of morphine and 6-AM is known to occur in low frequency. The aim of this study was to investigate this atypical pattern in more detail and to identify responsible substances for a possible inhibition of the conversion from 6-AM to morphine. Urine samples were selected from a routine flow of samples sent for drug testing. Out of 695 samples containing morphine and 6-acetylmorphine, 11.5% had the atypical pattern of a 6-AM to morphine ratio above 0.26 as derived from a bimodal frequency distribution. An in vitro study of the conversion of 6-acetylmorphine to morphine in human liver homogenates demonstrated that a number of known carboxylesterase inhibitors were able to inhibit the reaction mimicking the situation in vivo. Compound 3 (3,6-Dimethoxy-4-acetoxy-5-[2-(N-methylacetamido)ethyl]phenanthrene) a substance formed from thebaine during the production of heroin was found to be a strong inhibitor. Liquid chromatography-mass spectrometry was used to identify possible inhibitors present in vivo. This part of the investigation demonstrated that several components may contribute to the effect. It is concluded that inhibition of liver carboxylesterase activity is a possible mechanism causing the atypical pattern and that one candidate compound is the result of the heroin production process. An inhibition of 6-AM metabolism is likely to increase the pharmacological effect of heroin and may be related to a higher risk of lethal toxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

PubMed Central

McGhee, J. D.; Birchall, J. C.; Chung, M. A.; Cottrell, D. A.; Edgar, L. G.; Svendsen, P. C.; Ferrari, D. C.

1990-01-01

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter. PMID:2379823

Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

PubMed Central

Hosomi, Atsushi; Nakanishi, Takeo; Fujita, Takuya; Tamai, Ikumi

2012-01-01

Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats. PMID:22348008

Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

PubMed

Sato, Toshiro; Stange, Daniel E; Ferrante, Marc; Vries, Robert G J; Van Es, Johan H; Van den Brink, Stieneke; Van Houdt, Winan J; Pronk, Apollo; Van Gorp, Joost; Siersema, Peter D; Clevers, Hans

2011-11-01

We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Toxicological significance of azo dye metabolism by human intestinal microbiota

PubMed Central

Feng, Jinhui; Cerniglia, Carl E.; Chen, Huizhong

2018-01-01

Approximately 0.7 million tons of azo dyes are synthesized each year. Azo dyes are composed of one or more R1-N=N-R2 linkages. Studies have shown that both mammalian and microbial azoreductases cleave the azo bonds of the dyes to form compounds that are potentially genotoxic. The human gastrointestinal tract harbors a diverse microbiota comprised of at least several thousand species. Both water-soluble and water-insoluble azo dyes can be reduced by intestinal bacteria. Some of the metabolites produced by intestinal microbiota have been shown to be carcinogenic to humans although the parent azo dyes may not be classified as being carcinogenic. Azoreductase activity is commonly found in intestinal bacteria. Three types of azoreductases have been characterized in bacteria. They are flavin dependent NADH preferred azoreductase, flavin dependent NADPH preferred azoreductase, and flavin free NADPH preferred azoreductase. This review highlights how azo dyes are metabolized by intestinal bacteria, mechanisms of azo reduction, and the potential contribution in the carcinogenesis/mutagenesis of the reduction of the azo dyes by intestinal microbiota. PMID:22201895

Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

PubMed Central

Huys, Geert; Vanhoutte, Tom; Vandamme, Peter

2008-01-01

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated. PMID:19277102

Adherence of Clostridium perfringens spores to human intestinal epithelial Caco-2 cells.

PubMed

Sakanoue, Hideyo; Nakano, Takashi; Sano, Kouichi; Yasugi, Mayo; Monma, Chie; Miyake, Masami

2018-03-01

Clostridium perfringens is a gram-positive, spore-forming bacillus, and is a causative agent of foodborne infection, antibiotic-associated diarrhoea and sporadic diarrhoea in humans. In cases of antibiotic-associated and sporadic diarrhoea, C. perfringens colonises the intestine, proliferates and causes disease. However, bacterial colonisation of the intestine is not considered necessary in the pathogenesis of foodborne illness, because such pathogenesis can be explained by anchorage-independent production of diarrhoeic toxin by the bacterium in the intestine. In this study, we used an in vitro adherence assay to examine the adherence of C. perfringens spores to human intestinal Caco-2 cells. Adherence of spores from isolates of foodborne illness and nosocomial infection was observed within 15 min, and plateaued 60 min after inoculation. Electron microscopy revealed a tight association of spores with the surface of Caco-2 cells. The adherence of vegetative cells could not be confirmed by the same method, however. These results suggest that C. perfringens spores may adhere to intestinal epithelial cells in vivo, although its biological significance remains to be determined.

Characterizing intestinal strictures with acoustic resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Lei, Hao; Xu, Guan; Liu, Shengchun; Johnson, Laura A.; Moons, David S.; Higgins, Peter D. R.; Rice, Michael D.; Ni, Jun; Wang, Xueding

2016-03-01

Crohn's disease (CD) is an autoimmune disease, which may cause obstructing intestinal strictures due to inflammation, fibrosis (deposition of collagen), or a combination of both. Identifying the different stages of the disease progression is still challenging. In this work, we indicated the feasibility of non-invasively characterizing intestinal strictures using photoacoustic imaging (PAI), utilizing the uniquely optical absorption of hemoglobin and collagen. Surgically removed human intestinal stricture specimens were investigated with a prototype PAI system. 2D PA images with acoustic resolution at wavelength 532, 1210 and 1310 nm were formulated, and furthermore, the PA histochemical components images which show the microscopic distributions of histochemical components were solved. Imaging experiments on surgically removed human intestinal specimens has demonstrated the solved PA images were significantly different associated with the presence of fibrosis, which could be applied to characterize the intestinal strictures for given specimens.

Encapsulation of anthocyanins from bilberries - Effects on bioavailability and intestinal accessibility in humans.

PubMed

Mueller, Dolores; Jung, Kathrin; Winter, Manuel; Rogoll, Dorothee; Melcher, Ralph; Kulozik, Ulrich; Schwarz, Karin; Richling, Elke

2018-05-15

Anthocyanins are flavonoids that have been suggested to provide beneficial health effects. The biological activity of anthocyanins is influenced by their pharmacokinetic properties, but anthocyanins are associated with limited bioavailability in humans. In the presented study, we investigated how the encapsulation of bilberry extract (BE), a source of anthocyanins, with either whey protein or citrus pectin influences the bioavailability and intestinal accessibility of anthocyanins in humans. We performed an intervention study that analyzed anthocyanins and their degradation products in the urine, plasma, and ileal effluent of healthy volunteers and ileostomists (subjects without an intact colon). We were able to show, that whey protein encapsulation modulated short-term bioavailability and that citrus pectin encapsulation increased intestinal accessibility during passage through the small intestine and modulated the formation of the degradation product phloroglucinol aldehyde (PGAL) in human plasma. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Porcine models of digestive disease: the future of large animal translational research

PubMed Central

Gonzalez, Liara M.; Moeser, Adam J.; Blikslager, Anthony T.

2015-01-01

There is increasing interest in non-rodent translational models for the study of human disease. The pig, in particular, serves as a useful animal model for the study of pathophysiological conditions relevant to the human intestine. This review assesses currently used porcine models of gastrointestinal physiology and disease and provides a rationale for the use of these models for future translational studies. The pig has proven its utility for the study of fundamental disease conditions such as ischemia/ reperfusion injury, stress-induced intestinal dysfunction, and short bowel syndrome. Pigs have also shown great promise for the study of intestinal barrier function, surgical tissue manipulation and intervention, as well as biomaterial implantation and tissue transplantation. Advantages of pig models highlighted by these studies include the physiological similarity to human intestine as well as to mechanisms of human disease. Emerging future directions for porcine models of human disease include the fields of transgenics and stem cell biology, with exciting implications for regenerative medicine. PMID:25655839

Intestinal Parasite Co-infection among Pulmonary Tuberculosis Cases without Human Immunodeficiency Virus Infection in a Rural County in China

PubMed Central

Li, Xin-Xu; Chen, Jia-Xu; Wang, Li-Xia; Tian, Li-Guang; Zhang, Yu-Ping; Dong, Shuang-Pin; Hu, Xue-Guang; Liu, Jian; Wang, Feng-Feng; Wang, Yue; Yin, Xiao-Mei; He, Li-Jun; Yan, Qiu-Ye; Zhang, Hong-Wei; Xu, Bian-Li; Zhou, Xiao-Nong

2014-01-01

Epidemiologic studies of co-infection with tuberculosis (TB) and intestinal parasites in humans have not been extensively investigated in China. A cross-section study was conducted in a rural county of Henan Province, China. Pulmonary TB (PTB) case-patients receiving treatment for infection with Mycobacterium tuberculosis and healthy controls matched for geographic area, age, and sex were surveyed by using questionnaires. Fecal and blood specimens were collected for detection of intestinal parasites, routine blood examination, and infection with human immunodeficiency virus. The chi-square test was used for univariate analysis and multivariate logistic regression models were used to adjust for potential confounding factors. A total of 369 persons with PTB and 366 healthy controls were included; all participants were negative for human immunodeficiency virus. The overall prevalence of intestinal parasites in persons with PTB was 14.9%, including intestinal protozoa (7.9%) and helminthes (7.6%). The infection spectrum of intestinal parasites was Entamoeba spp. (1.4%), Blastocystis hominis (6.2%), Trichomonas hominis (0.3%), Clonorchis sinensis (0.3%), Ascaris lumbricoides (0.5%), Trichuris trichiura (2.2%), and hookworm (4.6%). The prevalence of intestinal parasites showed no significant difference between persons with PTB and healthy controls after adjusting for potential confounding factors. There was no factor that affected infection rates for intestinal parasites between the two groups. Infection with intestinal parasites of persons with PTB was associated with female sex (adjusted odds ratio [AOR] = 2.05, 95% confidence interval [CI] = 1.01–4.17), body mass index ≤ 19 (AOR = 3.02, 95% CI = 1.47–6.20), and anemia (AOR = 2.43, 95% CI = 1.17–5.03). Infection of healthy controls was only associated with an annual labor time in farmlands > 2 months (AOR = 4.50, 95% CI = 2.03–10.00). In addition, there was no significant trend between rates of infection with intestinal parasites and duration of receiving treatment for infection with M. tuberculosis in persons with PTB. The prevalence of intestinal parasites was not higher in persons with PTB, and there was no evidence that PTB increased susceptibility to intestinal parasites in this study. However, for patients with PTB, women and patients with comorbidities were more likely to be infected with intestinal parasites. PMID:24166044

Supplementation of milk formula with galacto-oligosaccharides improves intestinal micro-flora and fermentation in term infants.

PubMed

Ben, Xiao-ming; Zhou, Xiao-yu; Zhao, Wei-hua; Yu, Wen-liang; Pan, Wei; Zhang, Wei-li; Wu, Sheng-mei; Van Beusekom, Christien M; Schaafsma, Anne

2004-06-01

Oligosaccharides in human milk may protect infants by improving the intestinal micro-flora and fermentation. This study was to investigate effects of infant formula milk consisting of galacto-oligosaccharide (GOS) on intestinal microbial populations and the fermentation characteristics in term infants in comparison with that of human milk. The test formula (Frisolac H, Friesland, Netherland) was supplemented with GOS at a concentration of 0.24 g/dl. Human milk and another formula without oligosaccharides (Frisolac H, Friesland, Netherland) were used as positive and negative control respectively. Growth, stool characteristics, and side effects of the recruited infants were recorded after 3 and 6 months' follow-up, and the fecal species were collected for the analysis of intestinal micro-flora, short chain fatty acid (SCFA) and pH. At the end of 3- and 6-month feeding period, intestinal Bifidobacteria and Lactobacilli were significantly increased in infants fed with GOS supplemented formula and human milk when compared with infants fed with negative control formula; however, there was no statistically significant difference between GOS supplemented formula and human milk groups. Stool characteristics were influenced by the supplement and main fecal SCFA (acetic), and stool frequency were significantly increased in infants fed with GOS supplemented formula and human milk, while the fecal pH was significantly decreased as compared with that of negative control (P < 0.05). Supplementation had no influence on incidence of side effects (including crying, regurgitation and vomiting). Supplementing infant formula with GOS at a concentration of 0.24 g/dl stimulates the growth of Bifidobacteria and Lactobacilli in the intestine and stool characteristics are similar to in term infants fed with human milk.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes.

PubMed

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline L M; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-11-10

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

PubMed Central

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline LM; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-01-01

Background Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Results Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. Conclusion The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants. PMID:19000307

Functional metagenomic profiling of intestinal microbiome in extreme ageing.

PubMed

Rampelli, Simone; Candela, Marco; Turroni, Silvia; Biagi, Elena; Collino, Sebastiano; Franceschi, Claudio; O'Toole, Paul W; Brigidi, Patrizia

2013-12-01

Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in "pathobionts", i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing.

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

PubMed

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-06-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip1[W

PubMed Central

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-01-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. PMID:22474185

Magnetic-Fe/Fe3O4-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages

PubMed Central

Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor

2012-01-01

Summary The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe3O4 magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system. PMID:23016149

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure.

PubMed

Küster, E; Altenburger, R

2006-01-01

The acute zebra fish embryo test (Danio rerio Hamilton-Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC50 below 1.2 micromol compared with an EC50 of 91 micromol. The dose-response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

PubMed

Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

2016-01-01

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32μmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Distinct intestinal adaptation for vitamin B12 and bile acid absorption revealed in a new mouse model of massive ileocecal resection.

PubMed

Matsumoto, Yuka; Mochizuki, Wakana; Akiyama, Shintaro; Matsumoto, Taichi; Nozaki, Kengo; Watanabe, Mamoru; Nakamura, Tetsuya

2017-09-15

Ileocecal resection (ICR), one of several types of intestinal resection that results in short bowel syndrome (SBS), causes severe clinical disease in humans. We here describe a mouse model of massive ICR in which 75% of the distal small intestine is removed. We demonstrate that mice underwent 75% ICR show severe clinical signs and high mortality, which may recapitulate severe forms of human SBS, despite an adaptive response throughout the remnant intestine. By using this model, we also investigated whether the epithelium of the remnant intestine shows enhanced expression of factors involved in region-specific functions of the ileum. Cubn mRNA and its protein product, which play an essential role in vitamin B12 absorption in the ileum, are not compensatory up-regulated in any part of the remnant intestine, demonstrating a clear contrast with post-operative up-regulation of genes involved in bile acid absorption. Our study suggests that functional adaptation by phenotypical changes in the intestinal epithelium is not a general feature for nutrient absorption systems that are confined to the ileum. We also propose that the mouse model developed in this study will become a unique system to facilitate studies on SBS with ICR in humans. © 2017. Published by The Company of Biologists Ltd.

In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

PubMed

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

2014-12-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute(-1)·mM(-1), respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

In Vitro Metabolism and Drug-Drug Interaction Potential of UTL-5g, a Novel Chemo- and Radioprotective Agent

PubMed Central

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick

2014-01-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. PMID:25249693

Mechanisms and function of autophagy in intestinal disease.

PubMed

Lassen, Kara G; Xavier, Ramnik J

2018-01-01

The discovery of numerous genetic variants in the human genome that are associated with inflammatory bowel disease (IBD) has revealed critical pathways that play important roles in intestinal homeostasis. These genetic studies have identified a critical role for macroautophagy/autophagy and more recently, lysosomal function, in maintaining the intestinal barrier and mucosal homeostasis. This review highlights recent work on the functional characterization of IBD-associated human genetic variants in cell type-specific functions for autophagy.

[Gut barrier in the critically ill patient: facts and trends].

PubMed

Velasco, Nicolás

2006-08-01

The disturbances of gut barrier in critically ill patients may influence their outcome and prognosis. Experiments in animals show that fasting and stress collaborate to produce intestinal atrophy and translocation of microorganisms and toxins. This fact is one of the main arguments to promote the use of early enteral feeding in critically ill patients. However, the intestinal barrier behaves differently in humans than in animals. The human enteral cells have a good tolerance to fasting and stress, mucosal atrophy is mild and it is not always associated with changes in intestinal permeability. Moreover, the relationship between intestinal permeability with sepsis and bacterial translocation is controversial. This last phenomenon also happens in normal subjects and may be a mechanism to build immunological memory. One of the most important factors that influence bacterial translocation is the microorganism, that under stress conditions can adhere to the intestinal cell and penetrate the intestinal barrier. Splanchnic ischemia and reperfusion is one of the main pathogenic factors in the failure of intestinal barrier. Finally, the fact that the small bowel is an inflammatory target of extra intestinal injuries, explains several clinical situations. The pathophysiology of the intestinal barrier definitely requires more research.

Intestinal disposition of quercetin and its phase-II metabolites after oral administration in healthy volunteers.

PubMed

Chalet, Clément; Rubbens, Jari; Tack, Jan; Duchateau, Guus S; Augustijns, Patrick

2018-05-15

Quercetin is one of the main dietary flavonoids and undergoes a substantial intestinal phase-II metabolism. Quercetin conjugates have been detected in plasma and in urine, but their presence in the small intestine has not been assessed. This study aimed to investigate the intestinal metabolism and metabolite excretion of quercetin by the human small intestinal wall after oral dosing. Six healthy volunteers were given a capsule of 500 mg of quercetin with 240 ml of water. Duodenal fluids were collected using the intraluminal sampling technique for 4 h and analysed by LC-MS/MS. Phase-II metabolites of quercetin were detected and quantified in aspirated intestinal fluids. Metabolites appeared almost immediately after administration, indicating an intestinal metabolism and apical excretion into the lumen. Quercetin-3'-O-glucuronide was found to be the main intestinal metabolite. Our results could not conclude on the enterohepatic recycling of quercetin or its metabolites, although several individual profiles showed distinctive peaks. This study highlights the intestinal metabolism and excretion of quercetin and its conjugates in humans and gives insights into the relevant concentrations which should be used to investigate potential food-drug interactions in vitro. © 2018 Royal Pharmaceutical Society.

Gender Difference of Hepatic and Intestinal CYP3A4 in CYP3AHumanized Mice Generated by a Human Chromosome-engineering Technique.

PubMed

Kobayashi, Kaoru; Abe, Chihiro; Endo, Mika; Kazuki, Yasuhiro; Oshimura, Mitsuo; Chiba, Kan

2017-11-17

Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable. To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4. We examined the expression and activity of hepatic and intestinal CYP3A4 in male and female CYP3A-HAC mice. CYP3A activity was determined as α- and 4-hydroxylation activity of triazolam in liver and intestinal microsomes. Expression level of CYP3A protein was determined by Western blot analysis. Expression level of CYP3A4 mRNA was measured by quantitative real-time PCR. The results showed that triazolam hydroxylation activities and protein levels of CYP3A in the liver were significantly higher in female than in male CYP3A-HAC mice, whereas those in the intestine were not significantly different between the genders. In addition, the expression of CYP3A4 mRNA showed a tendency similar to that found for the activity and expression of CYP3A protein in the liver and intestine of CYP3A-HAC mice. These findings suggest that the expression and activity levels of CYP3A4 in the liver are higher in females than in males, whereas there is no gender difference in the levels in the intestine of CYP3A-HAC mice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Enterohemorrhagic Escherichia coli senses low biotin status in the large intestine for colonization and infection

PubMed Central

Yang, Bin; Feng, Lu; Wang, Fang; Wang, Lei

2015-01-01

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. Here we identify a virulence-regulating pathway in which the biotin protein ligase BirA signals to the global regulator Fur, which in turn activates LEE (locus of enterocyte effacement) genes to promote EHEC adherence in the low-biotin large intestine. LEE genes are repressed in the high-biotin small intestine, thus preventing adherence and ensuring selective colonization of the large intestine. The presence of this pathway in all nine EHEC serotypes tested indicates that it is an important evolutionary strategy for EHEC. The pathway is incomplete in closely related small-intestinal enteropathogenic E. coli due to the lack of the Fur response to BirA. Mice fed with a biotin-rich diet show significantly reduced EHEC adherence, indicating that biotin might be useful to prevent EHEC infection in humans. PMID:25791315

Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues.

PubMed

Lundquist, P; Artursson, P

2016-11-15

In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro-in vivo extrapolation are reviewed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

PubMed

Arias-Jayo, Nerea; Alonso-Saez, Laura; Ramirez-Garcia, Andoni; Pardo, Miguel A

2018-04-01

The human intestine hosts a vast and complex microbial community that is vital for maintaining several functions related with host health. The processes that determine the gut microbiome composition are poorly understood, being the interaction between species, the external environment, and the relationship with the host the most feasible. Animal models offer the opportunity to understand the interactions between the host and the microbiota. There are different gnotobiotic mice or rat models colonized with the human microbiota, however, to our knowledge, there are no reports on the colonization of germ-free zebrafish with a complex human intestinal microbiota. In the present study, we have successfully colonized 5 days postfertilization germ-free zebrafish larvae with the human intestinal microbiota previously extracted from a donor and analyzed by high-throughput sequencing the composition of the transferred microbial communities that established inside the zebrafish gut. Thus, we describe for first time which human bacteria phylotypes are able to colonize the zebrafish digestive tract. Species with relevant interest because of their linkage to dysbiosis in different human diseases, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Prevotella spp., or Roseburia spp. have been successfully transferred inside the zebrafish digestive tract.

Kinetics of Carboxylesterase: An Experiment for Biochemistry and Physical Chemistry Laboratory.

ERIC Educational Resources Information Center

Nichols, C. S.; Cromartie, T. H.

1979-01-01

Describes a convenient, inexpensive experiment in enzyme kinetics developed for the undergraduate biochemistry laboratory at the University of Virginia. Required are a single beam visible spectrophotometer with output to a recorder, a constant temperature, a commercially available enzyme, substrates, and buffers. (BT)

Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

PubMed

Edogawa, Shoko; Peters, Stephanie A; Jenkins, Gregory D; Gurunathan, Sakteesh V; Sundt, Wendy J; Johnson, Stephen; Lennon, Ryan J; Dyer, Roy B; Camilleri, Michael; Kashyap, Purna C; Farrugia, Gianrico; Chen, Jun; Singh, Ravinder J; Grover, Madhusudan

2018-06-13

Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex-based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty-three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex-specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex-based differences in intestinal barrier function and microbiome should be considered in future studies.-Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Assessment of the mode of action underlying development of rodent small intestinal tumors following oral exposure to hexavalent chromium and relevance to humans

PubMed Central

Proctor, Deborah M.; Suh, Mina; Haws, Laurie C.; Kirman, Christopher R.; Harris, Mark A.

2013-01-01

Chronic exposure to high concentrations of hexavalent chromium (Cr(VI)) in drinking water causes intestinal adenomas and carcinomas in mice, but not in rats. Cr(VI) causes damage to intestinal villi and crypt hyperplasia in mice after only one week of exposure. After two years of exposure, intestinal damage and crypt hyperplasia are evident in mice (but not rats), as are intestinal tumors. Although Cr(VI) has genotoxic properties, these findings suggest that intestinal tumors in mice arise as a result of chronic mucosal injury. To better understand the mode of action (MOA) of Cr(VI) in the intestine, a 90-day drinking water study was conducted to collect histological, biochemical, toxicogenomic and pharmacokinetic data in intestinal tissues. Using MOA analyses and human relevance frameworks proposed by national and international regulatory agencies, the weight of evidence supports a cytotoxic MOA with the following key events: (a) absorption of Cr(VI) from the intestinal lumen, (b) toxicity to intestinal villi, (c) crypt regenerative hyperplasia and (d) clonal expansion of mutations within the crypt stem cells, resulting in late onset tumorigenesis. This article summarizes the data supporting each key event in the MOA, as well as data that argue against a mutagenic MOA for Cr(VI)-induced intestinal tumors. PMID:23445218

Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells.

PubMed

Si-Tahar, M; Merlin, D; Sitaraman, S; Madara, J L

2000-06-01

Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.

The human TLR4 variant D299G mediates inflammation-associated cancer progression in the intestinal epithelium

PubMed Central

2013-01-01

Homeostatic TLR4 signaling protects the intestinal epithelium in health. Evidence suggests that perturbed TLR4 signaling is linked to carcinogenesis. We have recently demonstrated that the common human TLR4 variant D299G exerts pro-inflammatory effects and drives malignant tumor progression in human colon cancer. PMID:24073372

The human TLR4 variant D299G mediates inflammation-associated cancer progression in the intestinal epithelium.

PubMed

Cario, Elke

2013-07-01

Homeostatic TLR4 signaling protects the intestinal epithelium in health. Evidence suggests that perturbed TLR4 signaling is linked to carcinogenesis. We have recently demonstrated that the common human TLR4 variant D299G exerts pro-inflammatory effects and drives malignant tumor progression in human colon cancer.

Intestinal epithelial wound healing assay in an epithelial-mesenchymal co-culture system.

PubMed

Seltana, Amira; Basora, Nuria; Beaulieu, Jean-François

2010-01-01

Rapid and efficient healing of epithelial damage is critical to the functional integrity of the small intestine. Epithelial repair is a complex process that has largely been studied in cultured epithelium but to a much lesser extent in mucosa. We describe a novel method for the study of wound healing using a co-culture system that combined an intestinal epithelial Caco-2/15 cell monolayer cultured on top of human intestinal myofibroblasts, which together formed a basement membrane-like structure that contained many of the major components found at the epithelial-mesenchymal interface in the human intestine. To investigate the mechanism of restitution, small lesions were generated in epithelial cell monolayers on plastic or in co-cultures without disturbing the underlying mesenchymal layer. Monitoring of wound healing showed that repair was more efficient in Caco-2/15-myofibroblast co-cultures than in Caco-2/15 monolayers and involved the deposition of basement membrane components. Functional experiments showed that the addition of type I collagen or human fibronectin to the culture medium significantly accelerated wound closure on epithelial cell co-cultures. This system may provide a new tool to investigate the mechanisms that regulate wound healing in the intestinal epithelium.

Unregulated smooth-muscle myosin in human intestinal neoplasia.

PubMed

Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Järvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S; Lucassen, Anneke; Tomlinson, Ian P M; Launonen, Virpi; Ristimäki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H Lee; Aaltonen, Lauri A

2008-04-08

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

42 CFR 121.13 - Definition of human organ under section 301 of the National Organ Transplant Act of 1984, as...

Code of Federal Regulations, 2014 CFR

2014-10-01

... 1984, as amended, means the human (including fetal) kidney, liver, heart, lung, pancreas, bone marrow, cornea, eye, bone, skin, intestine (including the esophagus, stomach, small and/or large intestine, or...

42 CFR 121.13 - Definition of human organ under section 301 of the National Organ Transplant Act of 1984, as...

Code of Federal Regulations, 2013 CFR

2013-10-01

... 1984, as amended, means the human (including fetal) kidney, liver, heart, lung, pancreas, bone marrow, cornea, eye, bone, skin, intestine (including the esophagus, stomach, small and/or large intestine, or...

Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium

PubMed Central

Hill, David R; Huang, Sha; Nagy, Melinda S; Yadagiri, Veda K; Fields, Courtney; Mukherjee, Dishari; Bons, Brooke; Dedhia, Priya H; Chin, Alana M; Tsai, Yu-Hwai; Thodla, Shrikar; Schmidt, Thomas M; Walk, Seth

2017-01-01

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine. PMID:29110754

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

PubMed

Gabastou, J M; Kernéis, S; Bernet-Camard, M F; Barbat, A; Coconnier, M H; Kaper, J B; Servin, A L

1995-09-01

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term detection of seasonal influenza RNA in faeces and intestine.

PubMed

Hirose, R; Daidoji, T; Naito, Y; Watanabe, Y; Arai, Y; Oda, T; Konishi, H; Yamawaki, M; Itoh, Y; Nakaya, T

2016-09-01

Some cases of seasonal influenza virus (human influenza A virus (IAV)/human influenza B virus (IBV)) are associated with abdominal symptoms. Although virus RNA has been detected in faeces, intestinal infection has not been clearly demonstrated. We aimed to provide evidence that IAV/IBV infects the human intestine. This prospective observational study measured virus RNA in faecal and sputum samples from 22 patients infected with IAV/IBV (19 IAV positive and three IBV positive). Nineteen patients were included in the analysis and were assigned to faecal IAV-positive and -negative groups. Virus kinetics were examined in faecal samples from an IAV-infected patient (patient 1) and an IBV-infected patient (patient 2). Finally, intestinal tissue from an IAV-diagnosed patient who developed haemorrhagic colitis and underwent colonoscopy was examined for the presence of replicating IAV (patient 3). Virus RNA was detected in faecal samples from 8/22 IAV/IBV-infected patients (36.4%). Diarrhoea occurred significantly more often in the faecal IAV-positive group (p 0.002). In patients 1 and 2, virus RNA became undetectable in sputum on days 7 and 10 after infection, respectively, but was detected in faeces for a further 2 weeks. Virus mRNA and antigens were detected in intestinal tissues (mucosal epithelium of the sigmoid colon) from patient 3. These findings suggest that IAV/IBV infects within the intestinal tract; thus, the human intestine may be an additional target organ for IAV/IBV infection. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Milk with and without lactoferrin can influence intestinal damage in a pig model of malnutrition.

PubMed

Garas, Lydia C; Feltrin, Cristiano; Hamilton, M Kristina; Hagey, Jill V; Murray, James D; Bertolini, Luciana R; Bertolini, Marcelo; Raybould, Helen E; Maga, Elizabeth A

2016-02-01

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of five worldwide. However, the underlying mechanisms are not well understood necessitating an appropriate animal model to answer fundamental questions and conduct translational research into optimal interventions. One potential intervention is milk from livestock that more closely mimics human milk by increased levels of bioactive components that can promote a healthy intestinal epithelium. We tested the ability of cow milk and milk from transgenic cows expressing human lactoferrin at levels found in human milk (hLF milk) to mitigate the effects of malnutrition at the level of the intestine in a pig model of malnutrition. Weaned pigs (3 weeks old) were fed a protein and calorie restricted diet for five weeks, receiving cow, hLF or no milk supplementation daily from weeks 3-5. After three weeks, the restricted diet induced changes in growth, blood chemistry and intestinal structure including villous atrophy, increased ex vivo permeability and decreased expression of tight junction proteins. Addition of both cow and hLF milk to the diet increased growth rate and calcium and glucose levels while promoting growth of the intestinal epithelium. In the jejunum hLF milk restored intestinal morphology, reduced permeability and increased expression of anti-inflammatory IL-10. Overall, this pig model of malnutrition mimics salient aspects of the human condition and demonstrates that cow milk can stimulate the repair of damage to the intestinal epithelium caused by protein and calorie restriction with hLF milk improving this recovery to a greater extent.

Variation of human intestinal gamma-glutamyl transpeptidase in ontogenetic development.

PubMed

Sobiech, K A; Szewczuk, A

1977-01-01

Activity of gamma-glutamyl transpeptidase in human intestines was measured against alpha-naphthylamide and 12 gamma-glutamyl amino acids and peptides as substrate. Distinctly altered activity was found to accompany ontogenetic development. The ratio of the transpeptidase activity tested against monoglutamyl substrates in the intestines of 7-month fetuses, newborns and adults was 15:1:4, whereas the ratio of gamma-glutamyl cyclotransferase activities in the same age groups was 1-0:1-2:1-6. Distinct differences were found in resistance to heating, sensitivity to L-serine plus borate, and other effectors, and electrophoretic mobility, between fetal gamma-glutamyl transpeptidase and the enzyme from adults, which supports the hypothesis of existence of two forms of the enzyme in the human intestines. The results suggest involvement of gamma-glutamyl transpeptidase in the pathomechanism of celiakia in children.

Oxime-Induced Reactivation of Carboxylesterase Inhibited by Organophosphorus Compounds

DTIC Science & Technology

1993-05-13

detoxication enzyme for OP compounds (Maxwell, 1992a), when in the presence of an uncharged oxime, becomes even more effective because it is easily...Wolring, 1984). Therefore, oxime-induced reactivation of OP-inhibited CaE for protection by enhancement of OP detoxication occurs at approximately the

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

USDA-ARS?s Scientific Manuscript database

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal ...

Review article: the human intestinal virome in health and disease.

PubMed

Carding, S R; Davis, N; Hoyles, L

2017-11-01

The human virome consists of animal-cell viruses causing transient infections, bacteriophage (phage) predators of bacteria and archaea, endogenous retroviruses and viruses causing persistent and latent infections. High-throughput, inexpensive, sensitive sequencing methods and metagenomics now make it possible to study the contribution dsDNA, ssDNA and RNA virus-like particles make to the human virome, and in particular the intestinal virome. To review and evaluate the pioneering studies that have attempted to characterise the human virome and generated an increased interest in understanding how the intestinal virome might contribute to maintaining health, and the pathogenesis of chronic diseases. Relevant virome-related articles were selected for review following extensive language- and date-unrestricted, electronic searches of the literature. The human intestinal virome is personalised and stable, and dominated by phages. It develops soon after birth in parallel with prokaryotic communities of the microbiota, becoming established during the first few years of life. By infecting specific populations of bacteria, phages can alter microbiota structure by killing host cells or altering their phenotype, enabling phages to contribute to maintaining intestinal homeostasis or microbial imbalance (dysbiosis), and the development of chronic infectious and autoimmune diseases including HIV infection and Crohn's disease, respectively. Our understanding of the intestinal virome is fragmented and requires standardised methods for virus isolation and sequencing to provide a more complete picture of the virome, which is key to explaining the basis of virome-disease associations, and how enteric viruses can contribute to disease aetiologies and be rationalised as targets for interventions. © 2017 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.

Functional metagenomic profiling of intestinal microbiome in extreme ageing

PubMed Central

Rampelli, Simone; Candela, Marco; Turroni, Silvia; Biagi, Elena; Collino, Sebastiano; Franceschi, Claudio; O'Toole, Paul W; Brigidi, Patrizia

2013-01-01

Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in “pathobionts”, i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing. PMID:24334635

Both direct and indirect effects account for the pro-inflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium: Stimulation of interleukin-8 secretion, potentiation of interleukin-1{beta} effect and increase in the transepithelial passage of commensal bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maresca, Marc; Yahi, Nouara; Younes-Sakr, Lama

Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator ofmore » intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1{beta}), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1{beta} on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.« less

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

PubMed Central

Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

2015-01-01

ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures. PMID:26446608

Kingdom-agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

PubMed Central

Norman, Jason M.; Handley, Scott A.; Virgin, Herbert W.

2014-01-01

Advanced sequencing techniques have shown that bacteria are not the only complex and important microbes in the human intestine. Non-bacterial organisms, particularly the virome and the mycobiome, are important regulators of intestinal immunity and inflammation. The virome is mucosal and systemic; it can alter the host response to bacteria and interact with host genes and bacteria to contribute to disease pathogenesis. The human mycobiome is also complex and can contribute to intestinal inflammation. We review what has recently been learned about the non-bacterial and non-archaeal microbes in the gastrointestinal tract, discussing their potential effects on health and disease and analytical approaches for their study. Studies of associations between the microbiome and intestinal pathology should incorporate kingdom-agnostic approaches if we are to fully understand intestinal health and disease. PMID:24508599

Convergence in probiotic Lactobacillus gut-adaptive responses in humans and mice.

PubMed

Marco, Maria L; de Vries, Maaike C; Wels, Michiel; Molenaar, Douwe; Mangell, Peter; Ahrne, Siv; de Vos, Willem M; Vaughan, Elaine E; Kleerebezem, Michiel

2010-11-01

Probiotic bacteria provide unique opportunities to study the global responses and molecular mechanisms underlying the effects of gut-associated microorganisms in the human digestive tract. In this study, we show by comparative transcriptome analysis using DNA microarrays that the established probiotic Lactobacillus plantarum 299v specifically adapts its metabolic capacity in the human intestine for carbohydrate acquisition and expression of exopolysaccharide and proteinaceous cell surface compounds. This report constitutes the first application of global gene expression profiling of a commensal microorganism in the human gut. A core L. plantarum transcriptome expressed in the mammalian intestine was also determined through comparisons of L. plantarum 299v activities in humans to those found for L. plantarum WCFS1 in germ-free mice. These results identify the niche-specific adaptations of a dietary microorganism to the intestinal ecosystem and provide novel targets for molecular analysis of microbial-host interactions which affect human health.

Human Milk Mucin 1 and Mucin 4 Inhibit Salmonella enterica Serovar Typhimurium Invasion of Human Intestinal Epithelial Cells In Vitro123

PubMed Central

Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E.; Newburg, David S.

2012-01-01

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate–labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens. PMID:22718031

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

PubMed

Barrera, G J; Tortolero, G Sanchez

2016-01-01

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokines and promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions among TFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulation of PAR-2 by siRNA resulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensins expression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

[Effect of control program of human intestinal parasitic diseases in Nanping City].

PubMed

Ming-Ying, Zhuo; Zhi-Ping, Zhang; Hong-Mei, Zhu; Hui, Zhang; Jia-Mei, Huang; Hui, Wen; Han-Guo, Xie

2016-01-22

To understand the epidemic status of human intestinal parasitic diseases and evaluate the effect of the control program in Nanping City, so as to provide an evidence for improving the disease control. The villages were selected by the stratified cluster sampling method and the residents in these villages were surveyed for human intestinal parasitic diseases, and kindergartens were also selected and the children in these kindergartens were surveyed for Enterobius vermicularis infection. In 2007, before the control program, 9 851 residents of Nanping City were surveyed, with the parasitic infection rate of 9.10% (896 infection cases), and the infection rate of E. vermicularis of children was 18.56% (328/1 767). From 2011 to 2014, when the control program was performed, 4 679 residents were surveyed, with the infection rate of 4.06% (190 infection cases), and the infection rate of E. vermicularis of children was 3.87% (33/853). After the control program was launched, the infection rates of human intestinal parasites were decreased. The overall parasitic infection rate and hookworm infection rate showed increasing trends by age ( χ 2 = 49.03 and 53.58 respectively, both P < 0.01). The infection situation of human intestinal parasites is decreased after the implementation of the control program but the infection rate is still at a high level, and the control work should be strengthened.

In Silico Prediction for Intestinal Absorption and Brain Penetration of Chemical Pesticides in Humans.

PubMed

Chedik, Lisa; Mias-Lucquin, Dominique; Bruyere, Arnaud; Fardel, Olivier

2017-06-30

Intestinal absorption and brain permeation constitute key parameters of toxicokinetics for pesticides, conditioning their toxicity, including neurotoxicity. However, they remain poorly characterized in humans. The present study was therefore designed to evaluate human intestine and brain permeation for a large set of pesticides ( n = 338) belonging to various chemical classes, using an in silico graphical BOILED-Egg/SwissADME online method based on lipophilicity and polarity that was initially developed for drugs. A high percentage of the pesticides (81.4%) was predicted to exhibit high intestinal absorption, with a high accuracy (96%), whereas a lower, but substantial, percentage (38.5%) displayed brain permeation. Among the pesticide classes, organochlorines ( n = 30) constitute the class with the lowest percentage of intestine-permeant members (40%), whereas that of the organophosphorus compounds ( n = 99) has the lowest percentage of brain-permeant chemicals (9%). The predictions of the permeations for the pesticides were additionally shown to be significantly associated with various molecular descriptors well-known to discriminate between permeant and non-permeant drugs. Overall, our in silico data suggest that human exposure to pesticides through the oral way is likely to result in an intake of these dietary contaminants for most of them and brain permeation for some of them, thus supporting the idea that they have toxic effects on human health, including neurotoxic effects.

In Silico Prediction for Intestinal Absorption and Brain Penetration of Chemical Pesticides in Humans

PubMed Central

Chedik, Lisa; Mias-Lucquin, Dominique; Bruyere, Arnaud; Fardel, Olivier

2017-01-01

Intestinal absorption and brain permeation constitute key parameters of toxicokinetics for pesticides, conditioning their toxicity, including neurotoxicity. However, they remain poorly characterized in humans. The present study was therefore designed to evaluate human intestine and brain permeation for a large set of pesticides (n = 338) belonging to various chemical classes, using an in silico graphical BOILED-Egg/SwissADME online method based on lipophilicity and polarity that was initially developed for drugs. A high percentage of the pesticides (81.4%) was predicted to exhibit high intestinal absorption, with a high accuracy (96%), whereas a lower, but substantial, percentage (38.5%) displayed brain permeation. Among the pesticide classes, organochlorines (n = 30) constitute the class with the lowest percentage of intestine-permeant members (40%), whereas that of the organophosphorus compounds (n = 99) has the lowest percentage of brain-permeant chemicals (9%). The predictions of the permeations for the pesticides were additionally shown to be significantly associated with various molecular descriptors well-known to discriminate between permeant and non-permeant drugs. Overall, our in silico data suggest that human exposure to pesticides through the oral way is likely to result in an intake of these dietary contaminants for most of them and brain permeation for some of them, thus supporting the idea that they have toxic effects on human health, including neurotoxic effects. PMID:28665355

Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut

PubMed Central

Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito B.; Mills, David A.

2015-01-01

Oligosaccharides are abundant in human milk. Production of these highly diverse structures requires significant energy expenditure by the mother and yet these human milk oligosaccharides offer no direct nutritive value to her infant. A primary function of human milk oligosaccharides is to shape the infant’s intestinal microbiota with life-long consequences. Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure, the result of a large repertoire of bacterial genes encoding an array of glycosidases and oligosaccharide transporters not found in other bacterial species. In vitro, B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides, displays anti-inflammatory activity in premature intestinal cells, and decreases intestinal permeability. In premature infants, B. infantis given in combination with human milk increases B. infantis and decreases Enterobacteriaceae in the feces. Probiotics containing B. infantis decrease the risk of necrotizing enterocolitis in premature infants. Colonization with B. infantis is also associated with increased vaccine responses. Probiotic organisms have historically been selected based on ease of production and stability. The advantages of B. infantis, selected through coevolution with human milk glycans, present an opportunity for focused manipulation of the infant intestinal microbiota. PMID:25303277

Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning.

PubMed

Staley, Christopher; Kaiser, Thomas; Beura, Lalit K; Hamilton, Matthew J; Weingarden, Alexa R; Bobr, Aleh; Kang, Johnthomas; Masopust, David; Sadowsky, Michael J; Khoruts, Alexander

2017-08-01

Human microbiota-associated (HMA) animal models relying on germ-free recipient mice are being used to study the relationship between intestinal microbiota and human disease. However, transfer of microbiota into germ-free animals also triggers global developmental changes in the recipient intestine, which can mask disease-specific attributes of the donor material. Therefore, a simple model of replacing microbiota into a developmentally mature intestinal environment remains highly desirable. Here we report on the development of a sequential, three-course antibiotic conditioning regimen that allows sustained engraftment of intestinal microorganisms following a single oral gavage with human donor microbiota. SourceTracker, a Bayesian, OTU-based algorithm, indicated that 59.3 ± 3.0% of the fecal bacterial communities in treated mice were attributable to the donor source. This overall degree of microbiota engraftment was similar in mice conditioned with antibiotics and germ-free mice. Limited surveys of systemic and mucosal immune sites did not show evidence of immune activation following introduction of human microbiota. The antibiotic treatment protocol described here followed by a single gavage of human microbiota may provide a useful, complimentary HMA model to that established in germ-free facilities. The model has the potential for further in-depth translational investigations of microbiota in a variety of human disease states.

Intestinal Helminths of Wild Bonobos in Forest-Savanna Mosaic: Risk Assessment of Cross-Species Transmission with Local People in the Democratic Republic of the Congo.

PubMed

Narat, Victor; Guillot, Jacques; Pennec, Flora; Lafosse, Sophie; Grüner, Anne Charlotte; Simmen, Bruno; Bokika Ngawolo, Jean Christophe; Krief, Sabrina

2015-12-01

Phylogenetic and geographic proximities between humans and apes pose a risk of zoonotic transmission of pathogens. Bonobos (Pan paniscus) of the Bolobo Territory, Democratic Republic of the Congo, live in a fragmented forest-savanna mosaic setting, a marginal habitat for this species used to living in dense forests. Human activities in the forest have increased the risk of contacts between humans and bonobos. Over 21 months (September 2010-October 2013), we monitored intestinal parasites in bonobo (n = 273) and in human (n = 79) fecal samples to acquire data on bonobo parasitology and to assess the risk of intestinal helminth transmission between these hosts. Coproscopy, DNA amplification, and sequencing of stored dried feces and larvae were performed to identify helminths. Little difference was observed in intestinal parasites of bonobos in this dryer habitat compared to those living in dense forests. Although Strongylids, Enterobius sp., and Capillaria sp. were found in both humans and bonobos, the species were different between the hosts according to egg size or molecular data. Thus, no evidence of helminth transmission between humans and bonobos was found. However, because humans and this threatened species share the same habitat, it is essential to continue to monitor this risk.

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

PubMed

Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

2006-02-17

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

PubMed

Schall, K A; Holoyda, K A; Grant, C N; Levin, D E; Torres, E R; Maxwell, A; Pollack, H A; Moats, R A; Frey, M R; Darehzereshki, A; Al Alam, D; Lien, C; Grikscheit, T C

2015-08-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. Copyright © 2015 the American Physiological Society.

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

PubMed Central

Schall, K. A.; Holoyda, K. A.; Grant, C. N.; Levin, D. E.; Torres, E. R.; Maxwell, A.; Pollack, H. A.; Moats, R. A.; Frey, M. R.; Darehzereshki, A.; Al Alam, D.; Lien, C.

2015-01-01

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. PMID:26089336

Augmented cholesterol absorption and sarcolemmal sterol enrichment slow small intestinal transit in mice, contributing to cholesterol cholelithogenesis

PubMed Central

Xie, Meimin; Kotecha, Vijay R; Andrade, Jon David P; Fox, James G; Carey, Martin C

2012-01-01

Cholesterol gallstones are associated with slow intestinal transit in humans as well as in animal models, but the molecular mechanism is unknown. We investigated in C57L/J mice whether the components of a lithogenic diet (LD; 1.0% cholesterol, 0.5% cholic acid and 17% triglycerides), as well as distal intestinal infection with Helicobacter hepaticus, influence small intestinal transit time. By quantifying the distribution of 3H-sitostanol along the length of the small intestine following intraduodenal instillation, we observed that, in both sexes, the geometric centre (dimensionless) was retarded significantly (P < 0.05) by LD but not slowed further by helicobacter infection (males, 9.4 ± 0.5 (uninfected), 9.6 ± 0.5 (infected) on LD compared with 12.5 ± 0.4 and 11.4 ± 0.5 on chow). The effect of the LD was reproduced only by the binary combination of cholesterol and cholic acid. We inferred that the LD-induced cholesterol enrichment of the sarcolemmae of intestinal smooth muscle cells produced hypomotility from signal-transduction decoupling of cholecystokinin (CCK), a physiological agonist for small intestinal propulsion in mice. Treatment with ezetimibe in an amount sufficient to block intestinal cholesterol absorption caused small intestinal transit time to return to normal. In most cholesterol gallstone-prone humans, lithogenic bile carries large quantities of hepatic cholesterol into the upper small intestine continuously, thereby reproducing this dietary effect in mice. Intestinal hypomotility promotes cholelithogenesis by augmenting formation of deoxycholate, a pro-lithogenic secondary bile salt, and increasing the fraction of intestinal cholesterol absorbed. PMID:22331417

The role of intestinal microbiota in the pathogenesis of metabolic diseases.

PubMed

Węgielska, Iwona; Suliburska, Joanna

2016-01-01

The incidence of metabolic diseases is increasing rapidly all over the world. This situation has led researchers to attempt to explain the pathomechanisms of these disorders and to develop specific recommendations for the prevention and treatment of diseases such as obesity, type-2 diabetes, and atherosclerosis. Recent studies show clear evidence of the role of human intestinal microbiota in health and in predispositions to diseases. Gut microbiota affect a number of complex metabolic reactions, significantly altering the functioning of the human body. Numerous experiments have shown the key role played by the formation process of the intestinal ecosystem in the early stages of human life for programming its metabolic health. The following article is a compilation of the literature available on the formation of the complex intestinal ecosystem and its impact on the incidence of diseases such as obesity, type-2 diabetes, and atherosclerosis.

Synthetic Hydrogels for Human Intestinal Organoid Generation and Colonic Wound Repair

PubMed Central

Cruz-Acuña, Ricardo; Quirós, Miguel; Farkas, Attila E.; Dedhia, Priya H.; Huang, Sha; Siuda, Dorothée; García-Hernández, Vicky; Miller, Alyssa J.; Spence, Jason R.; Nusrat, Asma; García, Andrés J.

2017-01-01

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multi-cellular 3D structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumor-derived extracellular matrix (ECM), which severely limits use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs such that 3D structures are never embedded in tumor-derived ECM. We also demonstrate that the hydrogel serves as an injectable HIO vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury. PMID:29058719

Smoking cessation alters intestinal microbiota: insights from quantitative investigations on human fecal samples using FISH.

PubMed

Biedermann, Luc; Brülisauer, Karin; Zeitz, Jonas; Frei, Pascal; Scharl, Michael; Vavricka, Stephan R; Fried, Michael; Loessner, Martin J; Rogler, Gerhard; Schuppler, Markus

2014-09-01

There has been a dramatic increase in investigations on the potential mechanistic role of the intestinal microbiota in various diseases and factors modulating intestinal microbial composition. We recently reported on intestinal microbial shifts after smoking cessation in humans. In this study, we aimed to conduct further microbial analyses and verify our previous results obtained by pyrosequencing using a direct quantitative microbial approach. Stool samples of healthy smoking human subjects undergoing controlled smoking cessation during a 9-week observational period were analyzed and compared with 2 control groups, ongoing smoking and nonsmoking subjects. Fluorescence in situ hybridization was applied to quantify specific bacterial groups. Intestinal microbiota composition was substantially altered after smoking cessation as characterized by an increase in key representatives from the phyla of Firmicutes (Clostridium coccoides, Eubacterium rectale, and Clostridium leptum subgroup) and Actinobacteria (HGC bacteria and Bifidobacteria) as well as a decrease in Bacteroidetes (Prevotella spp. and Bacteroides spp.) and Proteobacteria (β- and γ-subgroup of Proteobacteria). As determined by fluorescence in situ hybridization, an independent direct quantitative microbial approach, we could confirm that intestinal microbiota composition in humans is influenced by smoking. The characteristics of observed microbial shifts suggest a potential mechanistic association to alterations in body weight subsequent to smoking cessation. More importantly, regarding previously described microbial hallmarks of dysbiosis in inflammatory bowel diseases, a variety of observed microbial alterations after smoking cessation deserve further consideration in view of the divergent effect of smoking on the clinical course of Crohn's disease and ulcerative colitis.

Nutrient fortification of human donor milk affects intestinal function and protein metabolism in preterm pigs

USDA-ARS?s Scientific Manuscript database

Nutrient fortification of human milk is often required to secure adequate growth and organ development for very preterm infants. There is concern that formula-based fortifiers (FFs) induce intestinal dysfunction, feeding intolerance, and necrotizing enterocolitis (NEC). Bovine colostrum (BC) may be ...

Altered egos: antibiotic effects on food animal microbiomes

USDA-ARS?s Scientific Manuscript database

The human food chain begins with upwards of 1000 species of bacteria that inhabit the intestinal tracts of poultry and livestock. These intestinal denizens are responsible for the health and safety of a major protein source for humans. The use of antibiotics to treat animal diseases was followed b...

Plasma endocannabinoid levels in lean, overweight and obese humans: relationships with intestinal permeability markers, inflammation and incretin secretion.

PubMed

Little, Tanya J; Cvijanovic, Nada; DiPatrizio, Nicholas V; Argueta, Donovan A; Rayner, Christopher K; Feinle-Bisset, Christine; Young, Richard L

2018-02-13

Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation, however little is known of these effects in humans. This study aimed to: (i) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl-sn-glycerol (2-AG) and OEA in humans, and (ii) examine relationships with intestinal permeability, inflammation markers and incretin hormone secretion. 20 lean, 18 overweight and 19 obese participants underwent intraduodenal Intralipid® infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumour necrosis factor-α (TNF-α), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and toll-like receptor-4 (TLR4) (RT-PCR), were assessed. Fasting plasma AEA was increased in obese, compared with lean and overweight (P<0.05), with no effect of BMI group or ID lipid infusion on plasma 2-AG or OEA. Duodenal expression of IAP and ZO-1 was reduced in obese, compared with lean (P<0.05), and these levels related negatively to plasma AEA (P<0.05). The iAUC for AEA was positively related to iAUC GIP (r=0.384, P=0.005). Obese individuals have increased plasma AEA and decreased duodenal expression of ZO-1 and IAP, in comparison to lean and overweight. The relationships between plasma AEA with duodenal ZO-1 and IAP, and GIP, suggest that altered endocannabinoid signalling may contribute to changes in intestinal permeability, inflammation and incretin release in human obesity.

Malaria-Associated l-Arginine Deficiency Induces Mast Cell-Associated Disruption to Intestinal Barrier Defenses against Nontyphoidal Salmonella Bacteremia

PubMed Central

Chau, Jennifer Y.; Tiffany, Caitlin M.; Nimishakavi, Shilpa; Lawrence, Jessica A.; Pakpour, Nazzy; Mooney, Jason P.; Lokken, Kristen L.; Caughey, George H.; Tsolis, Renee M.

2013-01-01

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop l-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of l-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with l-arginine or l-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with l-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing l-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans. PMID:23690397

Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.

PubMed

Flora, Alyssa D; Teel, Louise D; Smith, Mark A; Sinclair, James F; Melton-Celsa, Angela R; O'Brien, Alison D

2013-01-01

Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

Ricin Crosses Polarized Human Intestinal Cells and Intestines of Ricin-Gavaged Mice without Evident Damage and Then Disseminates to Mouse Kidneys

PubMed Central

Flora, Alyssa D.; Teel, Louise D.; Smith, Mark A.; Sinclair, James F.; Melton-Celsa, Angela R.; O’Brien, Alison D.

2013-01-01

Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock. PMID:23874986

Precision-cut rat, mouse, and human intestinal slices as novel models for the early-onset of intestinal fibrosis.

PubMed

Pham, Bao Tung; van Haaften, Wouter Tobias; Oosterhuis, Dorenda; Nieken, Judith; de Graaf, Inge Anne Maria; Olinga, Peter

2015-04-01

Intestinal fibrosis (IF) is a major complication of inflammatory bowel disease. IF research is limited by the lack of relevant in vitro and in vivo models. We evaluated precision-cut intestinal slices (PCIS) prepared from human, rat, and mouse intestine as ex vivo models mimicking the early-onset of (human) IF. Precision-cut intestinal slices prepared from human (h), rat (r), and mouse (m) jejunum, were incubated up to 72 h, the viability of PCIS was assessed by ATP content and morphology, and the gene expression of several fibrosis markers was determined. The viability of rPCIS decreased after 24 h of incubation, whereas mPCIS and hPCIS were viable up to 72 h of culturing. Furthermore, during this period, gene expression of heat shock protein 47 and plasminogen activator inhibitor 1 increased in all PCIS in addition to augmented expression of synaptophysin in hPCIS, fibronectin (Fn2) and TGF-β1 in rPCIS, and Fn2 and connective tissue growth factor (Ctgf) in mPCIS. Addition of TGF-β1 to rPCIS or mPCIS induced the gene expression of the fibrosis markers Pro-collagen1a1, Fn2, and Ctgf in both species. However, none of the fibrosis markers was further elevated in hPCIS. We successfully developed a novel ex vivo model that can mimic the early-onset of fibrosis in the intestine using human, rat, and mouse PCIS. Furthermore, in rat and mouse PCIS, TGF-β1 was able to even further increase the gene expression of fibrosis markers. This indicates that PCIS can be used as a model for the early-onset of IF. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function, Antigen Trafficking, and Cytokine Release

PubMed Central

Fiorentino, Maria; Levine, Myron M.

2014-01-01

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response. PMID:24416363

Alkaline sphingomyelinase (NPP7) in hepatobiliary diseases: A field that needs to be closely studied.

PubMed

Duan, Rui-Dong

2018-02-27

Alkaline sphingomyelinase cleaves phosphocholine from sphingomyelin, platelet-activating factor, lysophosphatidylcholine, and less effectively phosphatidylcholine. The enzyme shares no structure similarities with acid or neutral sphingomyelinase but belongs to ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family and therefore is also called NPP7 nowadays. The enzyme is expressed in the intestinal mucosa in many species and additionally in human liver. The enzyme in the intestinal tract has been extensively studied but not that in human liver. Studies on intestinal alkaline sphingomyelinase show that it inhibits colonic tumorigenesis and inflammation, hydrolyses dietary sphingomyelin, and stimulates cholesterol absorption. The review aims to summarize the current knowledge on liver alkaline sphingomyelinase in human and strengthen the necessity for close study on this unique human enzyme in hepatobiliary diseases.

Gut microbiota: puppeteer of the host juvenile growth.

PubMed

Schwarzer, Martin

2018-05-01

This review focuses on the recent discoveries about the impact of intestinal microbiota on mammalian host juvenile growth. Intestinal microbiota is a powerful modulator of many facets of multicellular host's physiology. Recent results from human field studies and animal research have clearly shown that not only the nutrition, but also the intestinal microbiota impacts host postnatal growth kinetics. Absence of microbiome leads to stunted growth in mammalian gnotobiotic models and changes in the composition of the intestinal microbiota can impact the postnatal growth kinetics both positively and negatively under normal nutritional conditions as well as in undernutrition. Strikingly, specific bacterial strains are able to interact with GH/IGF-1 somatotropic axis activity, thus directly impacting host juvenile development. Intestinal microbiota dictates the pace of host postnatal growth. This newly described role envisages that therapy with specific bacterial strains, together with re-nutritional strategies, might successfully alleviate the long-term sequelae of undernutrition during childhood in humans.

Disruption of the Gut Ecosystem by Antibiotics

PubMed Central

2018-01-01

The intestinal microbiota is a complex ecosystem consisting of various microorganisms that expands human genetic repertoire and therefore affects human health and disease. The metabolic processes and signal transduction pathways of the host and intestinal microorganisms are intimately linked, and abnormal progression of each process leads to changes in the intestinal environment. Alterations in microbial communities lead to changes in functional structures based on the metabolites produced in the gut, and these environmental changes result in various bacterial infections and chronic enteric inflammatory diseases. Here, we illustrate how antibiotics are associated with an increased risk of antibiotic-associated diseases by driving intestinal environment changes that favor the proliferation and virulence of pathogens. Understanding the pathogenesis caused by antibiotics would be a crucial key to the treatment of antibiotic-associated diseases by mitigating changes in the intestinal environment and restoring it to its original state. PMID:29214770

Gastric acid reduction leads to an alteration in lower intestinal microflora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanno, Takayuki; Matsuki, Takahiro; Oka, Masashi

2009-04-17

To clarify the alterations in lower intestinal microflora induced by gastric acid reduction, the dynamics of 12 major genera or groups of bacteria comprising the microflora in feces and colonic contents were examined by quantitative real-time PCR in proton pump inhibitor-treated rats and in asymptomatic human subjects with hypochlorhydria. In both rat and human experiments, most genera or groups of intestinal microflora (facultative and obligate anaerobes) proliferated by gastric acid reduction, and marked and significant increases in the Lactobacilli group and Veillonella, oropharyngeal bacteria, were observed. In rats, potent gastric acid inhibition led to a marked and significant increase ofmore » intestinal bacteria, including the Bacteroidesfragilis group, while Bifidobacterium, a beneficial bacterial species, remained at a constant level. These results strongly indicate that the gastric acid barrier not only controls the colonization and growth of oropharyngeal bacteria, but also regulates the population and composition of lower intestinal microflora.« less

Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant.

PubMed

van der Sanden, Sabine M G; Sachs, Norman; Koekkoek, Sylvie M; Koen, Gerrit; Pajkrt, Dasja; Clevers, Hans; Wolthers, Katja C

2018-05-09

Human enteroviruses frequently cause severe diseases in children. Human enteroviruses are transmitted via the fecal-oral route and respiratory droplets, and primary replication occurs in the gastro-intestinal and respiratory tracts; however, how enteroviruses infect these sites is largely unknown. Human intestinal organoids have recently proven to be valuable tools for studying enterovirus-host interactions in the intestinal tract. In this study, we demonstrated the susceptibility of a newly developed human airway organoid model for enterovirus 71 (EV71) infection. We showed for the first time in a human physiological model that EV71 replication kinetics are strain-dependent. A glutamine at position 145 of the VP1 capsid protein was identified as a key determinant of infectivity, and residues VP1-98K and VP1-104D were identified as potential infectivity markers. The results from this study provide new insights into EV71 infectivity in the human airway epithelia and demonstrate the value of organoid technology for virus research.

Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells.

PubMed

Hirotani, Yoshihiko; Ikeda, Kenji; Kato, Ryuji; Myotoku, Michiaki; Umeda, Takashi; Ijiri, Yoshio; Tanaka, Kazuhiko

2008-09-01

Indirect evidence suggests that lactoferrin (Lf), a major iron-binding protein in human milk, induces enterocyte growth and proliferation, depending on its concentration and affects the function and permeability of the intestinal mucosa. The bacterial endotoxin (lipopolysaccharide, LPS) is known to cause mucosal hyperpermeability in vivo. However, protective effects of Lf against LPS-mediated intestinal mucosal damage and barrier function in epithelial cells are not yet fully clarified. The aim of this study was to investigate whether Lf can reduce the cellular injury and alter epithelial hyperpermeability caused by LPS in human intestinal Caco-2 cells. When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), the protective effects against LPS-induced damage to Caco-2 cells were observed at doses of 800 and 1000 microg/ml Lf. The barrier function of Caco-2 monolayer tight junctions was assessed by measuring transepithelial electrical resistance (TEER) and permeability of FITC-labeled dextran 4000 (FD-4). The treatment of Caco-2 cells with Lf at doses of 400 and 1000 microg/ml significantly increased TEER as compared to treatment with LPS alone for 2 h (p<0.05). Further, at doses of 400 and 1000 microg/ml, Lf inhibited the enhancement of LPS-mediated permeability in Caco-2 cell monolayer. The results of this study suggest that Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells.

Metabolism of gentiopicroside (gentiopicrin) by human intestinal bacteria.

PubMed

el-Sedawy, A I; Hattori, M; Kobashi, K; Namba, T

1989-09-01

As a part of our studies on the metabolism of crude drug components by intestinal bacteria, gentiopicroside (a secoiridoid glucoside isolated from Gentiana lutea), was anaerobically incubated with various defined strains of human intestinal bacteria. Many species had ability to transform it to a series of metabolites. Among them, Veillonella parvula ss parvula produced five metabolites, which were identified as erythrocentaurin, gentiopicral, 5-hydroxymethylisochroman-1-one,5-hydroxymethylisochromen-1- one and trans-5,6-dihydro-5-hydroxymethyl-6-methyl-1H,3H-pyrano[3,4-c]pyra n-1-one.

Dogs and intestinal parasites: a public health problem.

PubMed

Seah, S K; Hucal, G; Law, C

1975-05-17

The stools of 239 stray dogs were examined for intestinal parasites. Of the helminths found, Toxocara canis (43.5%), tapeworms (25.5%), Ascaris species (21.3%) and hookworms (12.5%) were the commonest. Of the protozoans found, Isospora species and Entamoeba coli were the most prevalent. An unusual feature of the present study was the finding of Ascaris species. The importance of the high prevalence of intestinal parasites in dogs, the close contact of humans with dogs' excreta and the possible role of this environmental pollution in the spread of human disease are discussed.

Editorial: Emerging approaches for typing, detection, characterization, and traceback of Escherichia coli

USDA-ARS?s Scientific Manuscript database

Commensal E. coli inhabit the large intestines of humans and animals and are important in maintaining normal intestinal homeostasis. There are also many groups of disease-causing E. coli, including diarrheagenic and extra-intestinal pathogenic E. coli (ExPEC). There are approximately O188 somatic O...

Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase

USDA-ARS?s Scientific Manuscript database

Inhibition of intestinal alpha-glucosidases and pancreatic alpha-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 ...

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.

PubMed

Matano, Mami; Date, Shoichi; Shimokawa, Mariko; Takano, Ai; Fujii, Masayuki; Ohta, Yuki; Watanabe, Toshiaki; Kanai, Takanori; Sato, Toshiro

2015-03-01

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier.

PubMed

Abal, Paula; Louzao, M Carmen; Fraga, María; Vilariño, Natalia; Ferreiro, Sara; Vieytes, Mercedes R; Botana, Luis M

2017-01-01

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellates genera Azadinium and Amphidoma. These toxins cause azaspiracid poisoning (AZP), characterized by severe gastrointestinal illness in humans after the consumption of bivalve molluscs contaminated with AZAs. The main aim of the present study was to examine the consequences of human exposure to AZA1 by the study of absorption and effects of the toxin on Caco-2 cells, a reliable model of the human intestine. The ability of AZA1 to cross the human intestinal epithelium has been evaluated by the Caco-2 transepithelial permeability assay. The toxin has been detected and quantified using a microsphere-based immunoassay. Cell alterations and ultrastructural effects has been observed with confocal and transmission electron microscopy Results: AZA1 was absorbed by Caco-2 cells in a dose-dependent way without affecting cell viability. However, modifications on occludin distribution detected by confocal microscopy imaging indicated a possible monolayer integrity disruption. Nevertheless, transmission electron microscopy imaging revealed ultrastructural damages at the nucleus and mitochondria with autophagosomes in the cytoplasm, however, tight junctions and microvilli remained unaffected. After the ingestion of molluscs with the AZA1, the toxin will be transported through the human intestinal barrier to blood causing damage on epithelial cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Broad diversity and newly cultured bacterial isolates from enrichment of pig feces on complex polysaccharides

USDA-ARS?s Scientific Manuscript database

Microbial fermentation of plant cell wall components to short chain fatty acids in the large intestine provides energy to both humans and pigs. To better understand plant cell wall fermentation in the pig and human intestine, we isolated cellulose, xylan, and pectin fermenting bacteria from pig and ...

Influence of resistant starch on the SCFA production and cell counts of butyrate-producing Eubacterium spp. in the human intestine.

PubMed

Schwiertz, A; Lehmann, U; Jacobasch, G; Blaut, M

2002-01-01

The genus Eubacterium, which is the second most common genus in the human intestine, includes several known butyrate producers. We hypothesized that Eubacterium species play a role in the intestinal butyrate production and are inducible by resistant starch. In a human pilot study species-specific and group-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labelled oligonucleotide probes were used to quantify butyrogenic species of the genera Eubacterium, Clostridium and Ruminococcus. Following the intake of RS type III a significant increase in faecal butyrate but not in total SCFA was observed. However, increase in butyrate was not accompanied by a proliferation in the targeted bacteria. The tested Eubacterium species have the capacity to produce butyrate but do not appear to play a major role for butyric acid production in the human intestine. In view of the fact that the bacteria responsible for butyrate production are largely unknown, it is still difficult to devise a dietary intervention to stimulate butyrogenic bacteria in a targeted way.

Transcriptional and functional profiling defines human small intestinal macrophage subsets.

PubMed

Bujko, Anna; Atlasy, Nader; Landsverk, Ole J B; Richter, Lisa; Yaqub, Sheraz; Horneland, Rune; Øyen, Ole; Aandahl, Einar Martin; Aabakken, Lars; Stunnenberg, Hendrik G; Bækkevold, Espen S; Jahnsen, Frode L

2018-02-05

Macrophages (Mfs) are instrumental in maintaining immune homeostasis in the intestine, yet studies on the origin and heterogeneity of human intestinal Mfs are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMos), were largely replaced within 3 wk, whereas two subsets with features of mature Mfs, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptional analysis showed that all Mf subsets were markedly distinct from PBMos and dendritic cells. Compared with PBMos, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMos. © 2018 Bujko et al.

Expression of an Intestine-Specific Transcription Factor (CDX1) in Intestinal Metaplasia and in Subsequently Developed Intestinal Type of Cholangiocarcinoma in Rat Liver

PubMed Central

Ren, Ping; Silberg, Debra G.; Sirica, Alphonse E.

2000-01-01

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478–486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats. PMID:10666391

Expression of an intestine-specific transcription factor (CDX1) in intestinal metaplasia and in subsequently developed intestinal type of cholangiocarcinoma in rat liver.

PubMed

Ren, P; Silberg, D G; Sirica, A E

2000-02-01

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478-486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats.

A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

PubMed

Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

2014-12-01

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

Comparative analysis of the genomes of intestinal spirochetes of human and animal origin.

PubMed Central

Coene, M; Agliano, A M; Paques, A T; Cattani, P; Dettori, G; Sanna, A; Cocito, C

1989-01-01

The aim of the present work was to compare the genomes of 21 strains of intestinal spirochetes, which were isolated from patients suffering intestinal disorders, with those of Treponema hyodysenteriae (strain P18), the known etiological agent of swine dysentery (bloody scours), and of a nonpathogenic strain (M1) of Treponema innocens. The percent guanine-plus-cytosine value of the 23 DNAs was found to be 25.5 to 30.1, as determined by a double-labeling procedure based on nick-translation by DNA polymerase I. The genome size of two spirochetal strains, of human and porcine origin, was found to be similar (4 x 10(6) base pairs) and close to that of the reference bacterium Escherichia coli (4.2 x 10(6) base pairs). Restriction analysis showed the presence of two modified bases in spirochetal DNA. Methyladenine was present in the GATC sequence of DNA from 15 spirochetes of human origin, and methylcytosine was present in several sequences occurring in all strains. The DNA of T. hyodysenteriae displayed a 30 to 100% homology with respect to that of 21 spirochetes from humans, thus suggesting the occurrence of a genetic heterogeneity in the latter group. These data indicate that the intestinal spirochetes analyzed in the present work are related; hence there is a possibility of domestic animals being reservoirs of microorganisms pathogenic for humans. A classification of intestinal treponemes into subgroups has been proposed on the basis of restriction analysis and hybridization experiments. Images PMID:2535832

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease.

PubMed

Giles, E M; Sanders, T J; McCarthy, N E; Lung, J; Pathak, M; MacDonald, T T; Lindsay, J O; Stagg, A J

2017-01-01

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

Use of Isoform-Specific UGT Metabolism to Determine and Describe Rates and Profiles of Glucuronidation of Wogonin and Oroxylin A by Human Liver and Intestinal Microsomes

PubMed Central

Zhou, Qiong; Zheng, Zhijie; Xia, Bijun; Tang, Lan; Lv, Chang; Liu, Wei; Liu, Zhongqiu; Hu, Ming

2010-01-01

Purposes Glucuronidation via UDP-glucuronosyltransferases (or UGTs) is a major metabolic pathway. The purposes of this study are to determine the UGT-isoform specific metabolic fingerprint (or GSMF) of wogonin and oroxylin A, and to use isoform-specific metabolism rates and kinetics to determine and describe their glucuronidation behaviors in tissue microsomes. Methods In vitro glucuronidation rates and profiles were measured using expressed UGTs and human intestinal and liver microsomes. Results GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoform-specific metabolism could describe their metabolism rates and profiles in human liver and intestinal microsomes. Conclusion GSMF and isoform-specific metabolism profiles can determine and describe glucuronidation rates and profiles in human tissue microsomes. PMID:20411407

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes, liver cytosol and plasma preparations.

PubMed

Hedges, Laura; Brown, Susan; Vardy, Audrey; Doyle, Edward; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-04-19

The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 15, 21 and 90 days and from adult humans. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.

[Stratified sampling survey of major human parasitic diseases in Henan province].

PubMed

Xu, B L; Zhang, H W; Deng, Y; Chen, Z L; Chen, W Q; Lu, D L; Zhang, Y L; Zhao, Y L; Lin, X M; Huang, Q; Yang, C Y; Liu, Y; Zhou, R M; Li, P; Chen, J S; He, L J; Qian, D

2018-03-10

Objective: To understand the prevalence of major human parasitic diseases and related factors in Henan province. Methods: This stratified sampling survey was carried out according to the requirement of national survey protocol of major human parasitic diseases, 2014-2015. The prevalence of soil-transmitted helminths infection, taeniasis and intestinal protozoiasis were surveyed in 104 sites selected from 35 counties (districts) and the prevalence of clonorchiasis was surveyed in 62 sites selected from 37 townships. In each survey spot, 250 persons were surveyed. A total of 26 866 persons and 15 893 persons were surveyed. Modified Kato-Katz thick smear was used to detect the eggs of intestinal helminthes. Tube fecal culture was used to identify the species of hookworm. The Enterobius eggs were detected in children aged 3 to 6 years by using adhesive tape. The cyst and trophozoite of intestinal protozoa were examined with physiological saline direct smear method and iodine stain method. Results: The overall infestation rate of intestinal parasites was2.02% in Henan, and the worm infection rate was higher than protozoa infection rate. Fourteen kinds of intestinal parasites were found, including nematode (5 species), trematode (2 species), and protozoan (7 species). The infection rate of Enterobius vermicularis was highest, and Qinba Mountain ecological area had the highest infestation rate of intestinal parasites in 4 ecological areas of Henan. There was no significant difference in intestinal parasite infection rate between males and females ( χ (2)=3.630, P =0.057), and the differences in intestinal parasite infection rate among different age groups had significance ( χ (2)=124.783, P =0.000 1). The infection rate reached the peak in age group ≤9 years and the major parasite was Enterobius vermicularis . Furthermore the overall human infection rate of parasite showed a downward trend with the increase of educational level of the people ( χ (2)=70.969, P =0.000 1), the differences had significance ( χ (2)=120.118, P =0.000 1). For different populations, the infection rate of intestinal parasites was highest among preschool children. The infection of intestinal helminth was mainly mild, only 2 severe cases were detected. The infection rate of Clonorchis sinensis in urban residents was only 0.006%. Logistic regression analysis showed that being preschool children ( χ (2)=15.765, P =0.000 1) and drinking well water ( χ (2)=45.589, P =0.000 1) were the risk factors for intestinal parasite infection, and annual income per capita of farmers was the protective factor against intestinal parasite infection. The infection rates of protozoa and intestinal parasites decreased sharply compared with the results of previous two surveys, and the rate of intestinal helminth infection also dropped sharply compared with the second survey. The numbers of protozoa, helminth and intestinal parasites detected in this survey were all less than the numbers found in the previous two surveys. Conclusions: Compared the results of three surveys in Henan, the infection rate of protozoa and intestinal parasites showed a downward trend. The prevention and treatment of Enterobius vermicularis infection in children should be the key point of parasitic disease control in the future.

Carboxylesterase 1 Is Regulated by Hepatocyte Nuclear Factor 4α and Protects Against Alcohol- and MCD diet-induced Liver Injury.

PubMed

Xu, Jiesi; Xu, Yang; Li, Yuanyuan; Jadhav, Kavita; You, Min; Yin, Liya; Zhang, Yanqiao

2016-04-14

The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4α (HNF4α) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4α and CES1 expression in primary hepatocytes. HNF4α regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury.

Metabolism of aspirin and procaine in mice pretreated with O-4-nitrophenyl methyl(phenyl)phosphinate or O-4-nitrophenyl diphenylphosphinate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joly, J.M.; Brown, T.M.

Concentrations of (carboxyl-/sup 14/C)procaine in blood of mice were increased threefold for 27 min by exposure to O-4-nitrophenyl diphenylphosphinate 2 hr prior to (carboxyl-/sup 14/C)procaine injection ip, while there was no effect of O-4-nitrophenyl methyl(phenyl)phosphinate pretreatment. There was no effect of either organophosphinate on the primary hydrolysis of (acetyl-l-/sup 14/C)aspirin when assessed by the expiration of (/sup 14/C)carbon dioxide; however, O-4-nitrophenyl diphenylphosphinate pretreatment produced transient increases in blood concentrations of both (carboxyl-/sup 14/C)aspirin and (carboxyl-/sup 14/C)salicylic acid following administration of (carboxyl-/sup 14/C)aspirin. Liver carboxylesterase activity in O-4-nitrophenyl diphenylphosphinate pretreated mice was 11% of control activity. These results indicate the potentialmore » for drug interaction with O-4-nitrophenyl diphenylphosphinate but not with O-4-nitrophenyl methyl(phenyl)phosphinate. It appears that liver carboxylesterase activity has a minor role in hydrolysis of aspirin in vivo, but may be more important in procaine metabolism.« less

Prevalence of intestinal parasites in Isfahan city, central Iran, 2014.

PubMed

Jafari, Rasool; Sharifi, Forough; Bagherpour, Bahram; Safari, Marzieh

2016-09-01

Intestinal parasites are important enteric pathogens. Poverty, low quality of food and water supply and poor sanitation systems are the important factors associated with intestinal parasitic infections. These kinds of infections can be a good index for hygienic and sanitation status of the society. This study aimed to determine the prevalence of intestinal parasitic infections among humans referred to Dr. Sharifi Clinical Laboratory, Isfahan, Iran, 2014. In this cross sectional study, 652 fecal samples (286 males and 366 females) from humans who had stool examination test from January to August 2014 were chosen. Microscopic examination for parasitic infections has been carried out using wet mount method. Indistinguishable samples underwent trichrome staining method for accurate identification of protozoa. Intestinal parasitic infections were observed in 68 (10.42 %) out of 652 studied humans. Forty eight Blastocystis hominis (7.36 %), thirteen Endolimax nana (1.99 %), nine Giardia lamblia (1.38 %), five Entamoeba coli (0.76 %), four Chilomastix mesnili (0.61 %) and two Iodamoeba butschlii (0.15 %) were the observed protozoa in the studied population. B. hominis, E. nana and C. mesnili were found to be significantly more prevalent in people with loose stool specimen. Considering the helminthic infections, only one case (0.15 %) that was excreted Taenia saginata proglottids has been documented among 652 studied humans. Based on the findings of the present study intestinal parasitic infections in Isfahan city has been dramatically decreased over the past years and shows a good hygienic and sanitation status of the city.

A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium.

PubMed

Wang, Yuli; Gunasekara, Dulan B; Reed, Mark I; DiSalvo, Matthew; Bultman, Scott J; Sims, Christopher E; Magness, Scott T; Allbritton, Nancy L

2017-06-01

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biopharmaceutical classification of drugs using intrinsic dissolution rate (IDR) and rat intestinal permeability.

PubMed

Zakeri-Milani, Parvin; Barzegar-Jalali, Mohammad; Azimi, Mandana; Valizadeh, Hadi

2009-09-01

The solubility and dissolution rate of active ingredients are of major importance in preformulation studies of pharmaceutical dosage forms. In the present study, passively absorbed drugs are classified based on their intrinsic dissolution rate (IDR) and their intestinal permeabilities. IDR was determined by measuring the dissolution of a non-disintegrating disk of drug, and effective intestinal permeability of tested drugs in rat jejunum was determined using single perfusion technique. The obtained intrinsic dissolution rate values were in the range of 0.035-56.8 mg/min/cm(2) for tested drugs. The minimum and maximum intestinal permeabilities in rat intestine were determined to be 1.6 x 10(-5) and 2 x 10(-4)cm/s, respectively. Four classes of drugs were defined: Category I: P(eff,rat)>5 x 10(-5) (cm/s) or P(eff,human)>4.7 x 10(-5) (cm/s), IDR>1(mg/min/cm(2)), Category II: P(eff,rat)>5 x 10(-5) (cm/s) or P(eff,human)>4.7 x 10(-5) (cm/s), IDR<1(mg/min/cm(2)), Category III: P(eff,rat)<5 x 10(-5) (cm/s) or P(eff,human)<4.7 x 10(-5) (cm/s), IDR>1 (mg/min/cm(2)) and Category IV: P(eff,rat)<5 x 10(-5) (cm/s) or P(eff,human)<4.7 x 10(-5) (cm/s), IDR<1(mg/min/cm(2)). According to the results obtained and proposed classification of drugs, it is concluded that drugs could be categorized correctly based on their IDR and intestinal permeability values.

Intestinal microbiome landscaping: insight in community assemblage and implications for microbial modulation strategies

PubMed Central

Hugenholtz, Floor; Lahti, Leo; Smidt, Hauke; de Vos, Willem M.

2017-01-01

Abstract High individuality, large complexity and limited understanding of the mechanisms underlying human intestinal microbiome function remain the major challenges for designing beneficial modulation strategies. Exemplified by the analysis of intestinal bacteria in a thousand Western adults, we discuss key concepts of the human intestinal microbiome landscape, i.e. the compositional and functional ‘core’, the presence of community types and the existence of alternative stable states. Genomic investigation of core taxa revealed functional redundancy, which is expected to stabilize the ecosystem, as well as taxa with specialized functions that have the potential to shape the microbiome landscape. The contrast between Prevotella- and Bacteroides-dominated systems has been well described. However, less known is the effect of not so abundant bacteria, for example, Dialister spp. that have been proposed to exhibit distinct bistable dynamics. Studies employing time-series analysis have highlighted the dynamical variation in the microbiome landscape with and without the effect of defined perturbations, such as the use of antibiotics or dietary changes. We incorporate ecosystem-level observations of the human intestinal microbiota and its keystone species to suggest avenues for designing microbiome modulation strategies to improve host health. PMID:28364729

Multilayered epithelium in a rat model and human Barrett's esophagus: Similar expression patterns of transcription factors and differentiation markers

PubMed Central

Chen, Xiaoxin; Qin, Rong; Liu, Ba; Ma, Yan; Su, Yinghao; Yang, Chung S; Glickman, Jonathan N; Odze, Robert D; Shaheen, Nicholas J

2008-01-01

Background In rats, esophagogastroduodenal anastomosis (EGDA) without concomitant chemical carcinogen treatment leads to gastroesophageal reflux disease, multilayered epithelium (MLE, a presumed precursor in intestinal metaplasia), columnar-lined esophagus, dysplasia, and esophageal adenocarcinoma. Previously we have shown that columnar-lined esophagus in EGDA rats resembled human Barrett's esophagus (BE) in its morphology, mucin features and expression of differentiation markers (Lab. Invest. 2004;84:753–765). The purpose of this study was to compare the phenotype of rat MLE with human MLE, in order to gain insight into the nature of MLE and its potential role in the development of BE. Methods Serial sectioning was performed on tissue samples from 32 EGDA rats and 13 patients with established BE. Tissue sections were immunohistochemically stained for a variety of transcription factors and differentiation markers of esophageal squamous epithelium and intestinal columnar epithelium. Results We detected MLE in 56.3% (18/32) of EGDA rats, and in all human samples. As expected, both rat and human squamous epithelium, but not intestinal metaplasia, expressed squamous transcription factors and differentiation markers (p63, Sox2, CK14 and CK4) in all cases. Both rat and human intestinal metaplasia, but not squamous epithelium, expressed intestinal transcription factors and differentiation markers (Cdx2, GATA4, HNF1α, villin and Muc2) in all cases. Rat MLE shared expression patterns of Sox2, CK4, Cdx2, GATA4, villin and Muc2 with human MLE. However, p63 and CK14 were expressed in a higher proportion of rat MLE compared to humans. Conclusion These data indicate that rat MLE shares similar properties to human MLE in its expression pattern of these markers, not withstanding small differences, and support the concept that MLE may be a transitional stage in the metaplastic conversion of squamous to columnar epithelium in BE. PMID:18190713

Multilayered epithelium in a rat model and human Barrett's esophagus: similar expression patterns of transcription factors and differentiation markers.

PubMed

Chen, Xiaoxin; Qin, Rong; Liu, Ba; Ma, Yan; Su, Yinghao; Yang, Chung S; Glickman, Jonathan N; Odze, Robert D; Shaheen, Nicholas J

2008-01-11

In rats, esophagogastroduodenal anastomosis (EGDA) without concomitant chemical carcinogen treatment leads to gastroesophageal reflux disease, multilayered epithelium (MLE, a presumed precursor in intestinal metaplasia), columnar-lined esophagus, dysplasia, and esophageal adenocarcinoma. Previously we have shown that columnar-lined esophagus in EGDA rats resembled human Barrett's esophagus (BE) in its morphology, mucin features and expression of differentiation markers (Lab. Invest. 2004;84:753-765). The purpose of this study was to compare the phenotype of rat MLE with human MLE, in order to gain insight into the nature of MLE and its potential role in the development of BE. Serial sectioning was performed on tissue samples from 32 EGDA rats and 13 patients with established BE. Tissue sections were immunohistochemically stained for a variety of transcription factors and differentiation markers of esophageal squamous epithelium and intestinal columnar epithelium. We detected MLE in 56.3% (18/32) of EGDA rats, and in all human samples. As expected, both rat and human squamous epithelium, but not intestinal metaplasia, expressed squamous transcription factors and differentiation markers (p63, Sox2, CK14 and CK4) in all cases. Both rat and human intestinal metaplasia, but not squamous epithelium, expressed intestinal transcription factors and differentiation markers (Cdx2, GATA4, HNF1alpha, villin and Muc2) in all cases. Rat MLE shared expression patterns of Sox2, CK4, Cdx2, GATA4, villin and Muc2 with human MLE. However, p63 and CK14 were expressed in a higher proportion of rat MLE compared to humans. These data indicate that rat MLE shares similar properties to human MLE in its expression pattern of these markers, not withstanding small differences, and support the concept that MLE may be a transitional stage in the metaplastic conversion of squamous to columnar epithelium in BE.

Micro array analysis of the intestinal host response in Giardia duodenalis assemblage E infected cattle

USDA-ARS?s Scientific Manuscript database

Giardia duodenalis is one of the most commonly found intestinal pathogens in humans and animals. However, little is known about the host-parasite interaction in its natural hosts. The objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, us...

Carbohydrates and the human gut microbiota.

PubMed

Chassard, Christophe; Lacroix, Christophe

2013-07-01

Due to its scale and its important role in maintaining health, the gut microbiota can be considered as a 'new organ' inside the human body. Many complex carbohydrates are degraded and fermented by the human gut microbiota in the large intestine to both yield basic energy salvage and impact gut health through produced metabolites. This review will focus on the gut microbes and microbial mechanisms responsible for polysaccharides degradation and fermentation in the large intestine. Gut microbes and bacterial metabolites impact the host at many levels, including modulation of inflammation, and glucose and lipid metabolisms. A complex relationship occurs in the intestine between the human gut microbiota, diet and the host. Research on carbohydrates and gut microbiota composition and functionality is fast developing and will open opportunities for prevention and treatment of obesity, diabetes and other related metabolic disorders through manipulation of the gut ecosystem.

Diversity of halophilic archaea in fermented foods and human intestines and their application.

PubMed

Lee, Han-Seung

2013-12-01

Archaea are prokaryotic organisms distinct from bacteria in the structural and molecular biological sense, and these microorganisms are known to thrive mostly at extreme environments. In particular, most studies on halophilic archaea have been focused on environmental and ecological researches. However, new species of halophilic archaea are being isolated and identified from high salt-fermented foods consumed by humans, and it has been found that various types of halophilic archaea exist in food products by culture-independent molecular biological methods. In addition, even if the numbers are not quite high, DNAs of various halophilic archaea are being detected in human intestines and much interest is given to their possible roles. This review aims to summarize the types and characteristics of halophilic archaea reported to be present in foods and human intestines and to discuss their application as well.

hPSC-derived lung and intestinal organoids as models of human fetal tissue

PubMed Central

Aurora, Megan; Spence, Jason R.

2016-01-01

In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC). PMID:27287882

Chip-based human liver-intestine and liver-skin co-cultures--A first step toward systemic repeated dose substance testing in vitro.

PubMed

Maschmeyer, Ilka; Hasenberg, Tobias; Jaenicke, Annika; Lindner, Marcus; Lorenz, Alexandra Katharina; Zech, Julie; Garbe, Leif-Alexander; Sonntag, Frank; Hayden, Patrick; Ayehunie, Seyoum; Lauster, Roland; Marx, Uwe; Materne, Eva-Maria

2015-09-01

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures. Copyright © 2015. Published by Elsevier B.V.

Effect of Digestion and Storage of Human Milk on Free Fatty Acid Concentration and Cytotoxicity

PubMed Central

Penn, Alexander H.; Altshuler, Angelina E.; Small, James W.; Taylor, Sharon F.; Dobkins, Karen R.; Schmid-Schönbein, Geert W.

2014-01-01

Objectives Fat is digested in the intestine into free fatty acids (FFAs), which are detergents and therefore toxic to cells at micromolar concentration. The mucosal barrier protects cells in the adult intestine, but this barrier may not be fully developed in premature infants. Lipase-digested infant formula, but not fresh human milk, has elevated FFAs and is cytotoxic to intestinal cells, and therefore could contribute to intestinal injury in necrotizing enterocolitis (NEC). But even infants exclusively fed breast milk may develop NEC. Our objective was to determine if stored milk and milk from donor milk banks (DM) could also become cytotoxic, especially after digestion. Methods We exposed cultured rat intestinal epithelial cells or human neutrophils to DM and milk collected fresh and stored at 4 or −20 °C for up to 12 weeks and then treated for 2 hours (37°C) with 0.1 or 1 mg/ml pancreatic lipase and/or trypsin and chymotrypsin. Results DM and milk stored 3 days (at 4 or −20 °C) and then digested were cytotoxic. Storage at −20 °C for 8 and 12 weeks resulted in an additional increase in cytotoxicity. Protease digestion decreased, but did not eliminate cell death. Conclusions Current storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC. PMID:24840512

Delphinidin Reduces Glucose Uptake in Mice Jejunal Tissue and Human Intestinal Cells Lines through FFA1/GPR40.

PubMed

Hidalgo, Jorge; Teuber, Stefanie; Morera, Francisco J; Ojeda, Camila; Flores, Carlos A; Hidalgo, María A; Núñez, Lucía; Villalobos, Carlos; Burgos, Rafael A

2017-04-05

Anthocyanins are pigments with antihyperglycemic properties, and they are potential candidates for developing functional foods for the therapy or prevention of Diabetes mellitus type 2 (DM2). The mechanism of these beneficial effects of anthocyanins are, however, hard to explain, given their very low bioavailability due to poor intestinal absorption. We propose that free fatty acid receptor 1 (FFA1, also named GPR40), is involved in an inhibitory effect of the anthocyanidin delphinidin over intestinal glucose absorption. We show the direct effects of delphinidin on the intestine using jejunum samples from RF/J mice, and the human intestinal cell lines HT-29, Caco-2, and NCM460. By the use of specific pharmacological antagonists, we determined that delphinidin inhibits glucose absorption in both mouse jejunum and a human enterocytic cell line in a FFA1-dependent manner. Delphinidin also affects the function of sodium-glucose cotransporter 1 (SGLT1). Intracellular signaling after FFA1 activation involved cAMP increase and cytosolic Ca 2+ oscillations originated from intracellular Ca 2+ stores and were followed by store-operated Ca 2+ entry. Taken together, our results suggest a new GPR-40 mediated local mechanism of action for delphinidin over intestinal cells that may in part explain its antidiabetic effect. These findings are promising for the search for new prevention and pharmacological treatment strategies for DM2 management.

Improvement of the human intestinal flora by ingestion of the probiotic strain Lactobacillus johnsonii La1.

PubMed

Yamano, Toshihiko; Iino, Hisakazu; Takada, Mamiko; Blum, Stephanie; Rochat, Florence; Fukushima, Yoichi

2006-02-01

To exert beneficial effects for the host, for example, improving the intestinal microflora, a probiotic must reach the intestine as a viable strain. These properties must be demonstrated by in vitro as well as in vivo methods. However, only a few well-designed human clinical studies have shown these properties. Lactobacillus johnsonii La1 has been shown to give many beneficial effects for the host, but it is unclear whether a viable strain of L. johnsonii La1 has the effect of improving host intestinal microflora. In the present study, a randomised double-blind placebo-controlled cross-over trial was conducted to elucidate the effect of L. johnsonii La1 on human intestinal microflora. Twenty-two young healthy Japanese women were randomly divided into two groups, and either received fermented milk with L. johnsonii La1 or a fermented milk without L. johnsonii La1 (placebo) daily for 21 d. Consumption of the fermented milk: (a) increased total Bifidobacterium and Lactobacillus, and decreased lecithinase-positive Clostridium in the faeces; (b) increased the faecal lactic acid concentrations; (c) decreased the faecal pH; (d) increased the defecation frequency. These changes were stronger than those observed with the placebo. L. johnsonii La1 was identified in all subjects only after the consumption of the fermented milk. These results suggest that L. johnsonii La1 can contribute to improve intestinal microflora with probiotic properties.

Insecticide resistance of Anopheles sinensis and An. vagus in Hainan Island, a malaria-endemic area of China.

PubMed

Qin, Qian; Li, Yiji; Zhong, Daibin; Zhou, Ning; Chang, Xuelian; Li, Chunyuan; Cui, Liwang; Yan, Guiyun; Chen, Xiao-Guang

2014-03-03

Malaria is one of the most important public health problems in Southeast Asia, including Hainan Island, China. Vector control is the main malaria control measure, and insecticide resistance is a major concern for the effectiveness of chemical insecticide control programs. The objective of this study is to determine the resistance status of the main malaria vector species to pyrethroids and other insecticides recommended by the World Health Organization (WHO) for indoor residual sprays. The larvae and pupae of Anopheles mosquitoes were sampled from multiple sites in Hainan Island, and five sites yielded sufficient mosquitoes for insecticide susceptibility bioassays. Bioassays of female adult mosquitoes three days after emergence were conducted in the two most abundant species, Anopheles sinensis and An. vagus, using three insecticides (0.05% deltamethrin, 4% DDT, and 5% malathion) and following the WHO standard tube assay procedure. P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were measured. Mutations at the knockdown resistance (kdr) gene and the ace-1 gene were detected by DNA sequencing and PCR-RFLP analysis, respectively. An. sinensis and An. vagus were the predominant Anopheles mosquito species. An. sinensis was found to be resistant to DDT and deltamethrin. An. vagus was susceptible to deltamethrin but resistant to DDT and malathion. Low kdr mutation (L1014F) frequency (<10%) was detected in An. sinensis, but no kdr mutation was detected in An. vagus populations. Modest to high (45%-75%) ace-1 mutation frequency was found in An. sinensis populations, but no ace-1 mutation was detected in An. vagus populations. Significantly higher P450 monooxygenase and carboxylesterase activities were detected in deltamethrin-resistant An. sinensis, and significantly higher P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were found in malathion-resistant An. vagus mosquitoes. Multiple insecticide resistance was found in An. sinensis and An. vagus in Hainan Island, a malaria-endemic area of China. Cost-effective integrated vector control programs that go beyond synthetic insecticides are urgently needed.

Gastrointestinal Degradation of Fumonisin B1 by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine

PubMed Central

Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

2016-01-01

The mycotoxin fumonisin B1 (FB1) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB1 to its less toxic metabolite hydrolyzed FB1 (HFB1) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB1 was completely degraded to HFB1 in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB1+FB2; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB1 in excreta, whereas HFB1 concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB1+FB2 for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB1 and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB1 in the digestive tract of turkeys and pigs. PMID:27007395

Gastrointestinal Degradation of Fumonisin B₁ by Carboxylesterase FumD Prevents Fumonisin Induced Alteration of Sphingolipid Metabolism in Turkey and Swine.

PubMed

Masching, Sabine; Naehrer, Karin; Schwartz-Zimmermann, Heidi-Elisabeth; Sărăndan, Mihai; Schaumberger, Simone; Dohnal, Ilse; Nagl, Veronika; Schatzmayr, Dian

2016-03-21

The mycotoxin fumonisin B₁ (FB₁) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB₁ to its less toxic metabolite hydrolyzed FB₁ (HFB₁) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB₁ was completely degraded to HFB₁ in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB₁+FB₂; FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB₁ in excreta, whereas HFB₁ concentrations were significantly increased. Compared to the FB group (0.24 ± 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 ± 0.02), thus resembling values of the CON group (0.16 ± 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB₁+FB₂ for 42 days caused significantly elevated serum Sa/So ratios (0.39 ± 0.15) compared to the CON group (0.14 ± 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB₁ and unaffected Sa/So ratios (0.16 ± 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB₁ in the digestive tract of turkeys and pigs.

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the human intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which ...

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus.

PubMed

Gómez-Muñoz, María Teresa; Fernández-Barredo, Salceda; Martínez-Díaz, Rafael Alberto; Pérez-Gracia, María Teresa; Ponce-Gordo, Francisco

2012-01-01

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.

Protein-Linked Glycan Degradation in Infants Fed Human Milk

PubMed Central

Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

2014-01-01

Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

Doxycycline protects human intestinal cells from hypoxia/reoxygenation injury: Implications from an in-vitro hypoxia model.

PubMed

Hummitzsch, Lars; Zitta, Karina; Berndt, Rouven; Kott, Matthias; Schildhauer, Christin; Parczany, Kerstin; Steinfath, Markus; Albrecht, Martin

2017-04-15

Intestinal ischemia/reperfusion (I/R) injury is a grave clinical emergency and associated with high morbidity and mortality rates. Based on the complex underlying mechanisms, a multimodal pharmacological approach seems necessary to prevent intestinal I/R injury. The antibiotic drug doxycycline, which exhibits a wide range of pleiotropic therapeutic properties, might be a promising candidate for also reducing I/R injury in the intestine. To investigate possible protective effects of doxycycline on intestinal I/R injury, human intestinal CaCo-2 cells were exposed to doxycycline at clinically relevant concentrations. In order to mimic I/R injury, CaCo-2 were thereafter subjected to hypoxia/reoxygenation by using our recently described two-enzyme in-vitro hypoxia model. Investigations of cell morphology, cell damage, apoptosis and hydrogen peroxide formation were performed 24h after the hypoxic insult. Hypoxia/reoxygenation injury resulted in morphological signs of cell damage, elevated LDH concentrations in the respective culture media (P<0.001) and increased protein expression of proapoptotic caspase-3 (P<0.05) in the intestinal cultures. These events were associated with increased levels hydrogen peroxide (P<0.001). Preincubation of CaCo-2 cells with different concentrations of doxycycline (5µM, 10µM, 50µM) reduced the hypoxia induced signs of cell damage and LDH release (P<0.001 for all concentrations). The reduction of cellular damage was associated with a reduced expression of caspase-3 (5µM, P<0.01; 10µM, P<0.01; 50µM, P<0.05), while hydrogen peroxide levels remained unchanged. In summary, doxycycline protects human intestinal cells from hypoxia/reoxygenation injury in-vitro. Further animal and clinical studies are required to prove the protective potential of doxycycline on intestinal I/R injury under in-vivo conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanical Intestinal Obstruction in a Porcine Model: Effects of Intra-Abdominal Hypertension. A Preliminary Study

PubMed Central

Sánchez-Margallo, F. M.; Latorre, R.; López-Albors, O.; Wise, R.; Malbrain, M. L. N. G.; Castellanos, G.

2016-01-01

Introduction Mechanical intestinal obstruction is a disorder associated with intra-abdominal hypertension and abdominal compartment syndrome. As the large intestine intraluminal and intra-abdominal pressures are increased, so the patient’s risk for intestinal ischaemia. Previous studies have focused on hypoperfusion and bacterial translocation without considering the concomitant effect of intra-abdominal hypertension. The objective of this study was to design and evaluate a mechanical intestinal obstruction model in pigs similar to the human pathophysiology. Materials and Methods Fifteen pigs were divided into three groups: a control group (n = 5) and two groups of 5 pigs with intra-abdominal hypertension induced by mechanical intestinal obstruction. The intra-abdominal pressures of 20 mmHg were maintained for 2 and 5 hours respectively. Hemodynamic, respiratory and gastric intramucosal pH values, as well as blood tests were recorded every 30 min. Results Significant differences between the control and mechanical intestinal obstruction groups were noted. The mean arterial pressure, cardiac index, dynamic pulmonary compliance and abdominal perfusion pressure decreased. The systemic vascular resistance index, central venous pressure, pulse pressure variation, airway resistance and lactate increased within 2 hours from starting intra-abdominal hypertension (p<0.05). In addition, we observed increased values for the peak and plateau airway pressures, and low values of gastric intramucosal pH in the mechanical intestinal obstruction groups that were significant after 3 hours. Conclusion The mechanical intestinal obstruction model appears to adequately simulate the pathophysiology of intestinal obstruction that occurs in humans. Monitoring abdominal perfusion pressure, dynamic pulmonary compliance, gastric intramucosal pH and lactate values may provide insight in predicting the effects on endorgan function in patients with mechanical intestinal obstruction. PMID:26849559

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers.

PubMed

Takaishi, Naoki; Yoshida, Kazutaka; Satsu, Hideo; Shimizu, Makoto

2007-06-27

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.

Dogs and intestinal parasites: a public health problem.

PubMed Central

Seah, S. K.; Hucal, G.; Law, C.

1975-01-01

The stools of 239 stray dogs were examined for intestinal parasites. Of the helminths found, Toxocara canis (43.5%), tapeworms (25.5%), Ascaris species (21.3%) and hookworms (12.5%) were the commonest. Of the protozoans found, Isospora species and Entamoeba coli were the most prevalent. An unusual feature of the present study was the finding of Ascaris species. The importance of the high prevalence of intestinal parasites in dogs, the close contact of humans with dogs' excreta and the possible role of this environmental pollution in the spread of human disease are discussed. PMID:1125888

Intestinal Microbiota in Healthy Adults: Temporal Analysis Reveals Individual and Common Core and Relation to Intestinal Symptoms

PubMed Central

Nikkilä, Janne; Immonen, Outi; Kekkonen, Riina; Lahti, Leo; Palva, Airi; de Vos, Willem M.

2011-01-01

Background While our knowledge of the intestinal microbiota during disease is accumulating, basic information of the microbiota in healthy subjects is still scarce. The aim of this study was to characterize the intestinal microbiota of healthy adults and specifically address its temporal stability, core microbiota and relation with intestinal symptoms. We carried out a longitudinal study by following a set of 15 healthy Finnish subjects for seven weeks and regularly assessed their intestinal bacteria and archaea with the Human Intestinal Tract (HIT)Chip, a phylogenetic microarray, in conjunction with qPCR analyses. The health perception and occurrence of intestinal symptoms was recorded by questionnaire at each sampling point. Principal Findings A high overall temporal stability of the microbiota was observed. Five subjects showed transient microbiota destabilization, which correlated not only with the intake of antibiotics but also with overseas travelling and temporary illness, expanding the hitherto known factors affecting the intestinal microbiota. We identified significant correlations between the microbiota and common intestinal symptoms, including abdominal pain and bloating. The most striking finding was the inverse correlation between Bifidobacteria and abdominal pain: subjects who experienced pain had over five-fold less Bifidobacteria compared to those without pain. Finally, a novel computational approach was used to define the common core microbiota, highlighting the role of the analysis depth in finding the phylogenetic core and estimating its size. The in-depth analysis suggested that we share a substantial number of our intestinal phylotypes but as they represent highly variable proportions of the total community, many of them often remain undetected. Conclusions/Significance A global and high-resolution microbiota analysis was carried out to determine the temporal stability, the associations with intestinal symptoms, and the individual and common core microbiota in healthy adults. The findings provide new approaches to define intestinal health and to further characterize the microbial communities inhabiting the human gut. PMID:21829582

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

PubMed

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

PubMed Central

Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S. R. Murthy; Prentki, Marc

2016-01-01

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. PMID:26423520

Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).

PubMed

Hammoudi, Abeer; Song, Fei; Reed, Karen R; Jenkins, Rosalind E; Meniel, Valerie S; Watson, Alastair J M; Pritchard, D Mark; Clarke, Alan R; Jenkins, John R

2013-10-25

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death worldwide. Accurate non-invasive screening for CRC would greatly enhance a population's health. Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas, leading to Wnt signalling pathway activation. Acute conditional transgenic deletion of Apc in murine intestinal epithelium (AhCre(+)Apc(fl)(/)(fl)) causes phenotypic changes similar to those found during colorectal tumourigenesis. This study comprised a proteomic analysis of murine small intestinal epithelial cells following acute Apc deletion to identify proteins that show altered expression during human colorectal carcinogenesis, thus identifying proteins that may prove clinically useful as blood/serum biomarkers of colorectal neoplasia. Eighty-one proteins showed significantly increased expression following iTRAQ analysis, and validation of nine of these by Ingenuity Pathaway Analysis showed they could be detected in blood or serum. Expression was assessed in AhCre(+)Apc(fl)(/)(fl) small intestinal epithelium by immunohistochemistry, western blot and quantitative real-time PCR; increased nucelolin concentrations were also detected in the serum of AhCre(+)Apc(fl)(/)(fl) and Apc(Min)(/)(+) mice by ELISA. Six proteins; heat shock 60kDa protein 1, Nucleolin, Prohibitin, Cytokeratin 18, Ribosomal protein L6 and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5,were selected for further investigation. Increased expression of 4 of these was confirmed in human CRC by qPCR. In conclusion, several novel candidate biomarkers have been identified from analysis of transgenic mice in which the Apc gene was deleted in the intestinal epithelium that also showed increased expression in human CRC. Some of these warrant further investigation as potential serum-based biomarkers of human CRC. Copyright © 2013 Elsevier Inc. All rights reserved.

Trefoil factor 3 isolated from human breast milk downregulates cytokines (IL8 and IL6) and promotes human beta defensin (hBD2 and hBD4) expression in intestinal epithelial cells HT-29

PubMed Central

Barrera, Girolamo Jose; Sanchez, Gabriela; Gonzalez, Jose Emanuele

2012-01-01

Trefoil factors (TFF) are secretory products of mucin producing cells. They play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF1), spasmolytic peptide (TFF2), and the intestinal trefoil factor (TFF3). They have an important and necessary role in epithelial restitution within the gastrointestinal tract. Significant amounts of TFF are present in human milk. This study aimed to determine a possible correlation between TFF3 isolated from human breast milk and levels of cytokines (IL8 and IL6) and defensins (hBD2 and hBD4) in intestinal epithelial cells HT-29 treated with trefoil. Samples of human milk were collected within 2-4 weeks postpartum from healthy human mothers (18-30-years-old) by manual breast massage, and TFF3 was purified by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel filtration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA) and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the first evidence of human milk TFF3 purification. Here we show that the presence of TFF3 isolated from milk strongly correlates with downregulation of IL8 and IL6 in human intestinal epithelial cells. On the other hand, TFF3 activated the epithelial cells in culture to produce beta defensins 2 (hBD2) and beta defensins 4 (hBD4). These findings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins. PMID:23198942

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

PubMed

Akazawa, Takanori; Uchida, Yasuo; Miyauchi, Eisuke; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

2018-01-02

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Comparative Metagenomics Revealed Commonly Enriched Gene Sets in Human Gut Microbiomes

PubMed Central

Kurokawa, Ken; Itoh, Takehiko; Kuwahara, Tomomi; Oshima, Kenshiro; Toh, Hidehiro; Toyoda, Atsushi; Takami, Hideto; Morita, Hidetoshi; Sharma, Vineet K.; Srivastava, Tulika P.; Taylor, Todd D.; Noguchi, Hideki; Mori, Hiroshi; Ogura, Yoshitoshi; Ehrlich, Dusko S.; Itoh, Kikuji; Takagi, Toshihisa; Sakaki, Yoshiyuki; Hayashi, Tetsuya; Hattori, Masahira

2007-01-01

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a ‘hot spot’ for horizontal gene transfer between microbes. PMID:17916580

Inhibition of Human and Rat Sucrase and Maltase Activities To Assess Antiglycemic Potential: Optimization of the Assay Using Acarbose and Polyphenols.

PubMed

Pyner, Alison; Nyambe-Silavwe, Hilda; Williamson, Gary

2017-10-04

We optimized the assays used to measure inhibition of rat and human α-glucosidases (sucrase and maltase activities), intestinal enzymes which catalyze the final steps of carbohydrate digestion. Cell-free extracts from fully differentiated intestinal Caco-2/TC7 monolayers were shown to be a suitable source of sucrase-isomaltase, with the same sequence as human small intestine, and were compared to a rat intestinal extract. The kinetic conditions of the assay were optimized, including comparison of enzymatic and chromatographic methods to detect the monosaccharide products. Human sucrase activity was more susceptible than the rat enzyme to inhibition by acarbose (IC 50 (concentration required for 50% inhibition) = 2.5 ± 0.5 and 12.3 ± 0.6 μM, respectively), by a polyphenol-rich green tea extract, and by pure (-)-epigallocatechin gallate (EGCG) (IC 50 = 657 ± 150 and 950 ± 86 μM respectively). In contrast, the reverse was observed when assessing maltase activity (e.g. IC 50 = 677 ± 241 and 14.0 ± 2.0 μM for human and rat maltase, respectively). 5-Caffeoylquinic acid did not significantly inhibit maltase and was only a very weak inhibitor of sucrase. The data show that for sucrase and maltase activities, inhibition patterns of rat and human enzymes are generally qualitatively similar but can be quantitatively different.

Nitroreduction and formation of hemoglobin adducts in rats with a human intestinal microflora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheepers, P.T.J.; Straetemans, M.M.E.; Koopman, J.P.

1994-10-01

In the covalent binding of nitroarenes to macromolecules, nitroreduction is an important step. The intestinal microflora represents an enormous potential of bacterial nitroreductase activity. As a consequence, the in vivo nitroreduction of orally administerednitroarenes is primarily located in the intestine. In this study, we have investigated the nitroreduction of 2-nitrofluorene (2-NF) by a human microflora in female Wistar rats. Germ-free (FG) rats were equipped with a bacterial flora derived from human feces. Nontreated GF rats and GF animals equipped with a conventional rat flora were used as controls. The composition of the human and the conventional microflora isolated from themore » rats were consistent with the microflora of the administered feces. In the rats receiving only sunflower seed oil, no adducts were detected. The animals equipped with a human or rat microflora that received 2-aminofluorene (2-AF) formed 2-AF hemoglobin (Hb)-adducts at average levels mean {+-} 0.003 and 0.043 {+-} 0.010 {mu}mole/g Hb, respectively. In the FG rats, an adduct level of 0.57 {+-} 0.09 was determined after 2-AF administration and non adducts were detected after 2-NF administration. The results show that nitroreduction by an acquired human intestinal microflora and subsequent adduct formation can be studied in the rate in vivo. 21 refs., 3 tabs.« less

Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.

PubMed

Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi; Zachos, Nicholas C; Kovbasnjuk, Olga; Estes, Mary K; de Jonge, Hugo; Donowitz, Mark

2016-03-01

Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na(+) absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to show their functional relevance to ion transport physiology and pathophysiology. Human intestinal tissue specimens were obtained from an endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using quantitative reverse-transcription polymerase chain reaction, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions. Enteroids differentiated upon withdrawal of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. Na(+)/H(+) exchanger 3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling was completely dependent on cystic fibrosis transmembrane conductance regulator and partially dependent on Na(+)/H(+) exchanger 3 and Na(+)/K(+)/2Cl(-) cotransporter 1 inhibition in undifferentiated and differentiated enteroids. Increases in cyclic adenosine monophosphate with forskolin caused enteroid intracellular acidification in HCO3(-)-free buffer. Cyclic adenosine monophosphate-induced enteroid intracellular pH acidification as part of duodenal HCO3(-) secretion appears to require cystic fibrosis transmembrane conductance regulator and electrogenic Na(+)/HCO3(-) cotransporter 1. Undifferentiated or crypt-like, and differentiated or villus-like, human enteroids represent distinct points along the crypt-villus axis; they can be used to characterize electrolyte transport processes along the vertical axis of the small intestine. The duodenal enteroid model showed that electrogenic Na(+)/HCO3(-) cotransporter 1 might be a target in the intestinal mucosa for treatment of secretory diarrheas. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

PubMed Central

Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

2014-01-01

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

Butyrate modulating effects on pro-inflammatory pathways in human intestinal epithelial cells.

PubMed

Elce, A; Amato, F; Zarrilli, F; Calignano, A; Troncone, R; Castaldo, G; Canani, R B

2017-10-13

Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.

PubMed

He, Xi C; Zhang, Jiwang; Tong, Wei-Gang; Tawfik, Ossama; Ross, Jason; Scoville, David H; Tian, Qiang; Zeng, Xin; He, Xi; Wiedemann, Leanne M; Mishina, Yuji; Li, Linheng

2004-10-01

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.

Human intervention study to investigate the intestinal accessibility and bioavailability of anthocyanins from bilberries.

PubMed

Mueller, Dolores; Jung, Kathrin; Winter, Manuel; Rogoll, Dorothee; Melcher, Ralph; Richling, Elke

2017-09-15

We investigated the importance of the large intestine on the bioavailability of anthocyanins from bilberries in humans with/without a colon. Low bioavailability of anthocyanins in plasma and urine was observed in the frame of this study. Anthocyanins reached the circulation mainly as glucuronides. Analysis of ileal effluents (at end of small intestine) demonstrated that 30% of ingested anthocyanins were stable during 8h passage through the upper intestine. Only 20% degradants were formed and mostly intact anthocyanins were absorbed from the small intestine. Higher amounts of degradants than anthocyanins reached the circulation after bilberry extract consumption in both groups of subjects. Comparison of the bioavailability of anthocyanins in healthy subjects versus ileostomists revealed substantially higher amounts of anthocyanins and degradants in the plasma/urine of subjects with an intact gut. The results suggested that the colon is a significant site for absorption of bioactive components such as anthocyanins and their degradation products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Induction of intestinalization in human esophageal keratinocytes is a multistep process.

PubMed

Kong, Jianping; Nakagawa, Hiroshi; Isariyawongse, Brandon K; Funakoshi, Shinsuke; Silberg, Debra G; Rustgi, Anil K; Lynch, John P

2009-01-01

Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.

Induction of intestinalization in human esophageal keratinocytes is a multistep process

PubMed Central

Kong, Jianping; Nakagawa, Hiroshi; Isariyawongse, Brandon K.; Funakoshi, Shinsuke; Silberg, Debra G.; Rustgi, Anil K.; Lynch, John P.

2009-01-01

Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE. PMID:18845559

Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation.

PubMed

Hegazy, Ahmed N; West, Nathaniel R; Stubbington, Michael J T; Wendt, Emily; Suijker, Kim I M; Datsi, Angeliki; This, Sebastien; Danne, Camille; Campion, Suzanne; Duncan, Sylvia H; Owens, Benjamin M J; Uhlig, Holm H; McMichael, Andrew; Bergthaler, Andreas; Teichmann, Sarah A; Keshav, Satish; Powrie, Fiona

2017-11-01

Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4 + T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4 + T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4 + T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction. Circulating and gut-resident CD4 + T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4 + T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4 + T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls. In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4 + T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

PubMed

Alfen, Johanna Sophie; Larghi, Paola; Facciotti, Federica; Gagliani, Nicola; Bosotti, Roberto; Paroni, Moira; Maglie, Stefano; Gruarin, Paola; Vasco, Chiara Maria; Ranzani, Valeria; Frusteri, Cristina; Iseppon, Andrea; Moro, Monica; Crosti, Maria Cristina; Gatti, Stefano; Pagani, Massimiliano; Caprioli, Flavio; Abrignani, Sergio; Flavell, Richard A; Geginat, Jens

2018-01-31

IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells. We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed. Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Modulation of gut perception in humans by spatial summation phenomena

PubMed Central

Serra, Jordi; Azpiroz, Fernando; Malagelada, Juan-R

1998-01-01

We have recently shown that perception of intestinal stimuli increases by spatial summation phenomena. Our aim was to determine in humans whether intestinal perception depends on (a) the length of gut stimulated, and (b) the distance between stimuli. In a first series of studies, we compared perception of isobaric intestinal distensions applied over a 3 cm segment and a 36 cm segment by means of two separate barostats (n = 8). In a second series of studies we compared perception of intestinal distensions applied simultaneously by two balloons sited 3, 12 or 48 cm apart (n = 6). Distension of the 36 cm segment induced significantly greater perception than distension of the 3 cm intestinal segment (discomfort perceived at 20 ± 2 mmHg and 31 ± 2 mmHg, respectively; P < 0.05). Perception of intestinal balloon distension increased when a second stimulus was simultaneously applied, independently of the distance between the two balloons (the discomfort thresholds were 30 ± 11, 20 ± 6 and 28 ± 7% lower with simultaneous distensions 3, 12 and 48 cm apart, respectively). We conclude that perception of intestinal distension is determined by the extension of the field of stimulation, and the summation effect is similar whether adjacent or distant fields are stimulated. PMID:9490880

Transporters for the Intestinal Absorption of Cholesterol, Vitamin E, and Vitamin K

PubMed Central

Yamanashi, Yoshihide; Kurauchi, Ryoya; Tanaka, Yusuke; Komine, Toko; Suzuki, Hiroshi

2017-01-01

Humans cannot synthesize fat-soluble vitamins such as vitamin E and vitamin K. For this reason, they must be obtained from the diet via intestinal absorption. As the deficiency or excess of these vitamins has been reported to cause several types of diseases and disorders in humans, the intestinal absorption of these nutrients must be properly regulated to ensure good health. However, the mechanism of their intestinal absorption remains poorly understood. Recent studies on cholesterol using genome-edited mice, genome-wide association approaches, gene mutation analyses, and the development of cholesterol absorption inhibitors have revealed that several membrane proteins play crucial roles in the intestinal absorption of cholesterol. Surprisingly, detailed analyses of these cholesterol transporters have revealed that they can also transport vitamin E and vitamin K, providing clues to uncover the molecular mechanisms underlying the intestinal absorption of these fat-soluble vitamins. In this review, we focus on the membrane proteins (Niemann-Pick C1 like 1, scavenger receptor class B type I, cluster of differentiation 36, and ATP-binding cassette transporter A1) that are (potentially) involved in the intestinal absorption of cholesterol, vitamin E, and vitamin K and discuss their physiological and pharmacological importance. We also discuss the related uncertainties that need to be explored in future studies. PMID:28100881

Isolation of Intestinal Parasites of Public Health Importance from Cockroaches (Blattella germanica) in Jimma Town, Southwestern Ethiopia.

PubMed

Hamu, Haji; Debalke, Serkadis; Zemene, Endalew; Birlie, Belay; Mekonnen, Zeleke; Yewhalaw, Delenasaw

2014-01-01

Cockroaches are claimed to be mechanical transmitters of disease causing microorganisms such as intestinal parasites, bacteria, fungi, and viruses. This study assessed the potential of the German cockroach Blattella germanica in the mechanical transmission of intestinal parasites of public health importance. A total of 2010 cockroaches were collected from 404 households in Jimma Town, southwestern Ethiopia. All the collected cockroaches were identified to species as B. germanica. The contents of their gut and external body parts were examined for the presence of intestinal parasites. Overall, 152 (75.6%) of the 210 batches were found to harbor at least one species of human intestinal parasite. Ascaris lumbricoides, Trichuris trichiura, Taenia spp, Strongyloides-like parasite, Entamoeba histolytica/dispar/moshkovski, Giardia duodenalis and Balantidium coli were detected from gut contents. Moreover, parasites were also isolated from the external surface in 22 (10.95%) of the batches. There was significant difference in parasite carriage rate of the cockroaches among the study sites (P = 0.013). In conclusion, B. germanica was found to harbor intestinal parasites of public health importance. Hence, awareness on the potential role of cockroaches in the mechanical transmission of human intestinal parasites needs to be created. Moreover, further identification of the Strongyloides-like worm is required using molecular diagnostics.

Transporters for the Intestinal Absorption of Cholesterol, Vitamin E, and Vitamin K.

PubMed

Yamanashi, Yoshihide; Takada, Tappei; Kurauchi, Ryoya; Tanaka, Yusuke; Komine, Toko; Suzuki, Hiroshi

2017-04-03

Humans cannot synthesize fat-soluble vitamins such as vitamin E and vitamin K. For this reason, they must be obtained from the diet via intestinal absorption. As the deficiency or excess of these vitamins has been reported to cause several types of diseases and disorders in humans, the intestinal absorption of these nutrients must be properly regulated to ensure good health. However, the mechanism of their intestinal absorption remains poorly understood. Recent studies on cholesterol using genome-edited mice, genome-wide association approaches, gene mutation analyses, and the development of cholesterol absorption inhibitors have revealed that several membrane proteins play crucial roles in the intestinal absorption of cholesterol. Surprisingly, detailed analyses of these cholesterol transporters have revealed that they can also transport vitamin E and vitamin K, providing clues to uncover the molecular mechanisms underlying the intestinal absorption of these fat-soluble vitamins. In this review, we focus on the membrane proteins (Niemann-Pick C1 like 1, scavenger receptor class B type I, cluster of differentiation 36, and ATP-binding cassette transporter A1) that are (potentially) involved in the intestinal absorption of cholesterol, vitamin E, and vitamin K and discuss their physiological and pharmacological importance. We also discuss the related uncertainties that need to be explored in future studies.

"Omic" investigations of protozoa and worms for a deeper understanding of the human gut "parasitome".

PubMed

Marzano, Valeria; Mancinelli, Livia; Bracaglia, Giorgia; Del Chierico, Federica; Vernocchi, Pamela; Di Girolamo, Francesco; Garrone, Stefano; Tchidjou Kuekou, Hyppolite; D'Argenio, Patrizia; Dallapiccola, Bruno; Urbani, Andrea; Putignani, Lorenza

2017-11-01

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic "citizens." In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut "parasitome" through "omic" technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology-based profiles of the gut "parasitome" under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine.

“Omic” investigations of protozoa and worms for a deeper understanding of the human gut “parasitome”

PubMed Central

Marzano, Valeria; Mancinelli, Livia; Bracaglia, Giorgia; Del Chierico, Federica; Vernocchi, Pamela; Di Girolamo, Francesco; Garrone, Stefano; Tchidjou Kuekou, Hyppolite; D’Argenio, Patrizia; Dallapiccola, Bruno; Urbani, Andrea

2017-01-01

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic “citizens.” In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut “parasitome” through “omic” technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology–based profiles of the gut “parasitome” under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine. PMID:29095820

Short- and long-term effects of oral vancomycin on the human intestinal microbiota.

PubMed

Isaac, Sandrine; Scher, Jose U; Djukovic, Ana; Jiménez, Nuria; Littman, Dan R; Abramson, Steven B; Pamer, Eric G; Ubeda, Carles

2017-01-01

Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhoea in developed countries. However, its short- and long-term effects on the human intestinal microbiota remain largely unknown. We utilized high-throughput sequencing to analyse the effects of vancomycin on the faecal human microbiota up to 22 weeks post-antibiotic cessation. The clinical relevance of the observed microbiota perturbations was studied in mice. During vancomycin therapy, most intestinal microbiota genera and operational taxonomic units (OTUs) were depleted in all analysed subjects, including all baseline OTUs from the phylum Bacteroidetes. This was accompanied by a vast expansion of genera associated with infections, including Klebsiella and Escherichia/Shigella. Following antibiotic cessation, marked differences in microbiota resilience were observed among subjects. While some individuals recovered a microbiota close to baseline composition, in others, up to 89% of abundant OTUs could no longer be detected. The clinical relevance of the observed microbiota changes was further demonstrated in mice, which developed analogous microbiota alterations. During vancomycin treatment, mice were highly susceptible to intestinal colonization by an antibiotic-resistant pathogen and, upon antibiotic cessation, a less-resilient microbiota allowed higher levels of pathogen colonization. Oral vancomycin induces drastic and consistent changes in the human intestinal microbiota. Upon vancomycin cessation, the microbiota recovery rate varied considerably among subjects, which could influence, as validated in mice, the level of susceptibility to pathogen intestinal colonization. Our results demonstrate the negative long-term effects of vancomycin, which should be considered as a fundamental aspect of the cost-benefit equation for antibiotic prescription. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Short- and long-term effects of oral vancomycin on the human intestinal microbiota

PubMed Central

Isaac, Sandrine; Scher, Jose U.; Djukovic, Ana; Jiménez, Nuria; Littman, Dan R.; Abramson, Steven B.; Pamer, Eric G.; Ubeda, Carles

2017-01-01

Background Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhoea in developed countries. However, its short- and long-term effects on the human intestinal microbiota remain largely unknown. Methods We utilized high-throughput sequencing to analyse the effects of vancomycin on the faecal human microbiota up to 22 weeks post-antibiotic cessation. The clinical relevance of the observed microbiota perturbations was studied in mice. Results During vancomycin therapy, most intestinal microbiota genera and operational taxonomic units (OTUs) were depleted in all analysed subjects, including all baseline OTUs from the phylum Bacteroidetes. This was accompanied by a vast expansion of genera associated with infections, including Klebsiella and Escherichia/Shigella. Following antibiotic cessation, marked differences in microbiota resilience were observed among subjects. While some individuals recovered a microbiota close to baseline composition, in others, up to 89% of abundant OTUs could no longer be detected. The clinical relevance of the observed microbiota changes was further demonstrated in mice, which developed analogous microbiota alterations. During vancomycin treatment, mice were highly susceptible to intestinal colonization by an antibiotic-resistant pathogen and, upon antibiotic cessation, a less-resilient microbiota allowed higher levels of pathogen colonization. Conclusions Oral vancomycin induces drastic and consistent changes in the human intestinal microbiota. Upon vancomycin cessation, the microbiota recovery rate varied considerably among subjects, which could influence, as validated in mice, the level of susceptibility to pathogen intestinal colonization. Our results demonstrate the negative long-term effects of vancomycin, which should be considered as a fundamental aspect of the cost–benefit equation for antibiotic prescription. PMID:27707993

The Roles of Glutamine in the Intestine and Its Implication in Intestinal Diseases

PubMed Central

Kim, Min-Hyun; Kim, Hyeyoung

2017-01-01

Glutamine, the most abundant free amino acid in the human body, is a major substrate utilized by intestinal cells. The roles of glutamine in intestinal physiology and management of multiple intestinal diseases have been reported. In gut physiology, glutamine promotes enterocyte proliferation, regulates tight junction proteins, suppresses pro-inflammatory signaling pathways, and protects cells against apoptosis and cellular stresses during normal and pathologic conditions. As glutamine stores are depleted during severe metabolic stress including trauma, sepsis, and inflammatory bowel diseases, glutamine supplementation has been examined in patients to improve their clinical outcomes. In this review, we discuss the physiological roles of glutamine for intestinal health and its underlying mechanisms. In addition, we discuss the current evidence for the efficacy of glutamine supplementation in intestinal diseases. PMID:28498331

Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

PubMed Central

Baker, Ann-Marie; Cereser, Biancastella; Melton, Samuel; Fletcher, Alexander G.; Rodriguez-Justo, Manuel; Tadrous, Paul J.; Humphries, Adam; Elia, George; McDonald, Stuart A.C.; Wright, Nicholas A.; Simons, Benjamin D.; Jansen, Marnix; Graham, Trevor A.

2014-01-01

Summary Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+). Furthermore, we show that, in adenomatous crypts (APC−/−), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics. PMID:25127143

Quantification of folic acid in human feces after administration of Bifidobacterium probiotic strains.

PubMed

Strozzi, G Paolo; Mogna, Luca

2008-09-01

Folic acid, or vitamin B9, is involved in appropriate regulation of DNA replication, synthesis of purines and deoxythymidine (dTMP), conversion of homocysteine to methionine, histidine catabolism, and correct differentiation of the neural tube during fetal organogenesis. Folic acid from food sources is almost completely absorbed in the small intestine, mostly in the jejunum, and does not reach the large intestine. The administration of probiotic strains able to synthesize folates de novo and release them in the extracellular space may provide an additional, constant endogenous source of this important vitamin in the intestinal lumen of humans. A pilot study involving 23 healthy volunteers was conducted to evaluate the ability of 3 probiotic strains, Bifidobacterium adolescentis DSM 18350, B. adolescentis DSM 18352, and Bifidobacterium pseudocatenulatum DSM 18353, to produce folates in the human intestine. Volunteers were randomly assigned to 1 of 3 groups for treatment with a specific probiotic strain (5 x 10(9) colony forming units/d). Strain effectiveness was evaluated by determination of the folate concentration in feces evacuated within 48 hours before and after administration of the probiotics. Quantification of microorganisms belonging to the genus Bifidobacterium was performed in parallel to folate analysis. Ingestion of these probiotic strains resulted in a significant increase of folic acid concentration in human feces in all treated groups. Analysis of the fecal Bifidobacteria confirmed the potential of all strains, especially B. adolescentis DSM 18352, to colonize the intestinal environment. The demonstrated ability of the probiotic microorganisms B. adolescentis DSM 18350, B. adolescentis DSM 18352, and B. pseudocatenulatum DSM 18353 to synthesize and secrete folates in the human intestinal environment may provide a complementary endogenous source of such molecules, which is especially useful for the homeostasis of mucosal enterocytes of the colon and, unlike oral administration of the vitamin, ensures its constant bioavailability.

Metabolomics Analysis of Cistus monspeliensis Leaf Extract on Energy Metabolism Activation in Human Intestinal Cells

PubMed Central

Shimoda, Yoichi; Han, Junkyu; Kawada, Kiyokazu; Smaoui, Abderrazak; Isoda, Hiroko

2012-01-01

Energy metabolism is a very important process to improve and maintain health from the point of view of physiology. It is well known that the intracellular ATP production is contributed to energy metabolism in cells. Cistus monspeliensis is widely used as tea, spices, and medical herb; however, it has not been focusing on the activation of energy metabolism. In this study, C. monspeliensis was investigated as the food resources by activation of energy metabolism in human intestinal epithelial cells. C. monspeliensis extract showed high antioxidant ability. In addition, the promotion of metabolites of glycolysis and TCA cycle was induced by C. monspeliensis treatment. These results suggest that C. monspeliensis extract has an ability to enhance the energy metabolism in human intestinal cells. PMID:22523469

Ontogeny of a novel pituitary protein (7B2) in the human fetal intestine.

PubMed

Suzuki, H; Christofides, N D; Adrian, T E; Chretien, M; Seidah, N G; Polak, J M; Bloom, S R

1985-11-28

The developmental profile of the concentration of a novel pituitary protein (7B2) was studied immunochemically in the human gastrointestinal tract from 12 weeks of gestation to 4 months after birth and was compared to the distribution in the adult. 7B2-like immunoreactivity (IR-7B2) was detected in all segments studied, but no gross changes were seen through fetal life. At term higher concentrations of IR-7B2 were found in the duodenum and the antrum, which is similar to the distribution of adult man. Gel permeation chromatography revealed that the main peak of 7B2 immunoreactivity in the fetal intestinal extract eluted with a Kav of 0.3. Similar elution profiles were also observed in extracts of human adult intestine.

Intestinal parasites in First World War German soldiers from "Kilianstollen", Carspach, France.

PubMed

Le Bailly, Matthieu; Landolt, Michaël; Mauchamp, Leslie; Dufour, Benjamin

2014-01-01

Paleoparasitological investigations revealed the presence of intestinal helminths in samples taken from the abdominal cavities of two German soldiers, recovered in the First World War site named "Kilianstollen" in Carspach, France. Eggs from roundworm, whipworm, tapeworm and capillariids were identified. The morphological and morphometrical comparison, followed by statistical analyses, showed that the Carspach capillariid eggs are similar to rodent parasites. Poor sanitary conditions in the trenches, the lack of knowledge of parasites, and the widespread presence of commensal animals, can explain the occurrence of such parasites in human intestines. This study is the second dealing with 20th century human samples. It confirms the presence of intestinal worms in First World War German soldiers. In this case study, the application of statistics to precise measurements facilitated the diagnosis of ancient helminth eggs and completed the microscopic approach.

Morphological, kinetic, membrane biochemical and genetic aspects of intestinal enteroplasticity

PubMed Central

Drozdowski, Laurie A; Clandinin, M Tom; Thomson, Alan BR

2009-01-01

The process of intestinal adaptation (“enteroplasticity”) is complex and multifaceted. Although a number of trophic nutrients and non-nutritive factors have been identified in animal studies, successful, reproducible clinical trials in humans are awaited. Understanding mechanisms underlying this adaptive process may direct research toward strategies that maximize intestinal function and impart a true clinical benefit to patients with short bowel syndrome, or to persons in whom nutrient absorption needs to be maximized. In this review, we consider the morphological, kinetic and membrane biochemical aspects of enteroplasticity, focus on the importance of nutritional factors, provide an overview of the many hormones that may alter the adaptive process, and consider some of the possible molecular profiles. While most of the data is derived from rodent studies, wherever possible, the results of human studies of intestinal enteroplasticity are provided. PMID:19230039

Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine

PubMed Central

Hoekstra, Nadia; Collins, Danielle; Collaco, Anne; Baird, Alan W.; Winter, Desmond C.; Ameen, Nadia; Geibel, John P.; Kopic, Sascha

2013-01-01

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness. PMID:23935921

Movers and shakers

PubMed Central

Mills, Susan; Shanahan, Fergus; Stanton, Catherine; Hill, Colin; Coffey, Aidan; Ross, R. Paul

2013-01-01

The human intestinal microbiota is one of the most densely populated ecosystems on Earth, containing up to 1013 bacteria/g and in some respects can be considered an organ itself given its role in human health. Bacteriophages (phages) are the most abundant replicating entities on the planet and thrive wherever their bacterial hosts exist. They undoubtedly influence the dominant microbial populations in many ecosystems including the human intestine. Within this setting, lysogeny appears to be the preferred life cycle, presumably due to nutrient limitations and lack of suitable hosts protected in biofilms, hence the predator/prey dynamic observed in many ecosystems is absent. On the other hand, free virulent phages in the gut are more common among sufferers of intestinal diseases and have been shown to increase with antibiotic usage. Many of these phages evolve from prophages of intestinal bacteria and emerge under conditions where their bacterial hosts encounter stress suggesting that prophages can significantly alter the microbial community composition. Based on these observations, we propose the “community shuffling” model which hypothesizes that prophage induction contributes to intestinal dysbiosis by altering the ratio of symbionts to pathobionts, enabling pathobiont niche reoccupation. The consequences of the increased phage load on the mammalian immune system are also addressed. While this is an area of intestinal biology which has received little attention, this review assembles evidence from the literature which supports the role of phages as one of the biological drivers behind the composition of the gut microbiota. PMID:23022738

Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montoudis, Alain; Delvin, Edgard; Canadian Institute of Health Research, Group of the Functional Development and Physiopathology of the Digestive Tract, and Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Que., Canada J1H 5N4

2006-01-06

Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferationmore » and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.« less

A chronic oral reference dose for hexavalent chromium-induced intestinal cancer†

PubMed Central

Thompson, Chad M; Kirman, Christopher R; Proctor, Deborah M; Haws, Laurie C; Suh, Mina; Hays, Sean M; Hixon, J Gregory; Harris, Mark A

2014-01-01

High concentrations of hexavalent chromium [Cr(VI)] in drinking water induce villous cytotoxicity and compensatory crypt hyperplasia in the small intestines of mice (but not rats). Lifetime exposure to such cytotoxic concentrations increases intestinal neoplasms in mice, suggesting that the mode of action for Cr(VI)-induced intestinal tumors involves chronic wounding and compensatory cell proliferation of the intestine. Therefore, we developed a chronic oral reference dose (RfD) designed to be protective of intestinal damage and thus intestinal cancer. A physiologically based pharmacokinetic model for chromium in mice was used to estimate the amount of Cr(VI) entering each intestinal tissue section (duodenum, jejunum and ileum) from the lumen per day (normalized to intestinal tissue weight). These internal dose metrics, together with corresponding incidences for diffuse hyperplasia, were used to derive points of departure using benchmark dose modeling and constrained nonlinear regression. Both modeling techniques resulted in similar points of departure, which were subsequently converted to human equivalent doses using a human physiologically based pharmacokinetic model. Applying appropriate uncertainty factors, an RfD of 0.006 mg kg–1 day–1 was derived for diffuse hyperplasia—an effect that precedes tumor formation. This RfD is protective of both noncancer and cancer effects in the small intestine and corresponds to a safe drinking water equivalent level of 210 µg l–1. This concentration is higher than the current federal maximum contaminant level for total Cr (100 µg l–1) and well above levels of Cr(VI) in US drinking water supplies (typically ≤ 5 µg l–1). © 2013 The Authors. Journal of Applied Toxicology published by John Wiley & Sons, Ltd. PMID:23943231

The piglet as a model for studying dietary components in infant diets: effects of galacto-oligosaccharides on intestinal functions.

PubMed

Alizadeh, A; Akbari, P; Difilippo, E; Schols, H A; Ulfman, L H; Schoterman, M H C; Garssen, J; Fink-Gremmels, J; Braber, S

2016-02-28

Prebiotic oligosaccharides, including galacto-oligosaccharides (GOS), are used in infant formula to mimic human milk oligosaccharides, which are known to have an important role in the development of the intestinal microbiota and the immune system in neonates. The maturation of the intestines in piglets closely resembles that of human neonates and infants. Hence, a neonatal piglet model was used to study the multi-faceted effect of dietary GOS in early life. Naturally farrowed piglets were separated from the mother sow 24-48 h postpartum and received a milk replacer with or without the addition of GOS for 3 or 26 d, whereafter several indicators of intestinal colonisation and maturation were measured. Dietary GOS was readily fermented in the colon, leading to a decreased pH, an increase in butyric acid in caecum digesta and an increase in lactobacilli and bifidobacteria numbers at day 26. Histomorphological changes were observed in the intestines of piglets fed a GOS diet for 3 or 26 d. In turn, differences in the intestinal disaccharidase activity were observed between control and GOS-fed piglets. The mRNA expression of various tight junction proteins was up-regulated in the intestines of piglet fed a GOS diet and was not accompanied by an increase in protein expression. GOS also increased defensin porcine β-defensin-2 in the colon and secretory IgA levels in saliva. In conclusion, by applying a neonatal piglet model, it could be demonstrated that a GOS-supplemented milk replacer promotes the balance of the developing intestinal microbiota, improves the intestinal architecture and seems to stimulate the intestinal defence mechanism.

Gluten-degrading bacteria are present in the human small intestine of healthy volunteers and celiac patients.

PubMed

Herrán, Alexandra R; Pérez-Andrés, Jénifer; Caminero, Alberto; Nistal, Esther; Vivas, Santiago; Ruiz de Morales, José María; Casqueiro, Javier

2017-09-01

Gluten is the only known environmental factor that triggers celiac disease. Several studies have described an imbalance between the intestinal microbiota of different individuals based on diagnoses. Moreover, recent studies have suggested that human bacteria may play an important role in gluten hydrolysis. However, there has been no research focusing on the small intestine. This study aimed to characterize the adult small intestine microbiota possibly implicated in gluten hydrolysis. Duodenal biopsies from different diagnosed individuals were cultured in a gluten-containing medium, and the grown microbiota was analyzed by culture dependent/independent methods. Results showed that gluten-degrading bacteria can be found in the human small intestine. Indeed, 114 bacterial strains belonging to 32 species were isolated; 85 strains were able to grow in a medium containing gluten as the sole nitrogen source, 31 strains showed extracellular proteolytic activity against gluten protein and 27 strains showed peptidolytic activity towards the 33 mer peptide, an immunogenic peptide for celiac disease patients. We found that there are no differences based on the diagnosis, but each individual has its own population of gluten-hydrolyzing bacteria. These bacteria or their gluten-degrading enzymes could help to improve the quality of life of celiac disease patients'. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Vitamin D and intestinal calcium transport after bariatric surgery.

PubMed

Schafer, Anne L

2017-10-01

Bariatric surgery is a highly effective treatment for obesity, but it may have detrimental effects on the skeleton. Skeletal effects are multifactorial but mediated in part by nutrient malabsorption. While there is increasing interest in non-nutritional mechanisms such as changes in fat-derived and gut-derived hormones, nutritional factors are modifiable and thus represent potential opportunities to prevent and treat skeletal complications. This review begins with a discussion of normal intestinal calcium transport, including recent advances in our understanding of its regulation by vitamin D, and areas of continued uncertainty. Human and animal studies of vitamin D and intestinal calcium transport after bariatric surgery are then summarized. In humans, even with optimized 25-hydroxyvitamin D levels and recommended calcium intake, fractional calcium absorption decreased dramatically after Roux-en-Y gastric bypass (RYGB). In rats, intestinal calcium absorption was lower after RYGB than after sham surgery, despite elevated 1,25-dihyroxyvitamin D levels and intestinal gene expression evidence of vitamin D responsiveness. Such studies have the potential to shed new light on the physiology of vitamin D and intestinal calcium transport. Moreover, understanding the effects of bariatric surgery on these processes may improve the clinical care of bariatric surgery patients. Published by Elsevier Ltd.

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

PubMed Central

Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

2002-01-01

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

Chronic exposure to low environmental concentrations and legal aquaculture doses of antibiotics cause systemic adverse effects in Nile tilapia and provoke differential human health risk.

PubMed

Limbu, Samwel M; Zhou, Li; Sun, Sheng-Xiang; Zhang, Mei-Ling; Du, Zhen-Yu

2018-06-01

Antibiotics used globally to treat human and animal diseases exist ubiquitously in the environment at low doses because of misuse, overdose and poor absorption after ingestion, coupled with their high-water solubility and degradation resistance. However, the systemic chronic effects of exposure to low environmental concentrations of antibiotics (LECAs) and legal aquaculture doses of antibiotics (LADAs) in fish and their human health risk are currently unknown. To investigate the in vivo chronic effects of exposure to LECAs and LADAs using oxytetracycline (OTC) and sulfamethoxazole (SMZ) in Nile tilapia (Oreochromis niloticus) and their human health risk. Twenty O. niloticus weighing 27.73 ± 0.81 g were exposed to water containing LECAs (OTC at 420 ng/L and SMZ at 260 ng/L) and diets supplemented with LADAs (OTC 80 mg/kg/day and SMZ 100 mg/kg/day) for twelve weeks. General physiological functions, metabolic activities, intestinal and hepatic health were systemically evaluated. The possible human health risks of the consumption of the experimental Nile tilapia fillets in adults and children were assessed by using risk quotient. After exposure, we observed retarded growth performance accompanied by reduced nutrients digestibility, feed efficiency, organ indices, and lipid body composition in treated fish. Antibiotics distorted intestinal morphological features subsequently induced microbiota dysbiosis and suppressed intestinal tight junction proteins. Exposure of fish to LECAs and LADAs induced oxidative stress, suppressed innate immunity, stimulated inflammatory and detoxification responses, concomitantly inhibited antioxidant capacity and caused lipid peroxidation in intestine and liver organs. Both LECAs and LADAs enhanced gluconeogenesis, inhibited lipogenesis and fatty acid beta oxidation in intestine and liver organs. The exposure of fish to LECAs and LADAs induced anaerobic glycolytic pathway and affected intestinal fat catabolism in intestine while halted aerobic glycolysis, increased hepatic fat catabolism, and induced DNA damage in liver. The hazard risk quotient in children for fish treated with OTCD was >1 indicating human health risk. Overall, both LECAs and LADAs impair general physiological functions, nutritional metabolism, and compromise fish immune system. Consumption of fish fed with legal OTC provokes health risk in children. Global stringent prohibition policy for use of antibiotics in aquaculture production and strategies to limit their release into the environment are urgently required to protect human health. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys.

PubMed

Shimizudani, Takeshi; Nagaoka, Kenjiro; Hanioka, Nobumitsu; Yamano, Shigeru; Narimatsu, Shizuo

2010-01-05

Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PLS-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.

Screening for in vitro metabolites of kakkalide and irisolidone in human and rat intestinal bacteria by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

PubMed

Zhang, Guozhe; Gong, Tianxing; Kano, Yoshihiro; Yuan, Dan

2014-02-01

Kakkalide and irisolidone, the main isoflavones of Flos Puerariae, exhibit a wide spectrum of bioactivities. Intestinal bacteria biotransformation plays an important role in the metabolic pathways of flavones, and is directly related to the bioactivities of the prodrugs after oral administration. To the best of our knowledge, the metabolic pathways of kakkalide and irisolidone in vitro have not been comprehensively studied yet. This paper describes the strategy using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) for the rapid analysis of the metabolic profiles of kakkalide and irisolidone after incubated with human and rat intestinal bacteria. Bacteria incubated samples were prepared and analyzed after incubated under anaerobic conditions for 48 h. A total of 17 metabolites, including parent compounds, were detected in human and rat intestinal bacteria incubated samples. The results obtained indicate that hydrolysis, dehydroxylation, demethoxylation, demethylation, hydroxylation, decarbonylation, and reduction were the detected metabolic pathways of kakkalide and irisolidone in vitro. The conversion rate of irisolidone in human and rat bacteria was 8.57% and 6.51%, respectively. Biochanin A was the relatively main metabolite of irisolidone, and the content of biochanin A in human and rat bacteria was 3.68% and 4.25%, respectively. The conversion rate of kakkalide in human and rat bacteria was 99.92% and 98.58%, respectively. Irisolidone was the main metabolite of kakkalide, and the content of irisolidone in human and rat bacteria was 89.58% and 89.38%, respectively. This work not only provides the evidence of kakkalide and irisolidone metabolites in vivo, but also demonstrates a simple, fast, sensitive, and inexpensive method for identification of metabolites of other compounds transformed by intestinal bacteria. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

PubMed Central

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-01-01

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

PubMed

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-08-29

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction

PubMed Central

Gufford, Brandon T.; Chen, Gang; Vergara, Ana G.; Lazarus, Philip; Oberlies, Nicholas H.

2015-01-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4′- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27–66 µM; UGT1A1, 3.2–8.3 µM; UGT1A8, 19–73 µM; and UGT1A10, 65–120 µM) encompassed reported intestinal tissue concentrations (20–310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product–drug interactions in the context of cancer prevention. PMID:26070840

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

PubMed

Gufford, Brandon T; Chen, Gang; Vergara, Ana G; Lazarus, Philip; Oberlies, Nicholas H; Paine, Mary F

2015-09-01

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Starved Guts: Morphologic and Functional Intestinal Changes in Malnutrition.

PubMed

Attia, Suzanna; Feenstra, Marjon; Swain, Nathan; Cuesta, Melina; Bandsma, Robert H J

2017-11-01

Malnutrition contributes significantly to death and illness worldwide and especially to the deaths of children younger than 5 years. The relation between intestinal changes in malnutrition and morbidity and mortality has not been well characterized; however, recent research indicates that the functional and morphologic changes of the intestine secondary to malnutrition itself contribute significantly to these negative clinical outcomes and may be potent targets of intervention. The aim of this review was to summarize current knowledge of experimental and clinically observed changes in the intestine from malnutrition preclinical models and human studies. Limited clinical studies have shown villous blunting, intestinal inflammation, and changes in the intestinal microbiome of malnourished children. In addition to these findings, experimental data using various animal models of malnutrition have found evidence of increased intestinal permeability, upregulated intestinal inflammation, and loss of goblet cells. More mechanistic studies are urgently needed to improve our understanding of malnutrition-related intestinal dysfunction and to identify potential novel targets for intervention.

Interactions Between the Intestinal Microbiome and Liver Diseases

PubMed Central

Schnabl, Bernd; Brenner, David A.

2014-01-01

The human intestine harbors a diverse community of microbes that promote metabolism and digestion in their symbiotic relationship with the host. Disturbance of its homeostasis can result in disease. We review factors that disrupt intestinal homeostasis and contribute to non-alcoholic fatty liver disease (NAFLD), steatohepatitis (NASH), alcoholic liver disease, and cirrhosis. Liver disease has long been associated with qualitative and quantitative (overgrowth) dysbiotic changes in the intestinal microbiota. Extrinsic factors, such as the Western diet and alcohol, contribute to these changes. Dysbiosis results in intestinal inflammation, a breakdown of the intestinal barrier, and translocation of microbial products in animal models. However, the contribution of the intestinal microbiome to liver disease goes beyond simple translocation of bacterial products that promote hepatic injury and inflammation. Microbial metabolites produced in a dysbiotic intestinal environment and host factors are equally important in the pathogenesis of liver disease. We review how the combination of liver insult and disruptions in intestinal homeostasis contribute to liver disease. PMID:24440671

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

PubMed

Lu, Linling; Qian, Dawei; Yang, Jing; Jiang, Shu; Guo, Jianming; Shang, Er-xin; Duan, Jin-ao

2013-04-01

In this paper, ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and the MetaboLynx™ software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC-Q-TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.

Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells—A Review

PubMed Central

Kamiloglu, Senem; Capanoglu, Esra; Grootaert, Charlotte; Van Camp, John

2015-01-01

Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells. PMID:26370977

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to pro-inflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages1

PubMed Central

Zheng, Shasha; Hedl, Matija; Abraham, Clara

2014-01-01

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of NOD2, the Crohn’s disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor (PRR) stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl and Mer (TAM) receptors in regulating chronic PRR stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and pro-inflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGFβ-dependent TAM upregulation in human macrophages, which in turn, upregulated SOCS3 expression. Restoring SOCS3 expression under TAM knockdown conditions restored chronic NOD2-mediated pro-inflammatory cytokine downregulation. In contrast to the upregulated pro-inflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, MAFK and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating pro-inflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment. PMID:25567680

Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

PubMed Central

Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

2013-01-01

Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID:23502354

Purification and fermentation in vitro of sesaminol triglucoside from sesame cake by human intestinal microbiota.

PubMed

Zhu, Xiuling; Zhang, Xin; Sun, Yongkang; Su, Di; Sun, Yi; Hu, Bing; Zeng, Xiaoxiong

2013-02-27

Sesaminol triglucoside (STG), the most abundant lignan glycoside existing in sesame cake/meal, has exhibited various biological activities. However, little information about its in vitro fermentation with intestinal microbiota is available. Therefore, the effect of STG from sesame cake on the fermentation of human fecal microbiota was evaluated. First, high-purity STG was successfully prepared from defatted sesame cake by extraction with 80% ethanol and simple purification procedures of polyamide column chromatography and Toyopearl HW-40S column chromatography. Then the influence of STG on intestinal microbiota was conducted by monitoring bacterial populations and analyzing the concentrations of short-chain fatty acids (SCFA). We found that STG could significantly induce an increase in numbers of Lactobacillus - Enterococcus group and Bifidobacterium in fermentation in vitro with human fecal microbiota, while it did not stimulate the bacterial growth of Eubacterium rectale - Clostridium coccoides group, Clostridium histolyticum group, and Bacteroides - Prevotella group. Furthermore, it was found that concentrations of formic, acetic, propionic, and butyric acids in STG culture increased significantly during the fermentation, and its total SCFA concentration was relatively higher than those of the control and glucose cultures at 6 and 12 h fermentation. Our findings provided further evidence for the importance of human intestinal bacteria in the bioactivity of STG and its metabolites in the maintenance of human health.

An in vitro study to assess the impact of tetracycline on the human intestinal microbiome.

PubMed

Jung, Ji Young; Ahn, Youngbeom; Khare, Sangeeta; Gokulan, Kuppan; Piñeiro, Silvia A; Cerniglia, Carl E

2018-02-01

The human intestinal microbiome, a generally stable ecosystem, could be potentially altered by the ingestion of antimicrobial drug residues in foods derived from animals. Data and the scientific published literature on the effects of antimicrobial residues on the human intestinal microbiome are reviewed by national regulatory authorities as part of the human food safety evaluation of veterinary antimicrobial agents used in food-producing animals. In this study, we determined if tetracycline, at low residue concentrations, could impact the human intestinal microbiome structure and the resistance-gene profile, following acute and subchronic exposure. The effects of 0.15, 1.5, 15, and 150 μg/ml of tetracycline, after 24 h and 40 days of exposure, in 3% human fecal suspensions, collected from three individuals (A, B, and C) were investigated using in vitro batch cultures. Results were variable, with either no change or minor changes in total bacterial 16S rRNA gene copies after exposure of fecal samples to tetracycline, because of the inter-individual variation of human gastrointestinal tract microbiota. Bacterial community analysis using rRNA-based pyrosequencing revealed that Firmicutes and Bacteroidetes were the predominant phyla in the three fecal samples; the ratio of phylotypes varied among individuals. The evaluation of bacterial community changes at the genus level, from control to tetracycline-treated fecal samples, suggested that tetracycline under the conditions of this study could lead to slight differences in the composition of intestinal microbiota. The genus Bacteroides (of the Bacteroidetes) was consistently altered from 1.68 to 5.70% and 4.82-8.22% at tetracycline concentrations of 0.15 μg/ml or above at both time points for individual A, respectively, and increased 5.13-13.50% and 10.92-22.18% for individual B, respectively. Clostridium family XI increased 3.50-25.34% in the presence of tetracycline at 40 days for individual C. Principal Component Analysis (PCA) confirmed the pyrosequencing findings of inter-individual variability of the ratio of phylotypes and the effect of tetracycline. Among the 23 tetracycline resistance genes (TRGs) screened, four tet genes (tetO, Q, W, and X) were major TRGs in control and tetracycline-dosed fecal samples. A variable to slight increase of copy number of TRGs appeared to be related to tetracycline treatment, interindividual variability and duration of exposure. Despite, the inherent variability of the intestinal microbiota observed among or within individuals, this pilot study contributes to the knowledge base of the impact of low residue concentrations of tetracycline on the human intestinal microbiome on the potential for antimicrobial resistance. Published by Elsevier Ltd.

Helminths and Intestinal Flora Team Up to Improve Gut Health.

PubMed

Giacomin, Paul; Agha, Zainab; Loukas, Alex

2016-09-01

Inflammatory bowel diseases (IBD) are associated with impaired intestinal barrier function, chronic inflammation, and microbial dysbiosis. In a recent publication in Science, Ramanan et al. used murine and human studies to demonstrate that infections with gastrointestinal helminths can protect against IBD by provoking immune responses that alter the balance of commensal and pathogenic bacteria in the intestine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activation of intestinal hypoxia-inducible factor 2α during obesity contributes to hepatic steatosis.

PubMed

Xie, Cen; Yagai, Tomoki; Luo, Yuhong; Liang, Xianyi; Chen, Tao; Wang, Qiong; Sun, Dongxue; Zhao, Jie; Ramakrishnan, Sadeesh K; Sun, Lulu; Jiang, Chunmei; Xue, Xiang; Tian, Yuan; Krausz, Kristopher W; Patterson, Andrew D; Shah, Yatrik M; Wu, Yue; Jiang, Changtao; Gonzalez, Frank J

2017-11-01

Nonalcoholic fatty liver disease is becoming the most common chronic liver disease in Western countries, and limited therapeutic options are available. Here we uncovered a role for intestinal hypoxia-inducible factor (HIF) in hepatic steatosis. Human-intestine biopsies from individuals with or without obesity revealed that intestinal HIF-2α signaling was positively correlated with body-mass index and hepatic toxicity. The causality of this correlation was verified in mice with an intestine-specific disruption of Hif2a, in which high-fat-diet-induced hepatic steatosis and obesity were substantially lower as compared to control mice. PT2385, a HIF-2α-specific inhibitor, had preventive and therapeutic effects on metabolic disorders that were dependent on intestine HIF-2α. Intestine HIF-2α inhibition markedly reduced intestine and serum ceramide levels. Mechanistically, intestine HIF-2α regulates ceramide metabolism mainly from the salvage pathway, by positively regulating the expression of Neu3, the gene encoding neuraminidase 3. These results suggest that intestinal HIF-2α could be a viable target for hepatic steatosis therapy.

Prom1 Function in Development, Intestinal Inflammation, and Intestinal Tumorigenesis

PubMed Central

Karim, Baktiar O.; Rhee, Ki-Jong; Liu, Guosheng; Yun, Kyuson; Brant, Steven R.

2014-01-01

Prom1/CD133 has been identified in colorectal, hepatocellular, and pancreatic cancer as a cancer stem cell marker and has been used as such to predict colon cancer recurrence in humans. Its potential molecular function as well as its role as a marker of intestinal regeneration is still not fully known. We evaluated the role of Prom1 in intestinal regeneration in inflammatory bowel disease (IBD), determined the function of Prom1, and characterized the effect of a lack of Prom1 on intestinal tumor formation in animal models. Our results suggest that Apc mutations lead to an increase in Prom1 expressing cells in the intestinal crypt stem cell compartment and in early intestinal adenomas. Also, Prom1 knockout mice are more susceptible to intestinal tumor formation. We conclude that Prom1 likely plays a role in regulating intestinal homeostasis and that these results clearly illustrate the role of Prom1 in intestinal regeneration. We further conclude that Prom1 may provide a novel therapeutic target for patients with gastrointestinal conditions such as IBD, short bowel syndrome, and colorectal cancer. PMID:25452936

Regulation of intestinal health by branched-chain amino acids.

PubMed

Zhou, Hua; Yu, Bing; Gao, Jun; Htoo, John Khun; Chen, Daiwen

2018-01-01

Besides its primary role in the digestion and absorption of nutrients, the intestine also interacts with a complex external milieu, and is the first defense line against noxious pathogens and antigens. Dysfunction of the intestinal barrier is associated with enhanced intestinal permeability and development of various gastrointestinal diseases. The branched-chain amino acids (BCAAs) are important nutrients, which are the essential substrates for protein biosynthesis. Recently, emerging evidence showed that BCAAs are involved in maintaining intestinal barrier function. It has been reported that dietary supplementation with BCAAs promotes intestinal development, enhances enterocyte proliferation, increases intestinal absorption of amino acids (AA) and glucose, and improves the immune defenses of piglets. The underlying mechanism of these effects is mediated by regulating expression of genes and proteins associate with various signaling pathways. In addition, BCAAs promote the production of beneficial bacteria in the intestine of mice. Compelling evidence supports the notion that BCAAs play important roles in both nutrition and intestinal health. Therefore, as functional amino acids with various physiological effects, BCAAs hold key roles in promoting intestinal development and health in animals and humans. © 2017 Japanese Society of Animal Science.

Escherichia coli pathotypes

USDA-ARS?s Scientific Manuscript database

Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

PubMed Central

Liévin-Le Moal, Vanessa

2013-01-01

SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

Microbiota and innate immunity in intestinal inflammation and neoplasia.

PubMed

Cario, Elke

2013-01-01

This review focuses on recent advances and novel insights into the mechanistic events that may link commensal microbiota and host innate immunity in the pathophysiology of intestinal inflammation and neoplasia. Unanswered questions are discussed and future perspectives in the field are highlighted. Commensal microbiota, host innate immunity, and genetics form a multidimensional network that controls homeostasis of the mucosal barrier in the intestine. Large-scale sequencing projects have begun to catalog the healthy human microbiome. Converging evidence suggests that alterations in the regulation of the complex host environment [e.g., dysbiosis and overgrowth of select commensal bacterial species, dietary factors, copresence of facultative pathogens (including viruses), and changes in mucus characteristics] may trigger aberrant innate immune signaling, thereby contributing to the development of intestinal inflammation and associated colon cancer in the susceptible individual. Genetically determined innate immune malfunction may create an inflammatory environment that promotes tumor progression (such as the TLR4-D299G mutation). The next challenging steps to be taken are to decipher changes in the human microbiome (and virome) during well defined diseased states, and relate them to intestinal mucosal immune functions and host genotypes.

Animal models of intestinal fibrosis: new tools for the understanding of pathogenesis and therapy of human disease

PubMed Central

Rieder, Florian; Kessler, Sean; Sans, Miquel

2012-01-01

Fibrosis is a serious condition complicating chronic inflammatory processes affecting the intestinal tract. Advances in this field that rely on human studies have been slow and seriously restricted by practical and logistic reasons. As a consequence, well-characterized animal models of intestinal fibrosis have emerged as logical and essential systems to better define and understand the pathophysiology of fibrosis. In point of fact, animal models allow the execution of mechanistic studies as well as the implementation of clinical trials with novel, pathophysiology-based therapeutic approaches. This review provides an overview of the currently available animal models of intestinal fibrosis, taking into consideration the methods of induction, key characteristics of each model, and underlying mechanisms. Currently available models will be classified into seven categories: spontaneous, gene-targeted, chemical-, immune-, bacteria-, and radiation-induced as well as postoperative fibrosis. Each model will be discussed in regard to its potential to create research opportunities to gain insights into the mechanisms of intestinal fibrosis and stricture formation and assist in the development of effective and specific antifibrotic therapies. PMID:22878121

Genome sequence of Victivallis vadensis ATCC BAA-548, an anaerobic bacterium from the phylum Lentisphaerae, isolated from the human gastro-intestinal tract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Passel, Mark W.J.; Kant, Ravi; Palva, Airi

2011-01-01

Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastro-intestinal tract.

Human cytokine responses induced by Gram-positive cell walls of normal intestinal microbiota

PubMed Central

Chen, T; Isomäki, P; Rimpiläinen, M; Toivanen, P

1999-01-01

The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a.), Eubacterium limosum(Eu.l.), Lactobacillus casei(L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-γ). Monocytes are the main cell population in PBMC to produce TNF-α and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens. PMID:10540188

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

PubMed

Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Gut symbiotic microbes imprint intestinal immune cells with the innate receptor SLAMF4 which contributes to gut immune protection against enteric pathogens

PubMed Central

Cabinian, Allison; Sinsimer, Daniel; Tang, May; Jang, Youngsoon; Choi, Bongkum; Laouar, Yasmina; Laouar, Amale

2018-01-01

Background Interactions between host immune cells and gut microbiota are crucial for the integrity and function of the intestine. How these interactions regulate immune cell responses in the intestine remains a major gap in the field. Aim We have identified the signalling lymphocyte activation molecule family member 4 (SLAMF4) as an immunomodulator of the intestinal immunity. The aim is to determine how SLAMF4 is acquired in the gut and what its contribution to intestinal immunity is. Methods Expression of SLAMF4 was assessed in mice and humans. The mechanism of induction was studied using GFPtg bone marrow chimaera mice, lymphotoxin α and TNLG8A-deficient mice, as well as gnotobiotic mice. Role in immune protection was revealed using oral infection with Listeria monocytogenes and Cytobacter rodentium. Results SLAMF4 is a selective marker of intestinal immune cells of mice and humans. SLAMF4 induction occurs directly in the intestinal mucosa without the involvement of the gut-associated lymphoid tissue. Gut bacterial products, particularly those of gut anaerobes, and gut-resident antigen-presenting cell (APC)TNLG8A are key contributors of SLAMF4 induction in the intestine. Importantly, lack of SLAMF4 expression leads the increased susceptibility of mice to infection by oral pathogens culminating in their premature death. Conclusions SLAMF4 is a marker of intestinal immune cells which contributes to the protection against enteric pathogens and whose expression is dependent on the presence of the gut microbiota. This discovery provides a possible mechanism for answering the long-standing question of how the intertwining of the host and gut microbial biology regulates immune cell responses in the gut. PMID:28341747

Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

NASA Astrophysics Data System (ADS)

Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

2017-12-01

In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

3D stromal tissue equivalent affects intestinal epithelium morphogenesis in vitro.

PubMed

De Gregorio, Vincenza; Imparato, Giorgia; Urciuolo, Francesco; Netti, Paolo A

2018-04-01

Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment. © 2017 Wiley Periodicals, Inc.

Functional metagenomics to decipher food-microbe-host crosstalk.

PubMed

Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

2015-02-01

The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk.

Recent advances in transport of water-soluble vitamins in organs of the digestive system: a focus on the colon and the pancreas.

PubMed

Said, Hamid M

2013-11-01

This review focuses on recent advances in our understanding of the mechanisms and regulation of water-soluble vitamin (WSV) transport in the large intestine and pancreas, two important organs of the digestive system that have only recently received their fair share of attention. WSV, a group of structurally unrelated compounds, are essential for normal cell function and development and, thus, for overall health and survival of the organism. Humans cannot synthesize WSV endogenously; rather, WSV are obtained from exogenous sources via intestinal absorption. The intestine is exposed to two sources of WSV: a dietary source and a bacterial source (i.e., WSV generated by the large intestinal microbiota). Contribution of the latter source to human nutrition/health has been a subject of debate and doubt, mostly based on the absence of specialized systems for efficient uptake of WSV in the large intestine. However, recent studies utilizing a variety of human and animal colon preparations clearly demonstrate that such systems do exist in the large intestine. This has provided strong support for the idea that the microbiota-generated WSV are of nutritional value to the host, and especially to the nutritional needs of the local colonocytes and their health. In the pancreas, WSV are essential for normal metabolic activities of all its cell types and for its exocrine and endocrine functions. Significant progress has also been made in understanding the mechanisms involved in the uptake of WSV and the effect of chronic alcohol exposure on the uptake processes.

Recent advances in transport of water-soluble vitamins in organs of the digestive system: a focus on the colon and the pancreas

PubMed Central

2013-01-01

This review focuses on recent advances in our understanding of the mechanisms and regulation of water-soluble vitamin (WSV) transport in the large intestine and pancreas, two important organs of the digestive system that have only recently received their fair share of attention. WSV, a group of structurally unrelated compounds, are essential for normal cell function and development and, thus, for overall health and survival of the organism. Humans cannot synthesize WSV endogenously; rather, WSV are obtained from exogenous sources via intestinal absorption. The intestine is exposed to two sources of WSV: a dietary source and a bacterial source (i.e., WSV generated by the large intestinal microbiota). Contribution of the latter source to human nutrition/health has been a subject of debate and doubt, mostly based on the absence of specialized systems for efficient uptake of WSV in the large intestine. However, recent studies utilizing a variety of human and animal colon preparations clearly demonstrate that such systems do exist in the large intestine. This has provided strong support for the idea that the microbiota-generated WSV are of nutritional value to the host, and especially to the nutritional needs of the local colonocytes and their health. In the pancreas, WSV are essential for normal metabolic activities of all its cell types and for its exocrine and endocrine functions. Significant progress has also been made in understanding the mechanisms involved in the uptake of WSV and the effect of chronic alcohol exposure on the uptake processes. PMID:23989008

Clonal group distribution of fluoroquinolone-resistant Escherichia coli among humans and companion animals in Australia.

PubMed

Platell, Joanne L; Cobbold, Rowland N; Johnson, James R; Trott, Darren J

2010-09-01

To determine the phylogenetic group distribution and prevalence of three major globally disseminated clonal groups [clonal group A (CGA) and O15:K52:H1, associated with phylogenetic group D, and sequence type ST131, associated with phylogenetic group B2] among fluoroquinolone-resistant extra-intestinal Escherichia coli isolates from humans and companion animals in Australia. Clinical extra-intestinal fluoroquinolone-resistant E. coli isolates were obtained from humans (n = 582) and companion animals (n = 125), on Australia's east coast (October 2007-October 2009). Isolates were tested for susceptibility to seven antimicrobial agents, and for phylogenetic group, O type and clonal-group-specific single nucleotide polymorphisms by PCR. The fluoroquinolone-resistant isolates were typically resistant to multiple agents (median of four). Analysis revealed that clonal group ST131 accounted for a large subset of the human isolates (202/585, 35%), but for a much smaller proportion of the companion animal isolates (9/125, 7.2%; P

Water and solute absorption from carbohydrate-electrolyte solutions in the human proximal small intestine: a review and statistical analysis.

PubMed

Shi, Xiaocai; Passe, Dennis H

2010-10-01

The purpose of this study is to summarize water, carbohydrate (CHO), and electrolyte absorption from carbohydrate-electrolyte (CHO-E) solutions based on all of the triple-lumen-perfusion studies in humans since the early 1960s. The current statistical analysis included 30 reports from which were obtained information on water absorption, CHO absorption, total solute absorption, CHO concentration, CHO type, osmolality, sodium concentration, and sodium absorption in the different gut segments during exercise and at rest. Mean differences were assessed using independent-samples t tests. Exploratory multiple-regression analyses were conducted to create prediction models for intestinal water absorption. The factors influencing water and solute absorption are carefully evaluated and extensively discussed. The authors suggest that in the human proximal small intestine, water absorption is related to both total solute and CHO absorption; osmolality exerts various impacts on water absorption in the different segments; the multiple types of CHO in the ingested CHO-E solutions play a critical role in stimulating CHO, sodium, total solute, and water absorption; CHO concentration is negatively related to water absorption; and exercise may result in greater water absorption than rest. A potential regression model for predicting water absorption is also proposed for future research and practical application. In conclusion, water absorption in the human small intestine is influenced by osmolality, solute absorption, and the anatomical structures of gut segments. Multiple types of CHO in a CHO-E solution facilitate water absorption by stimulating CHO and solute absorption and lowering osmolality in the intestinal lumen.

Host-Microbe Interactions in the Neonatal Intestine: Role of Human Milk Oligosaccharides123

PubMed Central

Donovan, Sharon M.; Wang, Mei; Li, Min; Friedberg, Iddo; Schwartz, Scott L.; Chapkin, Robert S.

2012-01-01

The infant intestinal microbiota is shaped by genetics and environment, including the route of delivery and early dietary intake. Data from germ-free rodents and piglets support a critical role for the microbiota in regulating gastrointestinal and immune development. Human milk oligosaccharides (HMO) both directly and indirectly influence intestinal development by regulating cell proliferation, acting as prebiotics for beneficial bacteria and modulating immune development. We have shown that the gut microbiota, the microbial metatranscriptome, and metabolome differ between porcine milk–fed and formula-fed (FF) piglets. Our goal is to define how early nutrition, specifically HMO, shapes host-microbe interactions in breast-fed (BF) and FF human infants. We an established noninvasive method that uses stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in human infants. We hypothesized that a systems biology approach, combining i) HMO composition of the mother’s milk with the infant’s gut gene expression and fecal bacterial composition, ii) gene expression, and iii short-chain fatty acid profiles would identify important mechanistic pathways affecting intestinal development of BF and FF infants in the first few months of life. HMO composition was analyzed by HLPC Chip/time-of-flight MS and 3 HMO clusters were identified using principle component analysis. Initial findings indicated that both host epithelial cell mRNA expression and the microbial phylogenetic profiles provided strong feature sets that distinctly classified the BF and FF infants. Ongoing analyses are designed to integrate the host transcriptome, bacterial phylogenetic profiles, and functional metagenomic data using multivariate statistical analyses. PMID:22585924

The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human Intestinal Microbiota in Vitro.

PubMed

Zhang, Xin; Yang, Yang; Wu, Zufang; Weng, Peifang

2016-03-30

In order to investigate the modulatory effect of purple sweet potato anthocyanins (PSPAs) on human intestinal microbiota, PSPAs were prepared by column chromatography and their influence on intestinal microbiota was analyzed by monitoring the bacterial populations and analyzing short-chain fatty acid (SCFA) concentrations at different time points. The numbers (log10 cell/mL) of Bifidobacterium and Lactobacillus/Enterococcus spp., Bacteroides-Prevotella, Clostridium histolyticum, and total bacteria after 24 h of culture in anaerobic fermentation broth containing PSPAs were 8.44 ± 0.02, 8.30 ± 0.01, 7.80 ± 0.03, 7.60 ± 0.03, and 9.00 ± 0.02, respectively, compared with 8.21 ± 0.03, 8.12 ± 0.02, 7.95 ± 0.02, 7.77 ± 0.02, and 9.01 ± 0.03, respectively, in the controls. The results showed that PSPAs induced the proliferation of Bifidobacterium and Lactobacillus/Enterococcus spp., inhibited the growth of Bacteroides-Prevotella and Clostridium histolyticum, and did not affect the total bacteria number. Total SCFA concentrations in the cultures with PSPAs were significantly higher than in the controls (P < 0.05). Moreover, during the fermentation, the PSPAs were partially fragmented to phenolic acids, which may exert a better effect on intestinal microecology, suggesting that PSPAs may have prebiotic-like activity by generating SCFAs and modulating the intestinal microbiota, contributing to improvements in human health.

Characterization of intestinal inflammation and identification of related gene expression changes in mdr1a−/− mice

PubMed Central

Dommels, Y. E.M.; Zhu, S.; Davy, M.; Martell, S.; Hedderley, D.; Barnett, M. P.G.; McNabb, W. C.; Roy, N. C.

2007-01-01

Multidrug resistance targeted mutation (mdr1a−/−) mice spontaneously develop intestinal inflammation. The aim of this study was to further characterize the intestinal inflammation in mdr1a−/− mice. Intestinal samples were collected to measure inflammation and gene expression changes over time. The first signs of inflammation occurred around 16 weeks of age and most mdr1a−/− mice developed inflammation between 16 and 27 weeks of age. The total histological injury score was the highest in the colon. The inflammatory lesions were transmural and discontinuous, revealing similarities to human inflammatory bowel diseases (IBD). Genes involved in inflammatory response pathways were up-regulated whereas genes involved in biotransformation and transport were down-regulated in colonic epithelial cell scrapings of inflamed mdra1−/− mice at 25 weeks of age compared to non-inflamed FVB mice. These results show overlap to human IBD and strengthen the use of this in vivo model to study human IBD. The anti-inflammatory regenerating islet-derived genes were expressed at a lower level during inflammation initiation in non-inflamed colonic epithelial cell scrapings of mdr1a−/− mice at 12 weeks of age. This result suggests that an insufficiently suppressed immune response could be crucial to the initiation and development of intestinal inflammation in mdr1a−/− mice. PMID:18850176

Expression of recombinant human lysozyme in bacterial artificial chromosome transgenic mice promotes the growth of Bifidobacterium and inhibits the growth of Salmonella in the intestine.

PubMed

Dan, Lu; Liu, Shen; Shang, Shengzhe; Zhang, Huihua; Zhang, Ran; Li, Ning

2018-04-20

Targeted gene modification is a novel intervention strategy to increase disease resistance more quickly than traditional animal breeding. Human lysozyme, a natural, non-specific immune factor, participates in innate immunity, exerts a wide range of antimicrobial activities against pathogens, and has immuneregulatory effects. Therefore, it is a candidate gene for improved disease resistance in animals. In this study, we successfully generated a transgenic mouse model by microinjecting a modified bacterial artificial chromosome containing a recombinant human lysozyme (rhLZ) gene into the pronuclei of fertilized mouse embryos. rhLZ was expressed in serum, liver, spleen, lung, kidney, stomach, small intestine, and large intestine but not in milk. rhLZ protein concentrations in the serum of transgenic mice ranged from 2.09 to 2.60 mg/l. To examine the effect of rhLZ on intestinal microbiota, total aerobes, total anaerobes, Clostridium, Enterococcus, Streptococcus, Salmonella, Escherichia coli, Staphylococcus, Bifidobacterium, and Lactobacillus were measured in the intestines of transgenic and wild type mice. Results showed that Bifidobacteria were significantly increased (p < 0.001), whereas Salmonella were significantly decreased (p < 0.001) in transgenic mice compared to wild type mice. Our study suggests that rhLZ expression is a potential strategy to increase animal disease resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Intestinal parasites of owned dogs and cats from metropolitan and micropolitan areas: prevalence, zoonotic risks, and pet owner awareness in northern Italy.

PubMed

Zanzani, Sergio Aurelio; Gazzonis, Alessia Libera; Scarpa, Paola; Berrilli, Federica; Manfredi, Maria Teresa

2014-01-01

Intestinal parasites of dogs and cats are cosmopolitan pathogens with zoonotic potential for humans. Our investigation considered their diffusion in dogs and cats from northern Italy areas, specifically the metropolitan area of Milan and two micropolitan areas of neighboring provinces. It included the study of the level of awareness in pet owners of the zoonotic potential from these parasites. A total of 409 fresh fecal samples were collected from household dogs and cats for copromicroscopic analysis and detection of Giardia duodenalis coproantigens. The assemblages of Giardia were also identified. A questionnaire about intestinal parasites biology and zoonotic potential was submitted to 185 pet owners. The overall prevalence of intestinal parasites resulted higher in cats (47.37%-60.42%) and dogs (57.41%-43.02%) from micropolitan areas than that from the metropolis of Milan (dogs: P = 28.16%; cats: P = 32.58 %). The zoonotic parasites infecting pets under investigation were T. canis and T. cati, T. vulpis, Ancylostomatidae, and G. duodenalis assemblage A. Only 49.19% of pet owners showed to be aware of the risks for human health from canine and feline intestinal parasites. Parasitological results in pets and awareness determination in their owners clearly highlight how the role of veterinarians is important in indicating correct and widespread behaviors to reduce risks of infection for pets and humans in urban areas.

Characterization of porcine milk oligosaccharides during early lactation and their relation to the fecal microbiome

PubMed Central

Salcedo, J.; Frese, S. A.; Mills, D. A.; Barile, D.

2017-01-01

The composition of porcine milk oligosaccharides (PMO) was analyzed during early lactation and their relation to piglet gut microbiome was investigated. Pigs are considered ideal intestinal models to simulate humans because of the striking similarity in intestinal physiopathology to humans. The evolution of PMO was investigated in the milk from 3 healthy sows at prefarrowing, farrowing, and d 7 and 14 postpartum by Nano-LC Chip Quadrupole-Time-of-Flight mass spectrometer (Agilent Technologies, Santa Clara, CA). Previously sequenced metagenome libraries were reanalyzed to examine changes with specific gut bacterial populations. Over 30 oligosaccharides (OS) were identified in the milk, with 3′-sialyllactose, lacto-N-tetraose, α1–3,β1–4-d-galactotriose, 2′-fucosyllactose, and 6′-sialyllactose being the most abundant species (accounting for ~70% of the total OS). Porcine milk had lower OS diversity (number of unique structures) than human milk, and appeared closer to bovine and caprine milk. In agreement with previous studies, only 3 fucosylated OS were identified. Surprisingly, their contribution to total OS abundance was greater than in bovine milk (9 vs. 1%). Indeed, fucosylated PMO increased during lactation, mirroring a similar trend observed for neutral and type I OS content during early lactation. Taken together, these results suggest that, in terms of abundance, PMO are closer to human milk than other domestic species, such as bovine and caprine milks. Metagenomic sequencing revealed that fucose-consuming bacterial taxa in the gut microbiota of piglets were qualitatively but not quantitatively different between nursing and weaning stages, suggesting that both the composition and structure of dietary glycans may play a critical role in shaping the distal gut microbiome. The similarity of both intestinal physiopathology and milk OS composition in human and porcine species suggests similar effects on gastrointestinal development of early nutrition, reinforcing the use of the pig intestinal model to simulate human intestinal models in the clinical setting. PMID:27522435

Stem cell-derived human intestinal organoids as an infection model for rotaviruses.

PubMed

Finkbeiner, Stacy R; Zeng, Xi-Lei; Utama, Budi; Atmar, Robert L; Shroyer, Noah F; Estes, Mary K

2012-01-01

Directed differentiation of stem cell lines into intestine-like tissue called induced human intestinal organoids (iHIOs) is now possible (J. R. Spence, C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance, K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn, N. F. Shroyer, and J. M. Wells, Nature 470:105-109, 2011). We tested iHIOs as a new model to cultivate and study fecal viruses. Protocols for infection of iHIOs with a laboratory strain of rotavirus, simian SA11, were developed. Proof-of-principle analyses showed that iHIOs support replication of a gastrointestinal virus, rotavirus, on the basis of detection of nonstructural viral proteins (nonstructural protein 4 [NSP4] and NSP2) by immunofluorescence, increased levels of viral RNA by quantitative reverse transcription-PCR (qRT-PCR), and production of infectious progeny virus. iHIOs were also shown to support replication of 12/13 clinical rotavirus isolates directly from stool samples. An unexpected finding was the detection of rotavirus infection not only in the epithelial cells but also in the mesenchymal cell population of the iHIOs. This work demonstrates that iHIOs offer a promising new model to study rotaviruses and other gastrointestinal viruses. Gastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models.

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

PubMed

Roubos-van den Hil, P J; Nout, M J R; Beumer, R R; van der Meulen, J; Zwietering, M H

2009-03-01

This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

Flow and active mixing have a strong impact on bacterial growth dynamics in the proximal large intestine

NASA Astrophysics Data System (ADS)

Cremer, Jonas; Segota, Igor; Yang, Chih-Yu; Arnoldini, Markus; Groisman, Alex; Hwa, Terence

2016-11-01

More than half of fecal dry weight is bacterial mass with bacterial densities reaching up to 1012 cells per gram. Mostly, these bacteria grow in the proximal large intestine where lateral flow along the intestine is strong: flow can in principal lead to a washout of bacteria from the proximal large intestine. Active mixing by contractions of the intestinal wall together with bacterial growth might counteract such a washout and allow high bacterial densities to occur. As a step towards understanding bacterial growth in the presence of mixing and flow, we constructed an in-vitro setup where controlled wall-deformations of a channel emulate contractions. We investigate growth along the channel under a steady nutrient inflow. Depending on mixing and flow, we observe varying spatial gradients in bacterial density along the channel. Active mixing by deformations of the channel wall is shown to be crucial in maintaining a steady-state bacterial population in the presence of flow. The growth-dynamics is quantitatively captured by a simple mathematical model, with the effect of mixing described by an effective diffusion term. Based on this model, we discuss bacterial growth dynamics in the human large intestine using flow- and mixing-behavior having been observed for humans.

Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.

PubMed

Koeth, Robert A; Wang, Zeneng; Levison, Bruce S; Buffa, Jennifer A; Org, Elin; Sheehy, Brendan T; Britt, Earl B; Fu, Xiaoming; Wu, Yuping; Li, Lin; Smith, Jonathan D; DiDonato, Joseph A; Chen, Jun; Li, Hongzhe; Wu, Gary D; Lewis, James D; Warrier, Manya; Brown, J Mark; Krauss, Ronald M; Tang, W H Wilson; Bushman, Frederic D; Lusis, Aldons J; Hazen, Stanley L

2013-05-01

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of L-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

Gene expression in human small intestinal mucosa in vivo is mediated by iron-induced oxidative stress.

PubMed

Troost, Freddy J; Brummer, Robert-Jan M; Haenen, Guido R M M; Bast, Aalt; van Haaften, Rachel I; Evelo, Chris T; Saris, Wim H M

2006-04-13

Iron-induced oxidative stress in the small intestine may alter gene expression in the intestinal mucosa. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine. Eight healthy volunteers [22 yr(SD2)] were tested on two separate occasions in a randomized crossover design. After duodenal tissue sampling by gastroduodenoscopy, a perfusion catheter was inserted orogastrically to perfuse a 40-cm segment of the proximal small intestine with saline and, subsequently, with either 80 or 400 mg of iron as ferrous gluconate. After the intestinal perfusion, a second duodenal tissue sample was obtained. Thiobarbituric acid-reactive substances, an indicator of lipid peroxidation, in intestinal fluid samples increased significantly and dose dependently at 30 min after the start of perfusion with 80 or 400 mg of iron, respectively (P < 0.001). During the perfusion with 400 mg of iron, the increase in thiobarbituric acid-reactive substances was accompanied by a significant, momentary rise in trolox equivalent antioxidant capacity, an indicator of total antioxidant capacity (P < 0.05). The expression of 89 gene reporters was significantly altered by both iron interventions. Functional mapping showed that both iron dosages mediated six distinct processes. Three of those processes involved G-protein receptor coupled pathways. The other processes were associated with cell cycle, complement activation, and calcium channels. Iron administration in the small intestine induced dose-dependent lipid peroxidation and a momentary antioxidant response in the lumen, mediated the expression of at least 89 individual gene reporters, and affected at least six biological processes.

Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.

PubMed

Erickson, R H; Gum, J R; Lindstrom, M M; McKean, D; Kim, Y S

1995-11-02

RT-PCR was used to obtain rat small intestinal cDNAs for two peptide transporters, showing conclusively for the first time that both are present in normal intestinal mucosa. Sequencing of these cDNAs showed them to be highly homologous and similar to two different types of peptide transport proteins from either colorectal carcinoma cells (Caco-2) or human and rabbit intestine. An even distribution profile of steady state levels of mRNA for both peptide transporters was observed along the longitudinal axis of small intestine. Both were upregulated in the distal regions of intestine by a high protein diet. Also, high levels of the rat high affinity glutamate transporter EAAC1 were observed in the distal intestine. These results suggest that the distal regions of small intestine play an important role in the absorption of some amino acids and peptides. Furthermore this area appears to be a primary site where dietary-induced changes in peptide and amino acid transport occurs.

Pan-Nematoda Transcriptomic Elucidation of Essential Intestinal Functions and Therapeutic Targets With Broad Potential

PubMed Central

Wang, Qi; Rosa, Bruce A.; Jasmer, Douglas P.; Mitreva, Makedonka

2015-01-01

The nematode intestine is continuous with the outside environment, making it easily accessible to anthelmintics for parasite control, but the development of new therapeutics is impeded by limited knowledge of nematode intestinal cell biology. We established the most comprehensive nematode intestinal functional database to date by generating transcriptional data from the dissected intestines of three parasitic nematodes spanning the phylum, and integrating the results with the whole proteomes of 10 nematodes (including 9 pathogens of humans or animals) and 3 host species and 2 outgroup species. We resolved 10,772 predicted nematode intestinal protein families (IntFams), and studied their presence and absence within the different lineages (births and deaths) among nematodes. Conserved intestinal cell functions representing ancestral functions of evolutionary importance were delineated, and molecular features useful for selective therapeutic targeting were identified. Molecular patterns conserved among IntFam proteins demonstrated large potential as therapeutic targets to inhibit intestinal cell functions with broad applications towards treatment and control of parasitic nematodes. PMID:26501106

A Mathematical Model of the Human Small Intestine Following Acute Radiation and Burn Exposures

DTIC Science & Technology

2016-08-01

Acronyms and Symbols ARA Applied Research Associates, Inc. ARS Acute radiation syndrome d Days DE Differential Evolution DTRA Defense Threat...04-08-2016 Technical Report A Mathematical Model of the Human Small Intestine Following Acute Radiation and Burn Exposures HDTRA1...epithelial cells to acute radiation alone. The model has been modified for improved radiation response, and an addition to the model allows for thermal injury

Diagnosis of edema and inflammation in human intestines using ultrawideband radar

NASA Astrophysics Data System (ADS)

Smith, Sonny; Narayanan, Ram M.; Messaris, Evangelos

2015-05-01

Human intestines are vital organs, which are often subjected to chronic issues. In particular, Crohn's disease is a bowel aliment resulting in inflammation along the lining of one's digestive tract. Moreover, such an inflammatory condition causes changes in the thickness of the intestines; and we posit induce changes in the dielectric properties detectable by radar. This detection hinges on the increase in fluid content in the afflicted area, which is described by effective medium approximations (EMA). In this paper, we consider one of the constitutive parameters (i.e. relative permittivity) of different human tissues and introduce a simple numerical, electromagnetic multilayer model. We observe how the increase in water content in one layer can be approximated to predict the effective permittivity of that layer. Moreover, we note trends in how such an accumulation can influence the total effective reflection coefficient of the multiple layers.

A gastro-resistant ovalbumin bi-layered mini-tablet-in-tablet system for the delivery of Lactobacillus acidophilus probiotic to simulated human intestinal and colon conditions.

PubMed

Govender, Mershen; Choonara, Yahya Essop; van Vuuren, Sandy; Kumar, Pradeep; du Toit, Lisa Claire; Pillay, Viness

2015-07-01

The viability of probiotic bacteria during formulation processes and delivery is vital to ensure health benefits. This study focuses on the use of gastro-resistant denatured ovalbumin for the targeted delivery of probiotic Lactobacillus acidophilus to simulated human intestinal and colon conditions through a bi-layered mini-tablet-in-tablet system (BMTTS). The BMTTS consists of two gastro-resistant ovalbumin mini-tablets containing L. acidophilus suspended in lactose and eudragit S100 for targeted intestinal and colonic delivery respectively. Luminescence has been utilized to ensure probiotic viability during formulation processes in addition to determining all probiotic release profiles. The mechanism of probiotic release from the ovalbumin matrix was ascertained using mathematical modelling and molecular docking studies. Magnetic resonance imaging and differential scanning calorimetry are also included as part of the in-vitro characterization of the ovalbumin system. The BMTTS was effective in the delivery of L. acidophilus to simulated human intestinal and colon conditions. Formulation processes were furthermore determined to maintain probiotic viability. Statistical analysis of the release data noted a significant effect of pH denaturation on the release properties of ovalbumin. Magnetic resonance imaging results have indicated a decrease in ovalbumin matrix size upon exposure to simulated intestinal fluid. Molecular docking studies carried out depicted the interaction and binding positions inherent to the ovalbumin-pancreatic trypsin interaction complex indicating the possible enzymatic degradation of ovalbumin leading to the release of the probiotic from the protein matrix. The BMTTS has been determined to be effective in the protection and delivery of probiotic L. acidophilus to simulated human intestinal and colonic conditions. Molecular docking analysis has noted that pancreatin exerts a significant effect on probiotic release from the gastro-resistant ovalbumin matrix. © 2015 Royal Pharmaceutical Society.

Human Ghrelin Mitigates Intestinal Injury and Mortality after Whole Body Irradiation in Rats

PubMed Central

Wang, Zhimin; Yang, Weng Lang; Jacob, Asha; Aziz, Monowar; Wang, Ping

2015-01-01

Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury. PMID:25671547

Human ghrelin mitigates intestinal injury and mortality after whole body irradiation in rats.

PubMed

Wang, Zhimin; Yang, Weng Lang; Jacob, Asha; Aziz, Monowar; Wang, Ping

2015-01-01

Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

PubMed

Carvalho, Eunice B; Maga, Elizabeth A; Quetz, Josiane S; Lima, Ila F N; Magalhães, Hemerson Y F; Rodrigues, Felipe A R; Silva, Antônio V A; Prata, Mara M G; Cavalcante, Paloma A; Havt, Alexandre; Bertolini, Marcelo; Bertolini, Luciana R; Lima, Aldo A M

2012-08-11

Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers

PubMed Central

van Bokhorst-van de Veen, Hermien; van Swam, Iris; Wels, Michiel; Bron, Peter A.; Kleerebezem, Michiel

2012-01-01

Background An important trait of probiotics is their capability to reach their intestinal target sites alive to optimally exert their beneficial effects. Assessment of this trait in intestine-mimicking in vitro model systems has revealed differential survival of individual strains of a species. However, data on the in situ persistence characteristics of individual or mixtures of strains of the same species in the gastrointestinal tract of healthy human volunteers have not been reported to date. Methodology/Principal Findings The GI-tract survival of individual L. plantarum strains was determined using an intestine mimicking model system, revealing substantial inter-strain differences. The obtained data were correlated to genomic diversity of the strains using comparative genome hybridization (CGH) datasets, but this approach failed to discover specific genetic loci that explain the observed differences between the strains. Moreover, we developed a next-generation sequencing-based method that targets a variable intergenic region, and employed this method to assess the in vivo GI-tract persistence of different L. plantarum strains when administered in mixtures to healthy human volunteers. Remarkable consistency of the strain-specific persistence curves were observed between individual volunteers, which also correlated significantly with the GI-tract survival predicted on basis of the in vitro assay. Conclusion The survival of individual L. plantarum strains in the GI-tract could not be correlated to the absence or presence of specific genes compared to the reference strain L. plantarum WCFS1. Nevertheless, in vivo persistence analysis in the human GI-tract confirmed the strain-specific persistence, which appeared to be remarkably similar in different healthy volunteers. Moreover, the relative strain-specific persistence in vivo appeared to be accurately and significantly predicted by their relative survival in the intestine-mimicking in vitro assay, supporting the use of this assay for screening of strain-specific GI persistence. PMID:22970257

The role of genetic background in susceptibility to chemical warfare nerve agents across rodent and non-human primate models.

PubMed

Matson, Liana M; McCarren, Hilary S; Cadieux, C Linn; Cerasoli, Douglas M; McDonough, John H

2018-01-15

Genetics likely play a role in various responses to nerve agent exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses to environmental stimuli. Mouse strains or selected lines can be used to identify susceptibility based on background genetic features to nerve agent exposure. Additional genetic techniques can then be used to identify mechanisms underlying resistance and sensitivity, with the ultimate goal of developing more effective and targeted therapies. Here, we discuss the available literature on strain and selected line differences in cholinesterase activity levels and response to nerve agent-induced toxicity and seizures. We also discuss the available cholinesterase and toxicity literature across different non-human primate species. The available data suggest that robust genetic differences exist in cholinesterase activity, nerve agent-induced toxicity, and chemical-induced seizures. Available cholinesterase data suggest that acetylcholinesterase activity differs across strains, but are limited by the paucity of carboxylesterase data in strains and selected lines. Toxicity and seizures, two outcomes of nerve agent exposure, have not been fully evaluated for genetic differences, and thus further studies are required to understand baseline strain and selected line differences. Published by Elsevier B.V.

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

PubMed

Michalke, Klaus; Schmidt, Annette; Huber, Britta; Meyer, Jörg; Sulkowski, Margareta; Hirner, Alfred V; Boertz, Jens; Mosel, Frank; Dammann, Philip; Hilken, Gero; Hedrich, Hans J; Dorsch, Martina; Rettenmeier, Albert W; Hensel, Reinhard

2008-05-01

The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37 degrees C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH(3))(3)Bi] at rather low concentrations of bismuth (0.2 to 1 mumol kg(-1) [dry weight]). An increase of bismuth up to 2 to 14 mmol kg(-1) (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h(-1) kg(-1) (dry weight) to 2,100 pmol h(-1) kg(-1) (dry weight) in human feces samples and from approximately 5 pmol h(-1) kg(-1) (dry weight) to 120 pmol h(-1) kg(-1) (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH(3))(3)Bi.

The gastrointestinal-brain axis in humans as an evolutionary advance of the root-leaf axis in plants: A hypothesis linking quantum effects of light on serotonin and auxin.

PubMed

Tonello, Lucio; Gashi, Bekim; Scuotto, Alessandro; Cappello, Glenda; Cocchi, Massimo; Gabrielli, Fabio; Tuszynski, Jack A

2018-01-01

Living organisms tend to find viable strategies under ambient conditions that optimize their search for, and utilization of, life-sustaining resources. For plants, a leading role in this process is performed by auxin, a plant hormone that drives morphological development, dynamics, and movement to optimize the absorption of light (through branches and leaves) and chemical "food" (through roots). Similarly to auxin in plants, serotonin seems to play an important role in higher animals, especially humans. Here, it is proposed that morphological and functional similarities between (i) plant leaves and the animal/human brain and (ii) plant roots and the animal/human gastro-intestinal tract have general features in common. Plants interact with light and use it for biological energy, whereas, neurons in the central nervous system seem to interact with bio-photons and use them for proper brain function. Further, as auxin drives roots "arborescence" within the soil, similarly serotonin seems to facilitate enteric nervous system connectivity within the human gastro-intestinal tract. This auxin/serotonin parallel suggests the root-branches axis in plants may be an evolutionary precursor to the gastro-intestinal-brain axis in humans. Finally, we hypothesize that light might be an important factor, both in gastro-intestinal dynamics and brain function. Such a comparison may indicate a key role for the interaction of light and serotonin in neuronal physiology (possibly in both the central nervous system and the enteric nervous system), and according to recent work, mind and consciousness.

Enteroendocrine K and L cells in healthy and type 2 diabetic individuals.

PubMed

Jorsal, Tina; Rhee, Nicolai A; Pedersen, Jens; Wahlgren, Camilla D; Mortensen, Brynjulf; Jepsen, Sara L; Jelsing, Jacob; Dalbøge, Louise S; Vilmann, Peter; Hassan, Hazem; Hendel, Jakob W; Poulsen, Steen S; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

2018-02-01

Enteroendocrine K and L cells are pivotal in regulating appetite and glucose homeostasis. Knowledge of their distribution in humans is sparse and it is unknown whether alterations occur in type 2 diabetes. We aimed to evaluate the distribution of enteroendocrine K and L cells and relevant prohormone-processing enzymes (using immunohistochemical staining), and to evaluate the mRNA expression of the corresponding genes along the entire intestinal tract in individuals with type 2 diabetes and healthy participants. In this cross-sectional study, 12 individuals with type 2 diabetes and 12 age- and BMI-matched healthy individuals underwent upper and lower double-balloon enteroscopy with mucosal biopsy retrieval from approximately every 30 cm of the small intestine and from seven specific anatomical locations in the large intestine. Significantly different densities for cells positive for chromogranin A (CgA), glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, peptide YY, prohormone convertase (PC) 1/3 and PC2 were observed along the intestinal tract. The expression of CHGA did not vary along the intestinal tract, but the mRNA expression of GCG, GIP, PYY, PCSK1 and PCSK2 differed along the intestinal tract. Lower counts of CgA-positive and PC1/3-positive cells, respectively, were observed in the small intestine of individuals with type 2 diabetes compared with healthy participants. In individuals with type 2 diabetes compared with healthy participants, the expression of GCG and PYY was greater in the colon, while the expression of GIP and PCSK1 was greater in the small intestine and colon, and the expression of PCSK2 was greater in the small intestine. Our findings provide a detailed description of the distribution of enteroendocrine K and L cells and the expression of their products in the human intestinal tract and demonstrate significant differences between individuals with type 2 diabetes and healthy participants. NCT03044860.

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.

PubMed

Chandrashekharaiah, K S; Swamy, N Ramachandra; Murthy, K R Siddalinga

2011-12-01

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB). Copyright © 2011 Elsevier Ltd. All rights reserved.

In-situ intestinal rat perfusions for human Fabs prediction and BCS permeability class determination: Investigation of the single-pass vs. the Doluisio experimental approaches.

PubMed

Lozoya-Agullo, Isabel; Zur, Moran; Wolk, Omri; Beig, Avital; González-Álvarez, Isabel; González-Álvarez, Marta; Merino-Sanjuán, Matilde; Bermejo, Marival; Dahan, Arik

2015-03-01

Intestinal drug permeability has been recognized as a critical determinant of the fraction dose absorbed, with direct influence on bioavailability, bioequivalence and biowaiver. The purpose of this research was to compare intestinal permeability values obtained by two different intestinal rat perfusion methods: the single-pass intestinal perfusion (SPIP) model and the Doluisio (closed-loop) rat perfusion method. A list of 15 model drugs with different permeability characteristics (low, moderate, and high, as well as passively and actively absorbed) was constructed. We assessed the rat intestinal permeability of these 15 model drugs in both SPIP and the Doluisio methods, and evaluated the correlation between them. We then evaluated the ability of each of these methods to predict the fraction dose absorbed (Fabs) in humans, and to assign the correct BCS permeability class membership. Excellent correlation was obtained between the two experimental methods (r(2)=0.93). An excellent correlation was also shown between literature Fabs values and the predictions made by both rat perfusion techniques. Similar BCS permeability class membership was designated by literature data and by both SPIP and Doluisio methods for all compounds. In conclusion, the SPIP model and the Doluisio (closed-loop) rat perfusion method are both equally useful for obtaining intestinal permeability values that can be used for Fabs prediction and BCS classification. Copyright © 2015 Elsevier B.V. All rights reserved.

The utility of rat jejunal permeability for biopharmaceutics classification system.

PubMed

Zakeri-Milani, Parvin; Valizadeh, Hadi; Tajerzadeh, Hosnieh; Islambulchilar, Ziba

2009-12-01

The biopharmaceutical classification system has been developed to provide a scientific approach for classifying drug compounds based on their dose/solubility ratio and human intestinal permeability. Therefore in this study a new classification is presented, which is based on a correlation between rat and human intestinal permeability values. In situ technique in rat jejunum was used to determine the effective intestinal permeability of tested drugs. Then three dimensionless parameters--dose number, absorption number, and dissolution number (D(o), A(n), and D(n))--were calculated for each drug. Four classes of drugs were defined, that is, class I, D(0) < 0.5, P(eff(rat)) > 5.09 x 10(-5) cm/s; class II, D(o) > 1, P(eff(rat)) > 5.09 x 10( -5) cm/s; class III, D(0) < 0.5, P(eff(rat)) < 4.2 x 10(-5) cm/s; and class IV, D(o) > 1, P(eff(rat)) < 4.2 x 10(-5) cm/s. A region of borderline drugs (0.5 < D(o) < 1, 4.2 x 10(-5) < P(eff(rat)) < 5.09 x 10(-5) cm/s) was also defined. According to obtained results and proposed classification for drugs, it is concluded that drugs could be categorized correctly based on dose number and their intestinal permeability values in rat model using single-pass intestinal perfusion technique. This classification enables us to remark defined characteristics for intestinal absorption of all four classes using suitable cutoff points for both dose number and rat effective intestinal permeability values.

An Ecological Network of Polysaccharide Utilization Among Human Intestinal Symbionts

PubMed Central

Rakoff-Nahoum, Seth; Coyne, Michael J.; Comstock, Laurie E.

2013-01-01

Summary Background: The human intestine is colonized with trillions of microorganisms important to health and disease. There has been an intensive effort to catalog the species and genetic content of this microbial ecosystem. However, little is known of the ecological interactions between these microbes, a prerequisite to understanding the dynamics and stability of this host-associated microbial community. Here we perform a systematic investigation of public goods-based syntrophic interactions among the abundant human gut bacteria, the Bacteroidales. Results: We find evidence for a rich interaction network based on the breakdown and use of polysaccharides. Species that utilize a particular polysaccharide (producers) liberate polysaccharide breakdown products (PBP) that are consumed by other species unable to grow on the polysaccharide alone (recipients). Cross-species gene addition experiments demonstrate that recipients can grow on a polysaccharide if the producer-derived glycoside hydrolase, responsible for PBP generation, is provided. These producer-derived glycoside hydrolases are public goods transported extracellularly in outer membrane vesicles allowing for the creation of PBP and concomitant recipient growth spatially distant from the producer. Recipients can exploit these ecological interactions and conditionally outgrow producers. Finally, we show that these public good-based interactions occur among Bacteroidales species co-resident within a natural human intestinal community. Conclusions: This study examines public-goods based syntrophic interactions between bacterial members of the critically important gut microbial ecosystem. This polysaccharide-based network likely represents foundational relationships creating organized ecological units within the intestinal microbiota, knowledge of which can be applied to impact human health. PMID:24332541

Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli.

PubMed

Karve, Sayali S; Pradhan, Suman; Ward, Doyle V; Weiss, Alison A

2017-01-01

Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome, and currently only supportive therapy is available. Lack of suitable animal models has hindered study of this disease. Induced human intestinal organoids (iHIOs), generated by in vitro differentiation of pluripotent stem cells, represent differentiated human intestinal tissue. We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses. Commensal and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high numbers. Commensal E. coli did not cause damage, and were completely contained within the lumen, suggesting defenses, such as mucus production, can constrain non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew as filaments, consistent with activation of the bacterial SOS stress response. SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased ROS production. Transcriptional profiling (RNAseq) demonstrated that both commensal and O157:H7 upregulated genes associated with gastrointestinal maturation, while infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment, and infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO tissue.

Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

NASA Technical Reports Server (NTRS)

Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

2017-01-01

Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

Influence of human milk oligosaccharides on adherence of bifidobacteria and clostridia to cell lines.

PubMed

Musilova, Sarka; Modrackova, Nikol; Doskocil, Ivo; Svejstil, Roman; Rada, Vojtech

2017-12-01

Adhesion of gut bacteria to the intestinal epithelium is the first step in their colonization of the neonatal immature gut. Bacterial colonization of the infant gut is influenced by several factors, of which the most important are the mode of delivery and breast-feeding. Breast-fed infants ingest several grams of human milk oligosaccharides (HMOs) per day, which can become receptor decoys for intestinal bacteria. The most abundant intestinal bacteria in vaginally delivered infants are bifidobacteria, whereas infants born by cesarean section are colonized by clostridia. The influence of HMOs on the adhesion of five strains of intestinal bacteria (three bifidobacterial strains and two clostridial strains) to mucus-secreting and non-mucus-secreting human epithelial cells was investigated. Bifidobacterium bifidum 1 and Bifidobacterium longum displayed almost the same level of adhesion in the presence and absence of HMOs. By contrast, adhesion of Clostridium butyricum 1 and 2 decreased from 14.41% to 6.72% and from 41.54% to 30.91%, respectively, in the presence of HMOs. The results of this study indicate that HMOs affect bacterial adhesion and are an important factor influencing bacterial colonization of the gut. Adhesion of the tested bacteria correlates with their ability to autoaggregate.

Multispectral tissue characterization for intestinal anastomosis optimization.

PubMed

Cha, Jaepyeong; Shademan, Azad; Le, Hanh N D; Decker, Ryan; Kim, Peter C W; Kang, Jin U; Krieger, Axel

2015-10-01

Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement.

Multispectral tissue characterization for intestinal anastomosis optimization

PubMed Central

Cha, Jaepyeong; Shademan, Azad; Le, Hanh N. D.; Decker, Ryan; Kim, Peter C. W.; Kang, Jin U.; Krieger, Axel

2015-01-01

Abstract. Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement. PMID:26440616

Lactobacillus acidophilus attenuates Salmonella-induced intestinal inflammation via TGF-β signaling.

PubMed

Huang, I-Fei; Lin, I-Chun; Liu, Pei-Feng; Cheng, Ming-Fang; Liu, Yen-Chen; Hsieh, Yao-Dung; Chen, Jih-Jung; Chen, Chun-Lin; Chang, Hsueh-Wei; Shu, Chih-Wen

2015-10-07

Salmonella is a common intestinal pathogen that causes acute and chronic inflammatory response. Probiotics reduce inflammatory cytokine production and serve as beneficial commensal microorganisms in the human gastrointestinal tract. TGF-β (transforming growth factor β)/SMAD and NF-κB signaling play important roles in inflammation in intestinal cells. However, the involvement of the signaling in regulating inflammation between Salmonella and probiotics is not fully understood. L. acidophilus and prebiotic inulin were used to treat human intestinal Caco-2 cells prior to infection with Salmonella. The cells were harvested to examine the cytokines and MIR21 expression with immunoblotting and real-time PCR. NF-κB and SMAD3/4 reporter vectors were transfected into cells to monitor inflammation and TGF-β1 signaling, respectively. In this study, we showed that the probiotic L. acidophilus decreased Salmonella-induced NF-κB activation in human intestinal Caco-2 cells. Expression of the inflammatory cytokines, TNF-α and IL-8, in L. acidophilus-pretreated cells was also significantly lower than that in cells infected with Salmonella alone. Moreover, TGF-β1 and MIR21 expression was elevated in cells pretreated with L. acidophilus or synbiotic, a combination of inulin and L. acidophilus, compared to that in untreated cells or cells infected with S. typhimurium alone. By contrast, expression of SMAD7, a target of MIR21, was accordingly reduced in cells treated with L. acidophilus or synbiotics. Consistent with TGF-β1/MIR21 and SMAD7 expression, SMAD3/4 transcriptional activity was significantly higher in the cells treated with L. acidophilus or synbiotics. Furthermore, TGF-β1 antibody antagonized the SMAD3/4 and NF-κB transcriptional activity modulated by L. acidophilus in intestinal cells. Our results suggest that the TGF-β1/MIR21 signaling pathway may be involved in the suppressive effects of L. acidophilus on inflammation caused by S. typhimurium in intestinal Caco-2 cells.

Caco-2 accumulation of lutein is greater from human milk than from infant formula despite similar bioaccessibility.

PubMed

Lipkie, Tristan E; Banavara, Dattatreya; Shah, Bhavini; Morrow, Ardythe L; McMahon, Robert J; Jouni, Zeina E; Ferruzzi, Mario G

2014-10-01

Clinical evidence suggests that the bioavailability of lutein is lower from infant formula than from human milk. The purpose of this study was to assess characteristics of human milk and lutein-fortified infant formula that may impact carotenoid delivery. Carotenoid bioaccessibility and intestinal absorption were modeled by in vitro digestion coupled with Caco-2 human intestinal cell culture. Twelve human milk samples were assessed from 1-6 months postpartum, and 10 lutein-fortified infant formula samples from three lutein sources in both ready-to-use and reconstituted powder forms. The relative bioaccessibility of lutein was not different (p > 0.05) between human milk (29 ± 2%) and infant formula (36 ± 4%). However, lutein delivery was 4.5 times greater from human milk than infant formula when including Caco-2 accumulation efficiency. Caco-2 accumulation of lutein was increasingly efficient with decreasing concentration of lutein from milk. Carotenoid bioaccessibility and Caco-2 accumulation were not affected by lactation stage, total lipid content, lutein source, or form of infant formula (powder vs. liquid). These data suggest that the bioavailability of carotenoids is greater from human milk than infant formula primarily due to intestinal absorptive processes, and that absorption of lutein is potentiated by factors from human milk especially at low lutein concentration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

PubMed Central

Somsouk, Ma; Hunt, Peter W.

2017-01-01

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection. PMID:28241080

Biorelevant media resistant co-culture model mimicking permeability of human intestine.

PubMed

Antoine, Delphine; Pellequer, Yann; Tempesta, Camille; Lorscheidt, Stefan; Kettel, Bernadette; Tamaddon, Lana; Jannin, Vincent; Demarne, Frédéric; Lamprecht, Alf; Béduneau, Arnaud

2015-03-15

Cell culture models are currently used to predict absorption pattern of new compounds and formulations in the human gastro-intestinal tract (GIT). One major drawback is the lack of relevant apical incubation fluids allowing mimicking luminal conditions in the GIT. Here, we suggest a culture model compatible with biorelevant media, namely Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF). Co-culture was set up from Caco-2 and mucus-secreting HT29-MTX cells using an original seeding procedure. Viability and cytotoxicity assays were performed following incubation of FeSSIF and FaSSIF with co-culture. Influence of biorelevant fluids on paracellular permeability or transporter proteins were also evaluated. Results were compared with Caco-2 and HT29-MTX monocultures. While Caco-2 viability was strongly affected with FeSSIF, no toxic effect was detected for the co-cultures in terms of viability and lactate dehydrogenase release. The addition of FeSSIF to the basolateral compartment of the co-culture induced cytotoxic effects which suggested the apical mucus barrier being cell protective. In contrast to FeSSIF, FaSSIF induced a slight increase of the paracellular transport and both tested media inhibited partially the P-gp-mediated efflux in the co-culture. Additionally, the absorptive transport of propranolol hydrochloride, a lipophilic β-blocker, was strongly affected by biorelevant fluids. This study demonstrated the compatibility of the Caco-2/HT29-MTX model with some of the current biorelevant media. Combining biorelevant intestinal fluids with features such as mucus secretion, adjustable paracellular and P-gp mediated transports, is a step forward to more realistic in-vitro models of the human intestine. Copyright © 2015. Published by Elsevier B.V.

The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

PubMed

Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

2017-04-01

Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intestinal exposure to PCB 153 induces inflammation via the ATM/NEMO pathway.

PubMed

Phillips, Matthew C; Dheer, Rishu; Santaolalla, Rebeca; Davies, Julie M; Burgueño, Juan; Lang, Jessica K; Toborek, Michal; Abreu, Maria T

2018-01-15

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that adversely affect human health. PCBs bio-accumulate in organisms important for human consumption. PCBs accumulation in the body leads to activation of the transcription factor NF-κB, a major driver of inflammation. Despite dietary exposure being one of the main routes of exposure to PCBs, the gut has been widely ignored when studying the effects of PCBs. We investigated the effects of PCB 153 on the intestine and addressed whether PCB 153 affected intestinal permeability or inflammation and the mechanism by which this occurred. Mice were orally exposed to PCB 153 and gut permeability was assessed. Intestinal epithelial cells (IECs) were collected and evaluated for evidence of genotoxicity and inflammation. A human IEC line (SW480) was used to examine the direct effects of PCB 153 on epithelial function. NF-кB activation was measured using a reporter assay, DNA damage was assessed, and cytokine expression was ascertained with real-time PCR. Mice orally exposed to PCB 153 had an increase in intestinal permeability and inflammatory cytokine expression in their IECs; inhibition of NF-кB ameliorated both these effects. This inflammation was associated with genotoxic damage and NF-кB activation. Exposure of SW480 cells to PCB 153 led to similar effects as seen in vivo. We found that activation of the ATM/NEMO pathway by genotoxic stress was upstream of NF-kB activation. These results demonstrate that oral exposure to PCB 153 is genotoxic to IECs and induces downstream inflammation and barrier dysfunction in the intestinal epithelium. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of intestinal flora in colorectal cancer from the metabolite perspective: a systematic review

PubMed Central

Han, Shuwen; Gao, Jianlan; Zhou, Qing; Liu, Shanshan; Wen, Caixia

2018-01-01

Colorectal cancer is one of the most common human malignant tumors. Recent research has shown that colorectal cancer is a dysbacteriosis-induced disease; however, the role of intestinal bacteria in colorectal cancer is unclear. This review explores the role of intestinal flora in colorectal cancer. In total, 57 articles were included after identification and screening. The pertinent literature on floral metabolites in colorectal cancer from three metabolic perspectives – including carbohydrate, lipid, and amino acid metabolism – was analyzed. An association network regarding the role of intestinal flora from a metabolic perspective was constructed by analyzing the previous literature to provide direction and insight for further research on intestinal flora in colorectal cancer. PMID:29440929

Interleukin-22 Signaling in the Regulation of Intestinal Health and Disease

PubMed Central

Parks, Olivia B.; Pociask, Derek A.; Hodzic, Zerina; Kolls, Jay K.; Good, Misty

2016-01-01

Interleukin (IL)-22 is a member of the IL-10 family of cytokines that has been extensively studied since its discovery in 2000. This review article aims to describe the cellular sources and signaling pathways of this cytokine as well as the functions of IL-22 in the intestine. In addition, this article describes the roles of IL-22 in the pathogenesis of several gastrointestinal diseases, including inhibition of inflammation and barrier defense against pathogens within the intestine. Since many of the functions of IL-22 in the intestine are incompletely understood, this review is meant to assess our current understanding of the roles of IL-22 and provide new opportunities for inquiry to improve human intestinal health and disease. PMID:26793707

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Island biogeography effects on microbial evolution may contribute to Crohn's disease.

PubMed

Johnson, Mac A; Winquist, Raymond J

2011-12-15

Inflammatory bowel diseases (IBDs), such as Crohn's disease (CD), involve a poorly understood and complex immune response to both the biota of the human gut and the gut itself. The role of the gut biota in human health has been ill defined and attitudes toward the intestinal flora have ranged from judging them largely irrelevant to declaring them a human organ system. A better way to view the intestinal flora is as a group of evolutionarily self-interested species that form large, potentially interbreeding populations that utilize human beings as a series of semi-isolated habitats, like islands in an archipelago. Here we propose that the imposition of modern sanitation and hygiene standards has drastically attenuated the connection between the "islands" inhabited by the gut flora, and that existing work drawn from evolutionary biology studies of island ecosystems, rather than medicine, predicts that the evolution of gut flora should now be pushed toward limited-dispersion forms of intestinal microorganisms - a proposition borne out by the discovery of so-called "adherent invasive Escherichia coli." This pathogenic variant of the gut bacterium E. coli clings to and invades the intestinal epithelium and has been implicated in CD. Gut flora and diseases of the gut should arguably be studied as ecology as much as medicine, and treated within this context. Copyright © 2011 Elsevier Inc. All rights reserved.

Toward oral delivery of biopharmaceuticals: an assessment of the gastrointestinal stability of 17 peptide drugs.

PubMed

Wang, Jie; Yadav, Vipul; Smart, Alice L; Tajiri, Shinichiro; Basit, Abdul W

2015-03-02

A major barrier to successful oral delivery of peptide and protein molecules is their inherent instability in the lumen of the gastrointestinal tract. The aim of this study was to determine the stability of 17 disparate peptide drugs (insulin, calcitonin, glucagon, secretin, somatostatin, desmopressin, oxytocin, [Arg(8)]-vasopressin, octreotide, ciclosporin, leuprolide, nafarelin, buserelin, histrelin, [d-Ser](4)-gonadorelin, deslorelin, and goserelin) in gastric and small intestinal fluids from both humans and pigs, and in simulated gastric and intestinal fluids. In human gastric fluid, the larger peptides including somatostatin, calcitonin, secretin, glucagon, and insulin were metabolized rapidly, while the smaller peptides showed good stability. In human small intestinal fluid, however, both small and large peptides degraded rapidly with the exception of the cyclic peptide ciclosporin and the disulfide-bridge containing peptides octreotide and desmopressin, which showed good stability. The stability of peptides in both simulated gastric fluid and pig gastric fluid correlated well with stability in human gastric fluid. However, it was not possible to establish such a correlation with the small intestinal fluids because of the rapid rate of peptide degradation. This work has identified the molecular features in the structure of a wide range of peptides that influence their stability in the environment of the gastrointestinal tract, which in turn will allow for better selection of peptide candidates for oral delivery.

The Role of Carrageenan and Carboxymethylcellulose in the Development of Intestinal Inflammation

PubMed Central

Martino, John Vincent; Van Limbergen, Johan; Cahill, Leah E.

2017-01-01

Although the exact pathophysiology remains unknown, the development of inflammatory bowel disease (IBD) is influenced by the interplay between genetics, the immune system, and environmental factors such as diet. The commonly used food additives, carrageenan and carboxymethylcellulose (CMC), are used to develop intestinal inflammation in animal models. These food additives are excluded from current dietary approaches to induce disease remission in Crohn’s disease such as exclusive enteral nutrition (EEN) using a polymeric formula. By reviewing the existing scientific literature, this review aims to discuss the role that carrageenan and CMC may play in the development of IBD. Animal studies consistently report that carrageenan and CMC induce histopathological features that are typical of IBD while altering the microbiome, disrupting the intestinal epithelial barrier, inhibiting proteins that provide protection against microorganisms, and stimulating the elaboration of pro-inflammatory cytokines. Similar trials directly assessing the influence of carrageenan and CMC in humans are of course unethical to conduct, but recent studies of human epithelial cells and the human microbiome support the findings from animal studies. Carrageenan and CMC may trigger or magnify an inflammatory response in the human intestine but are unlikely to be identified as the sole environmental factor involved in the development of IBD or in disease recurrence after treatment. However, the widespread use of carrageenan and CMC in foods consumed by the pediatric population in a “Western” diet is on the rise alongside a corresponding increase in IBD incidence, and questions are being raised about the safety of frequent usage of these food additives. Therefore, further research is warranted to elucidate the role of carrageenan and CMC in intestinal inflammation, which may help identify novel nutritional strategies that hinder the development of the disease or prevent disease relapse post-EEN treatment. PMID:28507982

Species differences in the pharmacokinetics of cefadroxil as determined in wildtype and humanized PepT1 mice.

PubMed

Hu, Yongjun; Smith, David E

2016-05-01

PepT1 (SLC15A1) is a high-capacity low-affinity transporter that is important in the absorption of digested di/tripeptides from dietary protein in the small intestine. PepT1 is also crucial for the intestinal uptake and absorption of therapeutic agents such as the β-lactam aminocephalosporins and antiviral prodrugs. Species differences, however, have been observed in PepT1-mediated intestinal absorption and pharmacokinetics, thereby, making it more difficult to predict systemic drug exposure. In the present study, we evaluated the in situ intestinal permeability of the PepT1 substrate cefadroxil in wildtype and humanized PepT1 (huPepT1) mice, and the in vivo absorption and disposition of drug after escalating oral doses. The in situ perfusions indicated that cefadroxil had a twofold higher affinity (i.e., twofold lower Km) for jejunal PepT1 in huPepT1 mice, lower but substantial permeability in all regions of the small intestine, and low but measureable permeability in the colon as compared to wildtype animals. The in vivo experiments indicated almost superimposable pharmacokinetic profiles between the two genotypes after intravenous bolus dosing of cefadroxil. In contrast, after oral dose escalation, the systemic exposure of cefadroxil was reduced in huPepT1 mice as compared to wildtype animals. Moreover, the AUC and Cmax versus dose relationships were nonlinear for huPepT1 but not wildtype mice, and similar to that observed from human subjects. In conclusion, our findings indicate that huPepT1 mice may provide a valuable tool in the drug discovery process by better predicting the oral pharmacokinetic profiles of PepT1 substrates in humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Human Intestinal Fluid Layer Separation: The Effect On Colloidal Structures & Solubility Of Lipophilic Compounds.

PubMed

Danny, Riethorst; Amitava, Mitra; Filippos, Kesisoglou; Wei, Xu; Jan, Tack; Joachim, Brouwers; Patrick, Augustijns

2018-05-23

In addition to individual intestinal fluid components, colloidal structures are responsible for enhancing the solubility of lipophilic compounds. The present study investigated the link between as well as the variability in the ultrastructure of fed state human intestinal fluids (FeHIF) and their solubilizing capacity for lipophilic compounds. For this purpose, FeHIF samples from 10 healthy volunteers with known composition and ultrastructure were used to determine the solubility of four lipophilic compounds. In light of the focus on solubility and ultrastructure, the study carefully considered the methodology of solubility determination in relation to colloid composition and solubilizing capacity of FeHIF. To determine the solubilizing capacity of human and simulated intestinal fluids, the samples were saturated with the compound of interest, shaken for 24 h, and centrifuged. When using FeHIF, solubilities were determined in the micellar layer of FeHIF, i.e. after removing the upper (lipid) layer (standard procedure), as well as in 'full' FeHIF (without removal of the upper layer). Compound concentrations were determined using HPLC-UV/fluorescence. To link the solubilizing capacity with the ultrastructure, all human and simulated fluids were imaged using transmission electron microscopy (TEM) before and after centrifugation and top layer (lipid) removal. Comparing the ultrastructure and solubilizing capacity of individual FeHIF samples demonstrated a high intersubject variability in postprandial intestinal conditions. Imaging of FeHIF after removal of the upper layer clearly showed that only micellar structures remain in the lower layer. This observation suggests that larger colloids such as vesicles and lipid droplets are contained in the upper, lipid layer. The solubilizing capacity of most FeHIF samples substantially increased with inclusion of this lipid layer. The relative increase in solubilizing capacity upon inclusion of the lipid layer was most pronounced in samples that contained mainly vesicles alongside the micelles. Current fed state simulated intestinal fluids do not contain the larger colloids observed in the lipid layer of FeHIF and can only simulate the solubilizing capacity of the micellar layer of FeHIF. While the importance of drug molecules solubilized in the micellar layer of postprandial intestinal fluids for absorption has been extensively demonstrated previously, the in-vivo relevance of drug solubilization in the lipid layer is currently unclear. In the dynamic environment of the human gastrointestinal tract, drug initially entrapped in larger postprandial colloids may become available for absorption upon lipid digestion and uptake. The current study, demonstrating the substantial solubilization of lipophilic compounds in the larger colloids of postprandial intestinal fluids, warrants further research in this field. Copyright © 2018. Published by Elsevier B.V.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21*

PubMed Central

Basu, Nirmalya; Saha, Sayanti; Khan, Imran; Ramachandra, Subbaraya G.; Visweswariah, Sandhya S.

2014-01-01

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c−/−, mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence. PMID:24217248

Intimin, tir, and shiga toxin 1 do not influence enteropathogenic responses to shiga toxin-producing Escherichia coli in bovine ligated intestinal loops.

PubMed

Stevens, Mark P; Marchès, Olivier; Campbell, June; Huter, Veronika; Frankel, Gad; Phillips, Alan D; Oswald, Eric; Wallis, Timothy S

2002-02-01

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.

Bacillus megaterium SF185 induces stress pathways and affects the cell cycle distribution of human intestinal epithelial cells.

PubMed

Di Luccia, B; D'Apuzzo, E; Varriale, F; Baccigalupi, L; Ricca, E; Pollice, A

2016-09-01

The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

Kaiso overexpression promotes intestinal inflammation and potentiates intestinal tumorigenesis in Apc(Min/+) mice.

PubMed

Pierre, Christina C; Longo, Joseph; Mavor, Meaghan; Milosavljevic, Snezana B; Chaudhary, Roopali; Gilbreath, Ebony; Yates, Clayton; Daniel, Juliet M

2015-09-01

Constitutive Wnt/β-catenin signaling is a key contributor to colorectal cancer (CRC). Although inactivation of the tumor suppressor adenomatous polyposis coli (APC) is recognized as an early event in CRC development, it is the accumulation of multiple subsequent oncogenic insults facilitates malignant transformation. One potential contributor to colorectal carcinogenesis is the POZ-ZF transcription factor Kaiso, whose depletion extends lifespan and delays polyp onset in the widely used Apc(Min/+) mouse model of intestinal cancer. These findings suggested that Kaiso potentiates intestinal tumorigenesis, but this was paradoxical as Kaiso was previously implicated as a negative regulator of Wnt/β-catenin signaling. To resolve Kaiso's role in intestinal tumorigenesis and canonical Wnt signaling, we generated a transgenic mouse model (Kaiso(Tg/+)) expressing an intestinal-specific myc-tagged Kaiso transgene. We then mated Kaiso(Tg/+) and Apc(Min/+) mice to generate Kaiso(Tg/+):Apc(Min/+) mice for further characterization. Kaiso(Tg/+):Apc(Min/+) mice exhibited reduced lifespan and increased polyp multiplicity compared to Apc(Min/+) mice. Consistent with this murine phenotype, we found increased Kaiso expression in human CRC tissue, supporting a role for Kaiso in human CRC. Interestingly, Wnt target gene expression was increased in Kaiso(Tg/+):Apc(Min/+) mice, suggesting that Kaiso's function as a negative regulator of canonical Wnt signaling, as seen in Xenopus, is not maintained in this context. Notably, Kaiso(Tg/+):Apc(Min/+) mice exhibited increased inflammation and activation of NFκB signaling compared to their Apc(Min/+) counterparts. This phenotype was consistent with our previous report that Kaiso(Tg/+) mice exhibit chronic intestinal inflammation. Together our findings highlight a role for Kaiso in promoting Wnt signaling, inflammation and tumorigenesis in the mammalian intestine. Copyright © 2015 Elsevier B.V. All rights reserved.

Controlling Plasma Stability of Hydroxamic Acids: A MedChem Toolbox.

PubMed

Hermant, Paul; Bosc, Damien; Piveteau, Catherine; Gealageas, Ronan; Lam, BaoVy; Ronco, Cyril; Roignant, Matthieu; Tolojanahary, Hasina; Jean, Ludovic; Renard, Pierre-Yves; Lemdani, Mohamed; Bourotte, Marilyne; Herledan, Adrien; Bedart, Corentin; Biela, Alexandre; Leroux, Florence; Deprez, Benoit; Deprez-Poulain, Rebecca

2017-11-09

Hydroxamic acids are outstanding zinc chelating groups that can be used to design potent and selective metalloenzyme inhibitors in various therapeutic areas. Some hydroxamic acids display a high plasma clearance resulting in poor in vivo activity, though they may be very potent compounds in vitro. We designed a 57-member library of hydroxamic acids to explore the structure-plasma stability relationships in these series and to identify which enzyme(s) and which pharmacophores are critical for plasma stability. Arylesterases and carboxylesterases were identified as the main metabolic enzymes for hydroxamic acids. Finally, we suggest structural features to be introduced or removed to improve stability. This work thus provides the first medicinal chemistry toolbox (experimental procedures and structural guidance) to assess and control the plasma stability of hydroxamic acids and realize their full potential as in vivo pharmacological probes and therapeutic agents. This study is particularly relevant to preclinical development as it allows obtaining compounds equally stable in human and rodent models.

Role of T cell TGF beta signaling in intestinal cytokine responses and helminthic immune modulation

USDA-ARS?s Scientific Manuscript database

Colonization with helminthic parasites down-regulates inflammation in murine colitis and improves activity scores in human inflammatory bowel disease. Helminths induce mucosal regulatory T cells, which are important for intestinal immunologic homeostasis. Regulatory T cell function involves cytoki...

Reframing the Teenage Wasteland: Adolescent Microbiota-Gut-Brain Axis.

PubMed

McVey Neufeld, Karen-Anne; Luczynski, Pauline; Dinan, Timothy G; Cryan, John F

2016-04-01

Human adolescence is arguably one of the most challenging periods of development. The young adult is exposed to a variety of stressors and environmental stimuli on a backdrop of significant physiological change and development, which is especially apparent in the brain. It is therefore unsurprising that many psychiatric disorders are first observable during this time. The human intestine is inhabited by trillions of microorganisms, and evidence from both preclinical and clinical research focusing on the established microbiota-gut-brain axis suggests that the etiology and pathophysiology of psychiatric disorders may be influenced by intestinal dysbiosis. Provocatively, many if not all of the challenges faced by the developing teen have a documented impact on these intestinal commensal microbiota. In this review, we briefly summarize what is known about the developing adolescent brain and intestinal microbiota, discuss recent research investigating the microbiota-gut-brain axis during puberty, and propose that pre- and probiotics may prove useful in both the prevention and treatment of psychiatric disorders specifically benefitting the young adult. © The Author(s) 2016.

Reframing the Teenage Wasteland: Adolescent Microbiota-Gut-Brain Axis

PubMed Central

McVey Neufeld, Karen-Anne; Luczynski, Pauline; Dinan, Timothy G.

2016-01-01

Human adolescence is arguably one of the most challenging periods of development. The young adult is exposed to a variety of stressors and environmental stimuli on a backdrop of significant physiological change and development, which is especially apparent in the brain. It is therefore unsurprising that many psychiatric disorders are first observable during this time. The human intestine is inhabited by trillions of microorganisms, and evidence from both preclinical and clinical research focusing on the established microbiota-gut-brain axis suggests that the etiology and pathophysiology of psychiatric disorders may be influenced by intestinal dysbiosis. Provocatively, many if not all of the challenges faced by the developing teen have a documented impact on these intestinal commensal microbiota. In this review, we briefly summarize what is known about the developing adolescent brain and intestinal microbiota, discuss recent research investigating the microbiota-gut-brain axis during puberty, and propose that pre- and probiotics may prove useful in both the prevention and treatment of psychiatric disorders specifically benefitting the young adult. PMID:27254413

Effect of biopolymer encapsulation on the digestibility of lipid and cholesterol oxidation products in beef during in vitro human digestion.

PubMed

Hur, Sun Jin; Lee, Seung Yuan; Lee, Seung-Jae

2015-01-01

In this study, beef patties were encapsulated with 3% chitosan, pectin, onion powder, or green tea powder and the beef patties were then passed through an in vitro human digestion model. The total lipid digestibility was lowest (p<0.05) in beef patties encapsulated with chitosan and pectin after digestion in the small intestine. Thiobarbituric acid reactive substance (TBARS) values were significantly lower (p<0.05) for beef patties encapsulated with chitosan and pectin, when compared with the control, after digestion in the small intestine. In contrast, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical-scavenging activity was highest (p<0.05) in beef patties encapsulated with onion powder and green tea powder after digestion in the small intestine. The total cholesterol oxidation product (COP) content was significantly lower (p<0.05) in beef patties encapsulated with biopolymers than in the control after digestion in the small intestine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism.

PubMed

Jernberg, Cecilia; Sullivan, Asa; Edlund, Charlotta; Jansson, Janet K

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

PubMed Central

Jernberg, Cecilia; Sullivan, Åsa; Edlund, Charlotta; Jansson, Janet K.

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic. PMID:15640226

Antimicrobial Use, Human Gut Microbiota and Clostridium difficile Colonization and Infection

PubMed Central

Vincent, Caroline; Manges, Amee R.

2015-01-01

Clostridium difficile infection (CDI) is the most important cause of nosocomial diarrhea. Broad-spectrum antimicrobials have profound detrimental effects on the structure and diversity of the indigenous intestinal microbiota. These alterations often impair colonization resistance, allowing the establishment and proliferation of C. difficile in the gut. Studies involving animal models have begun to decipher the precise mechanisms by which the intestinal microbiota mediates colonization resistance against C. difficile and numerous investigations have described gut microbiota alterations associated with C. difficile colonization or infection in human subjects. Fecal microbiota transplantation (FMT) is a highly effective approach for the treatment of recurrent CDI that allows the restoration of a healthy intestinal ecosystem via infusion of fecal material from a healthy donor. The recovery of the intestinal microbiota after FMT has been examined in a few reports and work is being done to develop custom bacterial community preparations that could be used as a replacement for fecal material. PMID:27025623

Nardilysin controls intestinal tumorigenesis through HDAC1/p53-dependent transcriptional regulation.

PubMed

Kanda, Keitaro; Sakamoto, Jiro; Matsumoto, Yoshihide; Ikuta, Kozo; Goto, Norihiro; Morita, Yusuke; Ohno, Mikiko; Nishi, Kiyoto; Eto, Koji; Kimura, Yuto; Nakanishi, Yuki; Ikegami, Kanako; Yoshikawa, Takaaki; Fukuda, Akihisa; Kawada, Kenji; Sakai, Yoshiharu; Ito, Akihiro; Yoshida, Minoru; Kimura, Takeshi; Chiba, Tsutomu; Nishi, Eiichiro; Seno, Hiroshi

2018-04-19

Colon cancer is a complex disease affected by a combination of genetic and epigenetic factors. Here we demonstrate that nardilysin (N-arginine dibasic convertase; NRDC), a metalloendopeptidase of the M16 family, regulates intestinal tumorigenesis via its nuclear functions. NRDC is highly expressed in human colorectal cancers. Deletion of the Nrdc gene in ApcMin mice crucially suppressed intestinal tumor development. In ApcMin mice, epithelial cell-specific deletion of Nrdc recapitulated the tumor suppression observed in Nrdc-null mice. Moreover, epithelial cell-specific overexpression of Nrdc significantly enhanced tumor formation in ApcMin mice. Notably, epithelial NRDC controlled cell apoptosis in a gene dosage-dependent manner. In human colon cancer cells, nuclear NRDC directly associated with HDAC1, and controlled both acetylation and stabilization of p53, with alterations of p53 target apoptotic factors. These findings demonstrate that NRDC is critically involved in intestinal tumorigenesis through its epigenetic regulatory function, and targeting NRDC may lead to a novel prevention or therapeutic strategy against colon cancer.

SURVEY OF HOUSE RAT INTESTINAL PARASITES FROM SURABAYA DISTRICT, EAST JAVA, INDONESIA THAT CAN CAUSE OPPORTUNISTIC INFECTIONS IN HUMANS.

PubMed

Prasetyo, R H

2016-03-01

The purpose of this study was to investigate the prevalence of house rat zoonotic intestinal parasites from Surabaya District, East Java, Indonesia that have the potential to cause opportunistic infection in humans. House rat fecal samples were collected from an area of Surabaya District with a dense rat population during May 2015. Intestinal parasites were detected microscopically using direct smear of feces stained with Lugol's iodine and modified Ziehl-Neelsen stains. The fecal samples were also cultured for Strongyloides stercoralis. Ninety-eight house rat fecal samples were examined. The potential opportunistic infection parasite densities found in those samples were Strongyloides stercoralis in 53%, Hymenolepis nana in 42%, Cryptosporidium spp in 33%, and Blastocystis spp in 6%. This is the first report of this kind in Surabaya District. Measures need to be taken to control the house rat population in the study area to reduce the risk of the public health problem. Keywords: zoonotic intestinal parasites, opportunistic infection, house rat, densely populated area, Indonesia

Biotransformation of 1-nitropyrene to 1-aminopyrene and N-formyl-1-aminopyrene by the human intestinal microbiota

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manning, B.W.; Cerniglia, C.E.; Federle, T.W.

The nitropolycyclic aromatic hydrocarbon 1-nitropyrene (1-NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1-NP by the human intestinal microbiota was studied using a semicontinuous culture system that simulates the colonic lumen. (/sup 3/H)-1-Nitropyrene was metabolized by the intestinal microbiota to 1-aminopyrene (1-AP) and N-formyl-1-aminopyrene (FAP) as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. Twenty-four hours after the addition of (/sup 3/H)-1-NP, the formylated compound and 1-AP accounted for 20 and 80% of the total metabolism respectively. This percentage increased to 66% for FAP after 24 h following 10 dmore » of chronic exposure to unlabeled 1-NP, suggesting metabolic adaptation to 1-NP by the microbiota. Both 1-AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1-NP.« less

Engineered Commensal Bacteria Reprogram Intestinal Cells Into Glucose-Responsive Insulin-Secreting Cells for the Treatment of Diabetes

PubMed Central

Duan, Franklin F.; Liu, Joy H.

2015-01-01

The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1–secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25–33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)–secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo. PMID:25626737

Rifaximin Reduces the Number and Severity of Intestinal Lesions Associated With Use of Nonsteroidal Anti-Inflammatory Drugs in Humans.

PubMed

Scarpignato, Carmelo; Dolak, Werner; Lanas, Angel; Matzneller, Peter; Renzulli, Cecilia; Grimaldi, Maria; Zeitlinger, Markus; Bjarnason, Ingvar

2017-04-01

The intestinal microbiota might contribute to enteropathy associated with use of nonsteroidal anti-inflammatory drugs (NSAIDs), but there have been few human studies of this association. We performed a placebo-controlled study to determine whether a delayed-release antibiotic formulation (rifaximin-extended intestinal release [EIR]) prevents the development of intestinal lesions in subjects taking daily NSAIDs. Sixty healthy volunteers (median age, 26 y; 42% female) were given the NSAID diclofenac (75 mg twice daily) plus omeprazole (20 mg once daily), and either rifaximin-EIR (400 mg) or placebo, twice daily for 14 days. Subjects were assessed by videocapsule endoscopy at baseline and after 2 weeks of treatment. The primary end point was the proportion of subjects developing at least 1 small-bowel mucosal break at week 2. Secondary end points were the change in the mean number of mucosal lesions and the number of subjects with large erosions and/or ulcers after 14 days of exposure. We detected mucosal breaks in 20% of subjects given rifaximin and in 43% of subjects given placebo (P = .05 in the post hoc sensitivity analysis). None of the subjects in the rifaximin group developed large lesions, compared with 9 subjects in the placebo group (P < .001). Our findings indicate that intestinal bacteria contribute to the development of NSAID-associated enteropathy in human beings. Clinical trial no: EudraCT 2013-000730-36. Copyright © 2017. Published by Elsevier Inc.

Intestinal protozoa are hypothesized to stimulate immunosurveillance against colon cancer.

PubMed

Juckett, David A; Aylsworth, Charles F; Quensen, Janet Murphy

2008-01-01

Colon cancer in humans results in considerable morbidity and mortality throughout most of the world. During the twentieth century, there was a rapid rise in colon cancer within modernizing countries that has not been adequately explained, although the role of diet has been widely explored. Previously, we showed that the presence of the endemic Eimeria spp. protozoan in intestinal tissues is associated with regions of low tumorigenesis in the large and small bovine intestine and that an Eimeria surface protein is a potent activator of dendritic cells and a useful immunomodulator, with anti-cancer and anti-viral properties. Therefore, we hypothesize that the persistent presence of such an intestinal protozoan enhances immunosurveillance by elevating the intestinal alert status and that the loss of these organisms could lead to a higher incidence of colon cancer. Preliminary support of this hypothesis derives from the observations that domestic animals, known to maintain this protozoan, have very low colon cancer incidence. We propose that this also may occur in human populations that use human excrement (night soil) as a fertilizer, a practice that serves to complete the life cycle of this type of microbe. We examine some evidence for this hypothesis in Japan's mortality patterns, where we show that colon cancer increased after the cessation of night soil use, but before the change to a western diet. We conclude that this hypothesis, a variation of the hygiene hypothesis, is worth further consideration and continued elaboration.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Oral, gastric and intestinal influences on human feeding.

PubMed

French, S J; Cecil, J E

2001-01-01

Direct infusion of specific nutrients or foods into different areas of the gastrointestinal tract, and techniques to distend the stomach, are useful tools enabling eating behaviour to be studied without the influence of orosensory factors. Using these techniques, the role of nutrients on gastrointestinal mechanisms of satiation as well as the interactions between the various systems in the control of feeding can be examined. Recent research in humans investigating the relative contribution of signals arising from different areas of the gastrointestinal tract has demonstrated that optimal control of appetite occurs only when orosensory signals are coupled with signals arising from the stomach and intestine. Interactions between gastric and intestinal signals do however combine to produce a more potent suppression of appetite and food intake than when either of these sites is stimulated alone. Gastric distension probably exerts a predominate influence on appetite suppression compared with intestinal stimulation, whereas nutrient stimulation of the intestine may function to modulate the sensations arising from gastric distension to elicit a meal-like sensation of satiety. In addition, these studies have highlighted that previous hypotheses concerning differential fat and carbohydrate satiation may require an orosensory component and probably do not reflect an inherent difference in the physiological effects of these macronutrients at the level of the GI tract. The interaction of orosensory influences, such as palatability, with negative feedback signals arising from the GI tract can be further studied using a combination of these techniques with measurement of microstructure of feeding in humans.

Diabetes-induced mechanophysiological changes in the small intestine and colon

PubMed Central

Zhao, Mirabella; Liao, Donghua; Zhao, Jingbo

2017-01-01

The disorders of gastrointestinal (GI) tract including intestine and colon are common in the patients with diabetes mellitus (DM). DM induced intestinal and colonic structural and biomechanical remodeling in animals and humans. The remodeling is closely related to motor-sensory abnormalities of the intestine and colon which are associated with the symptoms frequently encountered in patients with DM such as diarrhea and constipation. In this review, firstly we review DM-induced histomorphological and biomechanical remodeling of intestine and colon. Secondly we review motor-sensory dysfunction and how they relate to intestinal and colonic abnormalities. Finally the clinical consequences of DM-induced changes in the intestine and colon including diarrhea, constipation, gut microbiota change and colon cancer are discussed. The final goal is to increase the understanding of DM-induced changes in the gut and the subsequent clinical consequences in order to provide the clinicians with a better understanding of the GI disorders in diabetic patients and facilitates treatments tailored to these patients. PMID:28694926

A role for the epidermal growth factor receptor signaling in development of intestinal serrated polyps in mice and humans.

PubMed

Bongers, Gerold; Muniz, Luciana R; Pacer, Michelle E; Iuga, Alina C; Thirunarayanan, Nanthakumar; Slinger, Erik; Smit, Martine J; Reddy, E Premkumar; Mayer, Lloyd; Furtado, Glaucia C; Harpaz, Noam; Lira, Sergio A

2012-09-01

Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to proinflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages.

PubMed

Zheng, Shasha; Hedl, Matija; Abraham, Clara

2015-02-15

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-β-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating proinflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment. Copyright © 2015 by The American Association of Immunologists, Inc.

Paracetamol absorption from different sites in the human small intestine.

PubMed Central

Gramatté, T; Richter, K

1994-01-01

Site-specificity in the small intestinal absorption of paracetamol was investigated using a segmental intestinal steady state perfusion technique (triple-lumen tubing system) combined with simultaneous measurements of serum drug concentrations. Dissolved paracetamol was perfused over 160 min into different parts of the small intestine (65-210 cm beyond the teeth). Each of the four healthy subjects was studied twice with a proximal and a more distal site of perfusion. Serum drug concentrations were similar after proximal and distal perfusions. Mean drug absorption rates calculated from intestinal aspirate concentrations were similar in both parts of the intestine--proximal: 869 micrograms 30 cm-1 min-1 (95% CI: 659-1079) vs distal: 941 micrograms 30 cm-1 min-1 (794-1088). The absorption rate was related directly to the amount of paracetamol perfused per unit time as well as to the rate of transmucosal water fluxes. PMID:7917782

Thyroid hormone regulation of adult intestinal stem cells: Implications on intestinal development and homeostasis.

PubMed

Sun, Guihong; Roediger, Julia; Shi, Yun-Bo

2016-12-01

Organ-specific adult stem cells are essential for organ homeostasis, tissue repair and regeneration. The formation of such stem cells often takes place during postembryonic development, a period around birth in mammals when plasma thyroid hormone concentration is high. The life-long self-renewal of the intestinal epithelium has made mammalian intestine a valuable model to study the function and regulation and adult stem cells. On the other hand, much less is known about how the adult intestinal stem cells are formed during vertebrate development. Here, we will review some recent progresses on this subject, focusing mainly on the formation of the adult intestine during Xenopus metamorphosis. We will discuss the role of thyroid hormone signaling pathway in the process and potential molecular conservations between amphibians and mammals as well as the implications in organ homeostasis and human diseases.

Interactions of Giardia sp. with the intestinal barrier: Epithelium, mucus, and microbiota

PubMed Central

Amat, Christina B.; Buret, André G.

2017-01-01

ABSTRACT Understanding how intestinal enteropathogens cause acute and chronic alterations has direct animal and human health perspectives. Significant advances have been made on this field by studies focusing on the dynamic crosstalk between the intestinal protozoan parasite model Giardia duodenalis and the host intestinal mucosa. The concept of intestinal barrier function is of the highest importance in the context of many gastrointestinal diseases such as infectious enteritis, inflammatory bowel disease, and post-infectious gastrointestinal disorders. This crucial function relies on 3 biotic and abiotic components, first the commensal microbiota organized as a biofilm, then an overlaying mucus layer, and finally the tightly structured intestinal epithelium. Herein we review multiple strategies used by Giardia parasite to circumvent these 3 components. We will summarize what is known and discuss preliminary observations suggesting how such enteropathogen directly and/ or indirectly impairs commensal microbiota biofilm architecture, disrupts mucus layer and damages host epithelium physiology and survival. PMID:28452685

Use of Methacrylic Acid-Containing Hydrogels to Increase Protein Transport Across the Intestinal Epithelium

NASA Astrophysics Data System (ADS)

Blanchette, James; Lopez, Jennifer; Park, Kinam; Peppas, Nicholas

2002-03-01

Oral protein delivery requires protection from the harsh environment of the stomach, release in the small intestine and passage from the intestinal lumen into the circulation. Hydrogels that swell in response to the pH change when passing from the stomach to the small intestine can accomplish the first two points. The ability to enhance the permeability of intestinal epithelial cells is currently under investigation. Methacrylic acid-containing hydrogels have shown the ability to bind calcium ions that decreases the concentration of free extracellular calcium for these epithelial cells. This change triggers a number of intracellular events including rearrangement of the cytoskeleton leading to increased permeability. Studies done on Caco-2 cells (human colon adenocarcinoma) measuring changes in transepithelial resistance are used to assess the effect of the polymer-cell interactions on the integrity of intestinal epithelial cell monolayers.

Molecular Hydrogen as an Emerging Therapeutic Medical Gas for Neurodegenerative and Other Diseases

PubMed Central

Ohno, Kinji; Ito, Mikako; Ichihara, Masatoshi; Ito, Masafumi

2012-01-01

Effects of molecular hydrogen on various diseases have been documented for 63 disease models and human diseases in the past four and a half years. Most studies have been performed on rodents including two models of Parkinson's disease and three models of Alzheimer's disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral hypoxia; Parkinson's disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome, hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects. Two enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further studies are required to elucidate molecular bases of prominent hydrogen effects and to determine the optimal frequency, amount, and method of hydrogen administration for each human disease. PMID:22720117

Anti-inflammatory and anti-allergic properties of donkey's and goat's milk.

PubMed

Jirillo, Felicita; Magrone, Thea

2014-03-01

Nowadays, donkey's and goat's milk consumption has been reevaluated for its potential benefits to human health. For example, in infants with intolerance to cow's milk, donkey's milk represents a good alternative due to its chemical characteristics similar to those of human milk. On the other hand, goat's milk in virtue of its higher content in short chain, medium chain, mono and polyunsaturated fatty acids than that of cow's milk, is more digestible than the bovine counterpart. From an immunological point of view, donkey's milk is able to induce release of inflammatory and anti-inflammatory cytokines from normal human peripheral blood lymphomononuclear cells, thus maintaining a condition of immune homeostasis. Similarly, goat's milk has been shown to trigger innate and adaptive immune responses in an in vitro human system, also inhibiting the endotoxin-induced activation of monocytes. Finally, in these milks the presence of their own microbiota may normalize the human intestinal microbiota with a cascade of protective effects at intestinal mucosal sites, even including triggering of intestinal T regulatory cells. In the light of the above considerations, donkey's and goat's milk should be recommended as a dietary supplement in individuals with inflammatory and allergic conditions, even including elderly people.

Roles of amino acids in preventing and treating intestinal diseases: recent studies with pig models.

PubMed

Liu, Yulan; Wang, Xiuying; Hou, Yongqing; Yin, Yulong; Qiu, Yinsheng; Wu, Guoyao; Hu, Chien-An Andy

2017-08-01

Animal models are needed to study and understand a human complex disease. Because of their similarities in anatomy, structure, physiology, and pathophysiology, the pig has proven its usefulness in studying human gastrointestinal diseases, such as inflammatory bowel disease, ischemia/reperfusion injury, diarrhea, and cancer. To understand the pathogenesis of these diseases, a number of experimental models generated in pigs are available, for example, through surgical manipulation, chemical induction, microbial infection, and genetic engineering. Our interests have been using amino acids as therapeutics in pig and human disease models. Amino acids not only play an important role in protein biosynthesis, but also exert significant physiological effects in regulating immunity, anti-oxidation, redox regulation, energy metabolism, signal transduction, and animal behavior. Recent studies in pigs have shown that specific dietary amino acids can improve intestinal integrity and function under normal and pathological conditions that protect the host from different diseases. In this review, we summarize several pig models in intestinal diseases and how amino acids can be used as therapeutics in treating pig and human diseases.

Human mesenchymal stromal cells decrease mortality after intestinal ischemia and reperfusion injury.

PubMed

Markel, Troy A; Crafts, Trevor D; Jensen, Amanda R; Hunsberger, Erin Bailey; Yoder, Mervin C

2015-11-01

Cellular therapy is a novel treatment option for intestinal ischemia. Bone marrow-derived mesenchymal stromal cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion (I/R) injury. We therefore hypothesized that (1) human BMSCs (hBMSCs) would produce more beneficial growth factors and lower levels of proinflammatory mediators compared to differentiated cells, (2) direct application of hBMSCs to ischemic intestine would decrease mortality after injury, and (3) decreased mortality would be associated with an altered intestinal and hepatic inflammatory response. Adult hBMSCs and keratinocytes were cultured on polystyrene flasks. For in vitro experiments, cells were exposed to tumor necrosis factor, lipopolysaccharides, or 2% oxygen for 24 h. Supernatants were then analyzed for growth factors and chemokines by multiplex assay. For in vivo experiments, 8- to 12-wk-old male C57Bl6J mice were anesthetized and underwent a midline laparotomy. Experimental groups were exposed to temporary superior mesenteric artery occlusion for 60 min. Immediately after ischemia, 2 × 10(6) hBMSCs or keratinocytes in phosphate-buffered saline were placed into the peritoneal cavity. Animals were then closed and allowed to recover for 6 h (molecular/histologic analysis) or 7 d (survival analysis). After 6-h reperfusion, animals were euthanized. Intestines and livers were harvested and analyzed for inflammatory chemokines, growth factors, and histologic changes. hBMSCs expressed higher levels of human interleukin (IL) 6, IL-8, vascular endothelial growth factor (VEGF), and epidermal growth factor and lower levels of IL-1, IL-3, IL-7, and granulocyte-monocyte colony-stimulating factor after stimulation. In vivo, I/R resulted in significant mortality (70% mortality), whereas application of hBMSCs after ischemia decreased mortality to 10% in a dose-dependent fashion (P = 0.004). Keratinocyte therapy offered no improvements in mortality above I/R. Histologic profiles were equivalent between ischemic groups, regardless of the application of hBMSCs or keratinocytes. Cellular therapy yielded significantly decreased murine intestinal levels of soluble activin receptor-like kinase 1, betacellulin, and endothelin, whereas increasing levels of eotaxin, monokine induced by gamma interferon (MIG), monocyte chemoattractant protein 1, IL-6, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein 10 (IP-10) from ischemia were appreciated. hBMSC therapy yielded significantly higher expression of murine intestinal VEGF and lower levels of intestinal MIG compared to keratinocyte therapy. Application of hBMSCs after ischemia yielded significantly lower murine levels of hepatic MIG, IP-10, and G-CSF compared to keratinocyte therapy. Human BMSCs produce multiple beneficial growth factors. Direct application of hBMSCs to the peritoneal cavity after intestinal I/R decreased mortality by 60%. Improved outcomes with hBMSC therapy were not associated with improved histologic profiles in this model. hBMSC therapy was associated with higher VEGF in intestines and lower levels of proinflammtory MIG, IP-10, and G-CSF in liver tissue after ischemia, suggesting that reperfusion with hBMSC therapy may alter survival by modulating the systemic inflammatory response to ischemia. Copyright © 2015 Elsevier Inc. All rights reserved.

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

PubMed

Duchmann, R; Kaiser, I; Hermann, E; Mayet, W; Ewe, K; Meyer zum Büschenfelde, K H

1995-12-01

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non-inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co-culture with sonicates of bacteria from autologous intestine (BsA). Proliferation was inhibitable by anti-MHC class II MoAb, suggesting that it was driven by antigen. LPMC from adjacent non-inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non-inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate. PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co-culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL-12, interferon-gamma (IFN-gamma), and IL-10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD.

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

PubMed Central

Duchmann, R; Kaiser, I; Hermann, E; Mayet, W; Ewe, K; Meyer zum Büschenfelde, K H

1995-01-01

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non-inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co-culture with sonicates of bacteria from autologous intestine (BsA). Proliferation was inhibitable by anti-MHC class II MoAb, suggesting that it was driven by antigen. LPMC from adjacent non-inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non-inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate. PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co-culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL-12, interferon-gamma (IFN-gamma), and IL-10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD. PMID:8536356

The colon revisited or the key to wellness, health and disease.

PubMed

Gonzalez-Correa, C A; Mulett-Vásquez, E; Miranda, D A; Gonzalez-Correa, C H; Gómez-Buitrago, P A

2017-10-01

The hypothesis being advanced in this paper is that there is a new medical paradigm emerging from the biomedical research carried out in this century, mainly due to the explosion of the so called "omics" and associated techniques. The main idea is that there is a common pathway from wellbeing and health to chronic disease ("chronopathy") and even to death, which comprises following steps: 1) unhealthy diet, sedentary life style and permanent exposition to xenobiotics and all kinds of noxious stimuli;→2) intestinal dysbiosis;→3) alteration of the intestinal mucus layer (especially that of the colon);→4) disruption of the endothelial tight junctions;→5) metabolic endotoxemia+bacterial translocation;→6) inflammation;→7) exacerbation of the enteric nervous system (ENS) and consequent maladaptation and malfunctioning of the colon;→8) epigenetic manifestations;→9) "chronopathy" and premature death. Therefore, in order to maintain a good health or to improve or even reverse chronic diseases in a person, the main outcome to look for is a homeostatic balance of the intestinal microbiota (eubiosis), most of which is located in the colon. Lynn Margulis was one of the main scientists to highlight the importance of the role played by bacteria not only in the origin of all biological species now present on earth, but also on their role in global homeostasis. Bacteria do not rely on other living beings for their existence, while the latter depend completely on the former. Humans are no exemption, and new evidence emerges each day about the pivotal role of intestinal microbiota in human health, disease and, in general, in its wellbeing. The following facts about intestinal microbiota are nowadays generally accepted: there are about 10 times more bacteria in the gut than human cells in every human being; the microbioma is about 100-150 times bigger that the human genome, and there is a clear link between intestinal microbiota and many of the most common chronic diseases, from obesity and diabetes to depression and Parkinson disease and different kinds of cancer. The main implication of this theory is that we should become a sort of microbiota farmers, that is, we ought to be more conscious of our intestinal microbiota, take care of it and monitor it permanently. Thus, as part of our good life habits (healthy eating, physical exercise), we should probably undergo periodic seasons of fasting and colon cleansing, as it was common in older times. Copyright © 2017 Elsevier Ltd. All rights reserved.

The transcriptomic profiles of Giardia duodenalis infected calves

USDA-ARS?s Scientific Manuscript database

Giardia duodenalis is one of the most commonly found intestinal pathogens in humans and animals. However, little is known about the host-parasite interaction in its natural hosts. The objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, us...

Effect of environmental contaminants in the Mississippi River Basin on carboxylesterases from four aquatic species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.; Huang, T.; Obih, P.

1995-12-31

The objectives of this study are to investigate the sensitivity of different classes of esterases in various aquatic species to environmental contaminants and the possible use of these enzymes as biomarkers for monitoring the effects of pollutants. Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the non-specific carboxylesterases (CaE) were analyzed in three fish species, Ictiobus bubalus (small mouth buffalo), Ictiobus cyprinellus (big mouth buffalo) and Lepisosteus oculatus (spotted gar) and the green tree frog, Hyla cinerea. These samples were collected from the Devil`s Swamp Site (DSS), an industrial site known to be highly contaminated at the Mississippi River Basin, and Lake Tunica,more » a nonindustrial site. ACHE and BuChE activities in the subcellular fractions of liver and brain were significantly lower in fishes and frogs obtained from DSS when compared to the same species obtained from Tunica swamp site. The greatest decrease was observed with ACHE activity in the liver and brain of Ictiobus bubalus from DSS. CaE activity analyzed with p-nitrophenyl acetate was found to be significantly lower in the liver of all three fish species collected from DSS when compared to the same fish species obtained from the Tunica swamp site.« less

Identification and Characterization of Carboxylesterases from Brachypodium distachyon Deacetylating Trichothecene Mycotoxins

PubMed Central

Schmeitzl, Clemens; Varga, Elisabeth; Warth, Benedikt; Kugler, Karl G.; Malachová, Alexandra; Michlmayr, Herbert; Wiesenberger, Gerlinde; Mayer, Klaus F. X.; Mewes, Hans-Werner; Krska, Rudolf; Schuhmacher, Rainer; Berthiller, Franz; Adam, Gerhard

2015-01-01

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease. PMID:26712789

The Intestinal Microbiota in Colorectal Cancer.

PubMed

Tilg, Herbert; Adolph, Timon E; Gerner, Romana R; Moschen, Alexander R

2018-06-11

Experimental evidence from the past years highlights a key role for the intestinal microbiota in inflammatory and malignant gastrointestinal diseases. Diet exhibits a strong impact on microbial composition and provides risk for developing colorectal carcinoma (CRC). Large metagenomic studies in human CRC associated microbiome signatures with the colorectal adenoma-carcinoma sequence, suggesting a fundamental role of the intestinal microbiota in the evolution of gastrointestinal malignancy. Basic science established a critical function for the intestinal microbiota in promoting tumorigenesis. Further studies are needed to decipher the mechanisms of tumor promotion and microbial co-evolution in CRC, which may be exploited therapeutically in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

INTESTINAL ALKALINE PHOSPHATASE: A SUMMARY OF ITS ROLE IN CLINICAL DISEASE

PubMed Central

Fawley, Jason; Gourlay, David

2016-01-01

Over the past few years, there is increasing evidence implicating a novel role for Intestinal Alkaline Phosphatase (IAP) in mitigating inflammatory mediated disorders. IAP is an endogenous protein expressed by the intestinal epithelium that is believed to play a vital role in maintaining gut homeostasis. Loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation and subsequently systemic inflammation. As these events are a cornerstone of the pathophysiology of many diseases relevant to surgeons, we sought to review recent research in both animal and humans on IAP’s physiologic function, mechanisms of action and current research in specific surgical diseases. PMID:27083970

Contribution of gut bacteria to the metabolism of cyanidin 3-glucoside in human microbiota-associated rats.

PubMed

Hanske, Laura; Engst, Wolfram; Loh, Gunnar; Sczesny, Silke; Blaut, Michael; Braune, Annett

2013-04-28

Cyanidin 3-glucoside (C3G) is one of the major dietary anthocyanins implicated in the prevention of chronic diseases. To evaluate the impact of human intestinal bacteria on the fate of C3G in the host, we studied the metabolism of C3G in human microbiota-associated (HMA) rats in comparison with germ-free (GF) rats. Urine and faeces of the rats were analysed for C3G and its metabolites within 48 h after the application of 92 μmol C3G/kg body weight. In addition, we tested the microbial C3G conversion in vitro by incubating C3G with human faecal slurries and selected human gut bacteria. The HMA rats excreted with faeces a three times higher percentage of unconjugated C3G products and a two times higher percentage of conjugated C3G products than the GF rats. These differences were mainly due to the increased excretion of 3,4-dihydroxybenzoic acid, 2,4,6-trihydroxybenzaldehyde and 2,4,6-trihydroxybenzoic acid. Only the urine of HMA rats contained peonidin and 3-hydroxycinnamic acid and the percentage of conjugated C3G products in the urine was decreased compared with the GF rats. Overall, the presence of intestinal microbiota resulted in a 3·7% recovery of the C3G dose in HMA rats compared with 1·7% in GF rats. Human intestinal bacteria rapidly degraded C3G in vitro. Most of the C3G products were also found in the absence of bacteria, but at considerably lower levels. The higher concentrations of phenolic acids observed in the presence of intestinal bacteria may contribute to the proposed beneficial health effects of C3G.

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

PubMed Central

Gufford, Brandon T.; Chen, Gang; Lazarus, Philip; Graf, Tyler N.; Oberlies, Nicholas H.

2014-01-01

Drug-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Furanocoumarins in grapefruit juice are cornerstone examples of diet-derived xenobiotics that perpetrate interactions with drugs via mechanism-based inhibition of intestinal CYP3A4. Relative to intestinal CYP3A4-mediated inhibition, alternate mechanisms underlying dietary substance–drug interactions remain understudied. A working systematic framework was applied to a panel of structurally diverse diet-derived constituents/extracts (n = 15) as inhibitors of intestinal UDP-glucuronosyl transferases (UGTs) to identify and characterize additional perpetrators of dietary substance–drug interactions. Using a screening assay involving the nonspecific UGT probe substrate 4-methylumbelliferone, human intestinal microsomes, and human embryonic kidney cell lysates overexpressing gut-relevant UGT1A isoforms, 14 diet-derived constituents/extracts inhibited UGT activity by >50% in at least one enzyme source, prompting IC50 determination. The IC50 values of 13 constituents/extracts (≤10 μM with at least one enzyme source) were well below intestinal tissue concentrations or concentrations in relevant juices, suggesting that these diet-derived substances can inhibit intestinal UGTs at clinically achievable concentrations. Evaluation of the effect of inhibitor depletion on IC50 determination demonstrated substantial impact (up to 2.8-fold shift) using silybin A and silybin B, two key flavonolignans from milk thistle (Silybum marianum) as exemplar inhibitors, highlighting an important consideration for interpretation of UGT inhibition in vitro. Results from this work will help refine a working systematic framework to identify dietary substance–drug interactions that warrant advanced modeling and simulation to inform clinical assessment. PMID:25008344

A Metagenomics Investigation of Carbohydrate-Active Enzymes along the Gastrointestinal Tract of Saudi Sheep.

PubMed

Al-Masaudi, Saad; El Kaoutari, Abdessamad; Drula, Elodie; Al-Mehdar, Hussein; Redwan, Elrashdy M; Lombard, Vincent; Henrissat, Bernard

2017-01-01

The digestive microbiota of humans and of a wide range of animals has recently become amenable to in-depth studies due to the emergence of DNA-based metagenomic techniques that do not require cultivation of gut microbes. These techniques are now commonly used to explore the feces of humans and animals under the assumption that such samples are faithful proxies for the intestinal microbiota. Sheep ( Ovis aries ) are ruminant animals particularly adapted to life in arid regions and in particular Najdi, Noaimi (Awassi), and Harrei (Harri) breeds that are raised in Saudi Arabia for milk and/or meat production. Here we report a metagenomics investigation of the distal digestive tract of one animal from each breed that (i) examines the microbiota at three intestinal subsites (small intestine, mid-colon, and rectum), (ii) performs an in-depth analysis of the carbohydrate-active enzymes genes encoded by the microbiota at the three subsites, and (iii) compares the microbiota and carbohydrate-active enzyme profile at the three subsites across the different breeds. For all animals we found that the small intestine is characterized by a lower taxonomic diversity than that of the large intestine and of the rectal samples. Mirroring this observation, we also find that the spectrum of encoded carbohydrate-active enzymes of the mid-colon and rectal sites is much richer than that of the small intestine. However, the number of encoded cellulases and xylanases in the various intestinal subsites was found to be surprisingly low, indicating that the bulk of the fiber digestion is performed upstream in the rumen, and that the carbon source for the intestinal flora is probably constituted of the rumen fungi and bacteria that pass in the intestines. In consequence we argue that ruminant feces, which are often analyzed for the search of microbial genes involved in plant cell wall degradation, are probably a poor proxy for the lignocellulolytic potential of the host.

Gut symbiotic microbes imprint intestinal immune cells with the innate receptor SLAMF4 which contributes to gut immune protection against enteric pathogens.

PubMed

Cabinian, Allison; Sinsimer, Daniel; Tang, May; Jang, Youngsoon; Choi, Bongkum; Laouar, Yasmina; Laouar, Amale

2018-05-01

Interactions between host immune cells and gut microbiota are crucial for the integrity and function of the intestine. How these interactions regulate immune cell responses in the intestine remains a major gap in the field. We have identified the signalling lymphocyte activation molecule family member 4 (SLAMF4) as an immunomodulator of the intestinal immunity. The aim is to determine how SLAMF4 is acquired in the gut and what its contribution to intestinal immunity is. Expression of SLAMF4 was assessed in mice and humans. The mechanism of induction was studied using GFP tg bone marrow chimaera mice, lymphotoxin α and TNLG8A-deficient mice, as well as gnotobiotic mice. Role in immune protection was revealed using oral infection with Listeria monocytogenes and Cytobacter rodentium . SLAMF4 is a selective marker of intestinal immune cells of mice and humans. SLAMF4 induction occurs directly in the intestinal mucosa without the involvement of the gut-associated lymphoid tissue. Gut bacterial products, particularly those of gut anaerobes, and gut-resident antigen-presenting cell (APC) TNLG8A are key contributors of SLAMF4 induction in the intestine. Importantly, lack of SLAMF4 expression leads the increased susceptibility of mice to infection by oral pathogens culminating in their premature death. SLAMF4 is a marker of intestinal immune cells which contributes to the protection against enteric pathogens and whose expression is dependent on the presence of the gut microbiota. This discovery provides a possible mechanism for answering the long-standing question of how the intertwining of the host and gut microbial biology regulates immune cell responses in the gut. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis

PubMed Central

Donaldson, David S.; Else, Kathryn J.

2015-01-01

ABSTRACT Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease. IMPORTANCE Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence. PMID:26157121

Human Intestinal Barrier Function in Health and Disease

PubMed Central

König, Julia; Wells, Jerry; Cani, Patrice D; García-Ródenas, Clara L; MacDonald, Tom; Mercenier, Annick; Whyte, Jacqueline; Troost, Freddy; Brummer, Robert-Jan

2016-01-01

The gastrointestinal tract consists of an enormous surface area that is optimized to efficiently absorb nutrients, water, and electrolytes from food. At the same time, it needs to provide a tight barrier against the ingress of harmful substances, and protect against a reaction to omnipresent harmless compounds. A dysfunctional intestinal barrier is associated with various diseases and disorders. In this review, the role of intestinal permeability in common disorders such as infections with intestinal pathogens, inflammatory bowel disease, irritable bowel syndrome, obesity, celiac disease, non-celiac gluten sensitivity, and food allergies will be discussed. In addition, the effect of the frequently prescribed drugs proton pump inhibitors and non-steroidal anti-inflammatory drugs on intestinal permeability, as well as commonly used methods to assess barrier function will be reviewed. PMID:27763627

Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

PubMed

Dominguez-Brauer, Carmen; Hao, Zhenyue; Elia, Andrew J; Fortin, Jérôme M; Nechanitzky, Robert; Brauer, Patrick M; Sheng, Yi; Mana, Miyeko D; Chio, Iok In Christine; Haight, Jillian; Pollett, Aaron; Cairns, Robert; Tworzyanski, Leanne; Inoue, Satoshi; Reardon, Colin; Marques, Ana; Silvester, Jennifer; Cox, Maureen A; Wakeham, Andrew; Yilmaz, Omer H; Sabatini, David M; van Es, Johan H; Clevers, Hans; Sato, Toshiro; Mak, Tak W

2016-08-04

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche. Copyright © 2016 Elsevier Inc. All rights reserved.

Th2 and Th1 Responses: Clear and Hidden Sides of Immunity Against Intestinal Helminths.

PubMed

Cortés, Alba; Muñoz-Antoli, Carla; Esteban, J Guillermo; Toledo, Rafael

2017-09-01

Intestinal helminthiases affect millions of people worldwide, mainly in developing regions, where they cause a significant negative impact on human health and socioeconomic growth of affected populations. However, intestinal helminthiases are still among the most neglected tropical diseases. Protective immunity against intestinal helminths is associated with development of type 2 responses. Nevertheless, in some host-intestinal helminth combinations, local Th1 responses are initiated, inducing chronicity. The usage of helminth-mouse models is useful for elucidating the mechanisms behind the initiation of each type of response. Herein, the current knowledge on these topics is reviewed, paying particular attention to the earliest events of the immune cascade which eventually lead to either susceptibility or resistance to infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

The magnitude and risk factors of intestinal parasitic infection in relation to Human Immunodeficiency Virus infection and immune status, at ALERT Hospital, Addis Ababa, Ethiopia.

PubMed

Taye, Biruhalem; Desta, Kassu; Ejigu, Selamawit; Dori, Geme Urge

2014-06-01

Human Immunodeficiency Virus (HIV) and intestinal parasitic infections are among the main health problems in developing countries like Ethiopia. Particularly, co-infections of these diseases would worsen the progression of HIV to Acquired Immunodeficiency Syndrome (AIDS). The purpose of this study was to determine the magnitude and risk factors for intestinal parasites in relation to HIV infection and immune status. The study was conducted in (1) HIV positive on antiretroviral therapy (ART) and (2) ART naïve HIV positive patients, and (3) HIV-negative individuals, at All African Leprosy and Tuberculosis (TB) Eradication and Rehabilitation Training Center (ALERT) hospital in Addis Ababa, Ethiopia. Study participants were interviewed using structured questionnaires to obtain socio-demographic characteristics and assess risk factors associated with intestinal parasitic infection. Intestinal parasites were identified from fecal samples by direct wet mount, formol ether concentration, and modified Ziehl-Neelsen staining techniques. The immune status was assessed by measuring whole blood CD4 T-cell count. The overall magnitude of intestinal parasite was 35.08%. This proportion was different among study groups with 39.2% (69/176), 38.83% (40/103) and 27.14% (38/140) in ART naïve HIV positives patients, in HIV negatives, and in HIV positive on ART patients respectively. HIV positive patients on ART had significantly lower magnitude of intestinal parasitic infection compared to HIV negative individuals. Intestinal helminths were significantly lower in HIV positive on ART and ART naïve patients than HIV negatives. Low monthly income, and being married, divorced or widowed were among the socio-demographic characteristics associated with intestinal parasitic infection. No association was observed between the magnitude of intestinal parasites and CD4 T-cell count. However, Cryptosporidium parvum, and Isospora belli were exclusively identified in individuals with CD4 T-cell count of ≤ 350 cells/mm(3). Regular provision of mass preventive chemotherapy and extended health education will curb the burden of intestinal parasitic infection in the community. Emphasis should also be given to laboratory diagnosis and identification of opportunistic intestinal parasites in patients with lower CD4-Tcell count. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Prevention of Necrotizing Enterocolitis Through Manipulation of the Intestinal Microbiota of the Premature Infant

PubMed Central

Vongbhavit, Kannikar; Underwood, Mark A.

2016-01-01

In spite of four decades of research, necrotizing enterocolitis (NEC) remains the most common gastrointestinal complication in premature infants with high mortality and long-term morbidity. The composition of the intestinal microbiota of the premature infant differs dramatically from that of the healthy term infant and appears to be an important risk factor for NEC. Promising NEC prevention strategies that alter the intestinal microbiota include probiotics, prebiotics, synbiotics, lacteroferrin, and human milk feeding. PMID:26872618

21 CFR 876.5900 - Ostomy pouch and accessories.

Code of Federal Regulations, 2012 CFR

2012-04-01

....5900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... created opening of the small intestine, large intestine, or the ureter on the surface of the body). This... bag, ostomy drainage bag with adhesive, stomal bag, ostomy protector, and the ostomy size selector...

21 CFR 876.5900 - Ostomy pouch and accessories.

Code of Federal Regulations, 2013 CFR

2013-04-01

....5900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... created opening of the small intestine, large intestine, or the ureter on the surface of the body). This... bag, ostomy drainage bag with adhesive, stomal bag, ostomy protector, and the ostomy size selector...

21 CFR 876.5900 - Ostomy pouch and accessories.

Code of Federal Regulations, 2014 CFR

2014-04-01

....5900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... created opening of the small intestine, large intestine, or the ureter on the surface of the body). This... bag, ostomy drainage bag with adhesive, stomal bag, ostomy protector, and the ostomy size selector...

21 CFR 876.5900 - Ostomy pouch and accessories.

Code of Federal Regulations, 2011 CFR

2011-04-01

....5900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... created opening of the small intestine, large intestine, or the ureter on the surface of the body). This... bag, ostomy drainage bag with adhesive, stomal bag, ostomy protector, and the ostomy size selector...

Intestinal threonine utilization for protein and mucin synthesis is decreased in formula-fed preterm pigs

USDA-ARS?s Scientific Manuscript database

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin (MUC2) to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against ...

TREATMENT OF LONG-EVANS RATS WITH A DEFINED MIXTURE OF DRINKING WATER DISINFECTION BY-PRODUCTS IMPACTS INTESTINAL MICROBIAL METABOLISM.

EPA Science Inventory

Water treatment results in the production of numerous halogenated disinfection by-products (DBPs), and has been associated with human colorectal cancer. Because the intestinal microbiota can bioactivate promutagens and procarcinogens, several studies have been done to examine the...

Omics for Understanding the Gut-Liver-Microbiome Axis and Precision Medicine

USDA-ARS?s Scientific Manuscript database

Human metabolic disease opens a new view to understanding the contribution of the intestinal microbiome to drug metabolism and drug-induced toxicity in gut-liver function. Gut microbiota, a key determinant of intestinal inflammation, also plays a direct role in chronic inflammation and liver disease...

Chemical form of selenium affects its uptake, transport, and glutathione peroxidase activity in the human intestinal Caco-2 cell model.

PubMed

Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

2011-11-01

Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

ILSI Southeast Asia Region conference proceedings: The gut, its microbes and health: relevance for Asia.

PubMed

Lee, Yuan Kun; Conway, Patricia; Pettersson, Sven; Nair, G Balakrish; Surono, Ingrid; Egayanti, Yusra; Amarra, Maria Sofia

2017-01-01

The human being is a complex entity, involving interaction between microbes and the human host. Evidence shows that the nutritional value of food is influenced in part by the structure and operations of an individual's gut microbial community, and food in turn shapes the individual's microbiome. A conference was held to promote understanding of the intestinal microbiome and its implications for health and disease, particularly among Asian populations. Papers describing 1) the intestinal ecosystem in Asian populations, 2) changes in intestinal microbiota through life and its effects, 3) the Asian gut microbiota in disease conditions, 4) indigenous probiotics to maintain a healthy gut microbiota, 5) probiotic regulation in an Asian country, and 6) the results of a panel discussion are included in this report. The gut microbial inhabitants of Asian people differ from those of Europe and North America. Geographic location, diet, and ethnic background influence intestinal microbial composition. Urbanization and economic development have brought changes in traditional Asian diets, which in turn affected the gut microbiome, contributing to a shift in the region's health burden from infectious diseases to non-communicable chronic diseases. Novel probiotic strains of Indonesian origin demonstrated significant enhancement of humoral immune response in human studies. Knowledge gaps and implications for research to further understand the Asian gut microbiome were discussed.

A role for antimicrobial peptides in intestinal microsporidiosis

PubMed Central

Leitch, Gordon J.; Ceballos, Carolina

2009-01-01

SUMMARY Clinical isolates from three microsporidia species, Encephalitozoon intestinalis and Encephalitozoon hellem, and the insect parasite Anncaliia (Brachiola, Nosema) algerae, were used in spore germination and enterocyte-like (C2Bbe1) cell infection assays to determine the effect of a panel of antimicrobial peptides. Spores were incubated with lactoferrin (Lf), lysozyme (Lz), and human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1), alone and in combination with Lz, prior to germination. Of the Encephalitozoon species only E. hellem spore germination was inhibited by HNP1, while A. algerae spore germination was inhibited by Lf, HBD2, HD5 and HNP1, although HBD2 and HD5 inhibition required the presence of Lz. The effects of HBD2 and HD5 on microsporidia enterocyte infection paralleled their effects on spore germination. Lysozyme alone only inhibited infection with A. algerae, while Lf inhibited infection by E. intestinalis and A. algerae. HNP1 significantly reduced enterocyte infection by all three parasite species and a combination of Lf, Lz and HNP1 caused a further reduced infection with A. algerae. These data suggest that intestinal antimicrobial peptides contribute to the defense of the intestine against infection by luminal microsporidia spores and may partially determine which parasite species infects the intestine. PMID:19079820

Interactions between parasites and microbial communities in the human gut.

PubMed

Berrilli, Federica; Di Cave, David; Cavallero, Serena; D'Amelio, Stefano

2012-01-01

The interactions between intestinal microbiota, immune system, and pathogens describe the human gut as a complex ecosystem, where all components play a relevant role in modulating each other and in the maintenance of homeostasis. The balance among the gut microbiota and the human body appear to be crucial for health maintenance. Intestinal parasites, both protozoans and helminths, interact with the microbial community modifying the balance between host and commensal microbiota. On the other hand, gut microbiota represents a relevant factor that may strongly interfere with the pathophysiology of the infections. In addition to the function that gut commensal microbiota may have in the processes that determine the survival and the outcome of many parasitic infections, including the production of nutritive macromolecules, also probiotics can play an important role in reducing the pathogenicity of many parasites. On these bases, there is a growing interest in explaining the rationale on the possible interactions between the microbiota, immune response, inflammatory processes, and intestinal parasites.

Interactions between parasites and microbial communities in the human gut

PubMed Central

Berrilli, Federica; Di Cave, David; Cavallero, Serena; D'Amelio, Stefano

2012-01-01

The interactions between intestinal microbiota, immune system, and pathogens describe the human gut as a complex ecosystem, where all components play a relevant role in modulating each other and in the maintenance of homeostasis. The balance among the gut microbiota and the human body appear to be crucial for health maintenance. Intestinal parasites, both protozoans and helminths, interact with the microbial community modifying the balance between host and commensal microbiota. On the other hand, gut microbiota represents a relevant factor that may strongly interfere with the pathophysiology of the infections. In addition to the function that gut commensal microbiota may have in the processes that determine the survival and the outcome of many parasitic infections, including the production of nutritive macromolecules, also probiotics can play an important role in reducing the pathogenicity of many parasites. On these bases, there is a growing interest in explaining the rationale on the possible interactions between the microbiota, immune response, inflammatory processes, and intestinal parasites. PMID:23162802

Generation of a human CDX2 knock-in reporter iPSC line (MHHi007-A-1) to model human trophoblast differentiation.

PubMed

Malysheva, Svitlana V; Wunderlich, Stephanie; Haase, Alexandra; Göhring, Gudrun; Martin, Ulrich; Merkert, Sylvia

2018-05-19

Caudal-type homeobox 2 (CDX2) transcription factor is an important marker for early trophoblast lineages and intestinal epithelium. Due to its nuclear expression the immunostaining and sorting of viable CDX2 pos cells is not possible. In this paper we report the generation and describe key characteristics of a CDX2 Venus knock-in reporter hiPSC-cell line (MHHi007-A-1) which can serve as an in vitro tool to study human trophoblast and intestinal differentiation. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

[Physiological patterns of intestinal microbiota. The role of dysbacteriosis in obesity, insulin resistance, diabetes and metabolic syndrome].

PubMed

Halmos, Tamás; Suba, Ilona

2016-01-03

The intestinal microbiota is well-known for a long time, but due to newly recognized functions, clinician's attention has turned to it again in the last decade. About 100 000 billion bacteria are present in the human intestines. The composition of bacteriota living in diverse parts of the intestinal tract is variable according to age, body weight, geological site, and diet as well. Normal bacteriota defend the organism against the penetration of harmful microorganisms, and has many other functions in the gut wall integrity, innate immunity, insulin sensitivity, metabolism, and it is in cross-talk with the brain functions as well. It's a recent recognition, that intestinal microbiota has a direct effect on the brain, and the brain also influences the microbiota. This two-way gut-brain axis consists of microbiota, immune and neuroendocrine system, as well as of the autonomic and central nervous system. Emerging from fermentation of carbohydrates, short-chain fatty acids develop into the intestines, which produce butyrates, acetates and propionates, having favorable effects on different metabolic processes. Composition of the intestinal microbiota is affected by the circadian rhythm, such as in shift workers. Dysruption of circadian rhythm may influence intestinal microbiota. The imbalance between the microbiota and host organism leads to dysbacteriosis. From the membrane of Gram-negative bacteria lipopolysacharides penetrate into the blood stream, via impaired permeability of the intestinal mucosa. These processes induce metabolic endotoxaemia, inflammation, impaired glucose metabolism, insulin resistance, obesity, and contribute to the development of metabolic syndrome, type 2 diabetes, inflammarory bowel diseases, autoimmunity and carcinogenesis. Encouraging therapeutic possibility is to restore the normal microbiota either using pro- or prebiotics, fecal transplantation or bariatric surgery. Human investigations seem to prove that fecal transplant from lean healthy individuals into obese diabetic patients improved all the pathological parameters. Wide spread use of bariatric surgery altered gut microbiota and improved metabolic parameters apart from surgery itself. Pathomechanism is not yet completely clarified. Clinicians hope, that deeper understanding of complex functions of intestinal microbiota will contribute to develop more effective therapeutic proceedings against diabetes, metabolic syndrome, and obesity.

New approaches to increase intestinal length: Methods used for intestinal regeneration and bioengineering

PubMed Central

Shirafkan, Ali; Montalbano, Mauro; McGuire, Joshua; Rastellini, Cristiana; Cicalese, Luca

2016-01-01

Inadequate absorptive surface area poses a great challenge to the patients suffering a variety of intestinal diseases causing short bowel syndrome. To date, these patients are managed with total parenteral nutrition or intestinal transplantation. However, these carry significant morbidity and mortality. Currently, by emergence of tissue engineering, anticipations to utilize an alternative method to increase the intestinal absorptive surface area are increasing. In this paper, we will review the improvements made over time in attempting elongating the intestine with surgical techniques as well as using intestinal bioengineering. Performing sequential intestinal lengthening was the preliminary method applied in humans. However, these methods did not reach widespread use and has limited outcome. Subsequent experimental methods were developed utilizing scaffolds to regenerate intestinal tissue and organoids unit from the intestinal epithelium. Stem cells also have been studied and applied in all types of tissue engineering. Biomaterials were utilized as a structural support for naive cells to produce bio-engineered tissue that can achieve a near-normal anatomical structure. A promising novel approach is the elongation of the intestine with an acellular biologic scaffold to generate a neo-formed intestinal tissue that showed, for the first time, evidence of absorption in vivo. In the large intestine, studies are more focused on regeneration and engineering of sphincters and will be briefly reviewed. From the review of the existing literature, it can be concluded that significant progress has been achieved in these experimental methods but that these now need to be fully translated into a pre-clinical and clinical experimentation to become a future viable therapeutic option. PMID:27011901

Comparison of the Effects of Iodine and Iodide on Thyroid Function in Humans

NASA Technical Reports Server (NTRS)

Robison, Linda M.; Bull, Richard J.; Sylvester, Paul W.; Birkenfeld, Paul; Lang, Jerome

1995-01-01

The present experiment in humans failed to confirm the differential effect of I(sub 2) on maintenance of serum T(sub 4) concentrations relative to the effects of I(-) that was observed in prior experiments in rats. The reaction of I(sub 2) with metabolites of thyroid hormones in the intestine that appears responsible for this effect in rats probably also exists at some level in humans. The present results suggest that the concentrations of such metabolites in the human intestinal tract are too small to significantly affect circulating concentration of T(sub 4). However, based on the elevations in TSH, there should be some concern over the potential impacts of chronic consumption of iodine in drinking water.

A Human Strain of Oxalobacter (HC-1) Promotes Enteric Oxalate Secretion in the Small Intestine of Mice and Reduces Urinary Oxalate Excretion

PubMed Central

Hatch, Marguerite; Freel, Robert W.

2013-01-01

Enteric oxalate secretion that correlated with reductions in urinary oxalate excretion was previously reported in a mouse model of Primary Hyperoxaluria, and in wild type (WT) mice colonized with a wild rat strain (OXWR) of Oxalobacter (Am J Physiol 300: G461-G469, 2011). Since a human strain of the bacterium is more likely to be clinically used as a probiotic therapeutic, we tested the effects of HC-1 in WT. Following artificial colonization of WT mice with HC-1, the bacteria were confirmed to be present in the large intestine and, unexpectedly, detected in the small intestine for varying periods of time. The main objective of the present study was to determine whether the presence of HC-1 promoted intestinal secretion in the more proximal segments of the gastrointestinal tract. In addition, we determined whether HC-1 colonization led to reductions in urinary oxalate excretion in these mice. The results show that the human Oxalobacter strain promotes a robust net secretion of oxalate in the distal ileum as well as in the caecum and distal colon and these changes in transport correlate with the beneficial effect of reducing renal excretion of oxalate. We conclude that OXWR effects on intestinal oxalate transport and oxalate homeostasis are not unique to the wild rat strain and that, mechanistically, HC-1 has significant potential for use as a probiotic treatment for hyperoxaluria especially if it is also targeted to the upper and lower gastrointestinal tract. PMID:23959075

The pancreas responds to remote damage and systemic stress by secretion of the pancreatic secretory proteins PSP/regI and PAP/regIII.

PubMed

Reding, Theresia; Palmiere, Cristian; Pazhepurackel, Clinsyjos; Schiesser, Marc; Bimmler, Daniel; Schlegel, Andrea; Süss, Ursula; Steiner, Sabrina; Mancina, Leandro; Seleznik, Gitta; Graf, Rolf

2017-05-02

In patients with infection and sepsis serum levels of Pancreatic Stone protein/regenerating protein I (PSP) are highly elevated. The origin of PSP during these conditions is presumably the pancreas, however, an intestinal origin cannot be excluded. Similarly, pancreatitis-associated protein (PAP) was identified in the pancreas. These proteins were also localized in intestinal organs. Here we aim to elucidate the bio-distribution of PSP and PAP in animal models of sepsis and in healthy humans. PSP and PAP responded to remote lesions in rats although the pancreatic response was much more pronounced than the intestinal. Tissue distribution of PSP demonstrated a 100-fold higher content in the pancreas compared to any other organ while PAP was most abundant in the small intestine. Both proteins responded to CLP or sham operation in the pancreas. PSP also increased in the intestine during CLP. The distribution of PSP and PAP in human tissue mirrored the distribution in the murine models. Distribution of PSP and PAP was visualized by immunohistochemistry. Rats and mice underwent midline laparotomies followed by mobilization of tissue and incision of the pancreatic duct or duodenum. Standard cecum-ligation-puncture (CLP) procedures or sham laparotomies were performed. Human tissue extracts were analyzed for PSP and PAP. The pancreas reacts to remote lesions and septic insults in mice and rats with increased PSP synthesis, while PAP is selectively responsive to septic events. Furthermore, our results suggest that serum PSP in septic patients is predominantly derived through an acute phase response of the pancreas.

Production of bioactive substances by intestinal bacteria as a basis for explaining probiotic mechanisms: bacteriocins and conjugated linoleic acid.

PubMed

O'Shea, Eileen F; Cotter, Paul D; Stanton, Catherine; Ross, R Paul; Hill, Colin

2012-01-16

The mechanisms by which intestinal bacteria achieve their associated health benefits can be complex and multifaceted. In this respect, the diverse microbial composition of the human gastrointestinal tract (GIT) provides an almost unlimited potential source of bioactive substances (pharmabiotics) which can directly or indirectly affect human health. Bacteriocins and fatty acids are just two examples of pharmabiotic substances which may contribute to probiotic functionality within the mammalian GIT. Bacteriocin production is believed to confer producing strains with a competitive advantage within complex microbial environments as a consequence of their associated antimicrobial activity. This has the potential to enable the establishment and prevalence of producing strains as well as directly inhibiting pathogens within the GIT. Consequently, these antimicrobial peptides and the associated intestinal producing strains may be exploited to beneficially influence microbial populations. Intestinal bacteria are also known to produce a diverse array of health-promoting fatty acids. Indeed, certain strains of intestinal bifidobacteria have been shown to produce conjugated linoleic acid (CLA), a fatty acid which has been associated with a variety of systemic health-promoting effects. Recently, the ability to modulate the fatty acid composition of the liver and adipose tissue of the host upon oral administration of CLA-producing bifidobacteria and lactobacilli was demonstrated in a murine model. Importantly, this implies a potential therapeutic role for probiotics in the treatment of certain metabolic and immunoinflammatory disorders. Such examples serve to highlight the potential contribution of pharmabiotic production to probiotic functionality in relation to human health maintenance. Copyright © 2011 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moon, Yuseok; Yang, Hyun; Kim, Yung Bu

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and inflammation and have also received considerable attention because of their preventive effects against human cancer. However, the drug application is sometimes limited by the severe gastrointestinal ulcers and mucosal complications. In the present study, NSAID sulindac sulfide was investigated for the cytotoxic injury in the intestinal epithelial cells in association with an immediate inducible factor, early growth response gene 1 (EGR-1). Previously we reported that sulindac sulfide can suppress tumor cell invasion by inducing EGR-1. Extending the previous study, EGR-1 induction by sulindac sulfide was observed both in the non-transformedmore » and transformed human intestinal epithelial cell lines. In terms of signaling pathway, ERK1/2 MAP kinases and its substrate Elk-1 transcription factor were involved in the sulindac sulfide-induced EGR-1 gene expression. Moreover, sulindac sulfide stimulated the nuclear translocation of the transcription factor EGR-1, which was also mediated by ERK1/2 signaling pathway. The roles of EGR-1 signals in the apoptotic cell death were assessed in the intestinal epithelial cells. Suppression of EGR-1 expression retarded cellular growth and colony forming activity in the intestinal epithelial cells. Moreover, induced EGR-1 ameliorated sulindac sulfide-mediated apoptotic cell death and enhanced the cellular survival. Taken all together, sulindac sulfide activated ERK1/2 MAP kinases which then mediated EGR-1 induction and nuclear translocation, all of which played important roles in the cellular survival from NSAID-mediated cytotoxicity in the human intestinal epithelial cells, implicating the protective roles of EGR-1 in the NSAID-mediated mucosal injuries.« less

The human milk oligosaccharide 2'-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine.

PubMed

Good, Misty; Sodhi, Chhinder P; Yamaguchi, Yukihiro; Jia, Hongpeng; Lu, Peng; Fulton, William B; Martin, Laura Y; Prindle, Thomas; Nino, Diego F; Zhou, Qinjie; Ma, Congrong; Ozolek, John A; Buck, Rachael H; Goehring, Karen C; Hackam, David J

2016-10-01

Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2'-fucosyllactose (2'FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2'FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2'FL to eNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2'FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2'FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2'FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2'FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC.

The human milk oligosaccharide 2′-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine

PubMed Central

Good, Misty; Sodhi, Chhinder P.; Yamaguchi, Yukihiro; Jia, Hongpeng; Lu, Peng; Fulton, William B.; Martin, Laura Y.; Prindle, Thomas; Nino, Diego F.; Zhou, Qinjie; Ma, Congrong; Ozolek, John A.; Buck, Rachael H.; Goehring, Karen C.; Hackam, David J.

2016-01-01

Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2′-fucosyllactose (2′FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2′FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2′FL to eNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2′FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2′FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2′FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2′FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC. PMID:27609061

Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota.

PubMed

Iraola, Gregorio; Forster, Samuel C; Kumar, Nitin; Lehours, Philippe; Bekal, Sadjia; García-Peña, Francisco J; Paolicchi, Fernando; Morsella, Claudia; Hotzel, Helmut; Hsueh, Po-Ren; Vidal, Ana; Lévesque, Simon; Yamazaki, Wataru; Balzan, Claudia; Vargas, Agueda; Piccirillo, Alessandra; Chaban, Bonnie; Hill, Janet E; Betancor, Laura; Collado, Luis; Truyers, Isabelle; Midwinter, Anne C; Dagi, Hatice T; Mégraud, Francis; Calleros, Lucía; Pérez, Ruben; Naya, Hugo; Lawley, Trevor D

2017-11-08

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.

Association between the gut microbiota and mineral metabolism.

PubMed

Skrypnik, Katarzyna; Suliburska, Joanna

2018-05-01

The aim of this review is to present the most recent scientific evidence of interactions between the intestinal microbiota and minerals, and the effect of this interaction on the health of the host. The Web of Science database from the years 2013-2017 on this topic was reviewed. Numerous in vitro studies have shown that iron significantly affects the intestinal microbiota. However, Bifidobacteriaceae are capable of binding iron in the large intestine, thereby limiting the formation of free radicals synthesized in the presence of iron, and thus reducing the risk of colorectal cancer. Animal studies have revealed that supplementation with probiotics, prebiotics and synbiotics has a significant effect on bone calcium, phosphate and bone metabolism. The dynamic interaction between microbiota and zinc was shown. Human studies have provided evidence of the influence of probiotic bacteria on parathormone, calcium and phosphate levels and thus on bone resorption. Recent studies have produced new information mainly on the impact of the intestinal bacteria on the metabolism of calcium and iron. From a scientific perspective, the most urgent fields that remain to be investigated are the identification of all human gut microbes and new therapies targeting the interaction between intestinal bacteria and minerals. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Modification of Intestinal Microbiota and Its Consequences for Innate Immune Response in the Pathogenesis of Campylobacteriosis

PubMed Central

Heimesaat, Markus M.; Bereswill, Stefan; Tareen, Abdul Malik; Lugert, Raimond; Groß, Uwe; Zautner, Andreas E.

2013-01-01

Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis in the world, and thus one of the most important public health concerns. The initial stage in its pathogenesis after ingestion is to overcome colonization resistance that is maintained by the human intestinal microbiota. But how it overcomes colonization resistance is unknown. Recently developed humanized gnotobiotic mouse models have provided deeper insights into this initial stage and host's immune response. These studies have found that a fat-rich diet modifies the composition of the conventional intestinal microbiota by increasing the Firmicutes and Proteobacteria loads while reducing the Actinobacteria and Bacteroidetes loads creating an imbalance that exposes the intestinal epithelial cells to adherence. Upon adherence, deoxycholic acid stimulates C. jejuni to synthesize Campylobacter invasion antigens, which invade the epithelial cells. In response, NF-κB triggers the maturation of dendritic cells. Chemokines produced by the activated dendritic cells initiate the clearance of C. jejuni cells by inducing the actions of neutrophils, B-lymphocytes, and various subsets of T-cells. This immune response causes inflammation. This review focuses on the progress that has been made on understanding the relationship between intestinal microbiota shift, establishment of C. jejuni infection, and consequent immune response. PMID:24324507

Lactobacillus paracasei subsp. paracasei LC01 positively modulates intestinal microflora in healthy young adults.

PubMed

Zhang, Hao; Sun, Jing; Liu, Xianting; Hong, Chuan; Zhu, Yuanbo; Liu, Aiping; Li, Siqi; Guo, Huiyuan; Ren, Fazheng

2013-12-01

Lactobacillus paracasei subsp. paracasei LC01 (LC01) can tolerate intestinal stresses and has antioxidant activity. To evaluate the effect of the bacterium on human intestinal microflora, a randomized, double-blind, placebo-controlled human trial was carried out. Fifty-two healthy adult volunteers were randomized equally to two groups. One group consumed 12% (wt/vol) skimmed milk supplemented with 10(10) CFU of LC01 each day for the 4-week treatment period, and then consumed placebo in the next treatment period, separated by a 2-week washout. The other group followed the reverse order. Group-specific real-time PCR and biochemical analyses was used to determine the intestinal bacterial composition of fecal samples collected at the end of every period, and the concentration of short-chain fatty acids and ammonia. A significant inhibition in fecal Escherichia coli and increase in Lactobacillus, Bifidobacterium, and Roseburia intestinalis were observed after consumption of LC01. Acetic acid and butyric acid were significantly higher in the probiotic stage and fecal ammonia was significantly lower. The results indicated a modulation effect of LC01 on the intestinal microflora of young adults, suggesting a beneficial effect on bowel health. LC01 may have potential value as a probiotic.

Identification of astilbin metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

PubMed

Zhao, Min; Xu, Jun; Qian, Dawei; Guo, Jianming; Jiang, Shu; Shang, Er-xin; Duan, Jin-ao

2014-07-01

Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(TM) software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8-2 and 9-2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2014 John Wiley & Sons, Ltd.

Decision trees to characterise the roles of permeability and solubility on the prediction of oral absorption.

PubMed

Newby, Danielle; Freitas, Alex A; Ghafourian, Taravat

2015-01-27

Oral absorption of compounds depends on many physiological, physiochemical and formulation factors. Two important properties that govern oral absorption are in vitro permeability and solubility, which are commonly used as indicators of human intestinal absorption. Despite this, the nature and exact characteristics of the relationship between these parameters are not well understood. In this study a large dataset of human intestinal absorption was collated along with in vitro permeability, aqueous solubility, melting point, and maximum dose for the same compounds. The dataset allowed a permeability threshold to be established objectively to predict high or low intestinal absorption. Using this permeability threshold, classification decision trees incorporating a solubility-related parameter such as experimental or predicted solubility, or the melting point based absorption potential (MPbAP), along with structural molecular descriptors were developed and validated to predict oral absorption class. The decision trees were able to determine the individual roles of permeability and solubility in oral absorption process. Poorly permeable compounds with high solubility show low intestinal absorption, whereas poorly water soluble compounds with high or low permeability may have high intestinal absorption provided that they have certain molecular characteristics such as a small polar surface or specific topology. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Intimin, Tir, and Shiga Toxin 1 Do Not Influence Enteropathogenic Responses to Shiga Toxin-Producing Escherichia coli in Bovine Ligated Intestinal Loops

PubMed Central

Stevens, Mark P.; Marchès, Olivier; Campbell, June; Huter, Veronika; Frankel, Gad; Phillips, Alan D.; Oswald, Eric; Wallis, Timothy S.

2002-01-01

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle. PMID:11796630

Helminths as an alternative therapy for intestinal diseases.

PubMed

Sipahi, Aytan Miranda; Baptista, Daniel Machado

2017-09-07

Animal models and clinical studies have shown that helminth infections exert immunomodulatory activity, altering intestinal permeability and providing a potential beneficial action on autoimmune and inflammatory disorders in human beings, such as inflammatory bowel disease (IBD) and celiac disease. This is consistent with the theory that intestinal microbiota is responsible for shaping human immunological responses. With the arrival of the immunobiologic era and the use of antibodies, we propose a distinctive pathway for treating patients with IBD and celiac disease. We have some evidence about the safety and tolerability of helminth use, but evidence about their impact on disease activity is lacking. Using worms to treat diseases could be a possible way to lower treatment costs, since the era of immunobiologic agents is responsible for a significant rise in expenses. Some questions remain to be investigated regarding the use of helminths in intestinal disease, such as the importance of the specific species of helminths used, appropriate dosing regimens, optimal timing of treatment, the role of host genetics, diet, environment, and the elucidation of the exact mechanisms of action. One promising approach is the use of helminth-derived anti-inflammatory molecules as drugs. Yet there are still many challenges with this method, especially with regard to safety. Studies on intestinal permeability point to Strongyloides stercoralis as a useful nematode for these purposes.

Opportunistic and non-opportunistic intestinal parasites in HIV/ AIDS patients in relation to their clinical and epidemiological status in a specialized medical service in Goiás, Brazil.

PubMed

Barcelos, Natane Barbosa; Silva, Lorena de Freitas E; Dias, Regyane Ferreira Guimarães; Menezes Filho, Hélio Ranes de; Rodrigues, Rosângela Maria

2018-03-08

Patients infected with the Human Immunodeficiency Virus (HIV) often have opportunistic infections, among which strongyloidiasis and coccidiosis are the most common parasitic infections that aggravate their health status. This study examined the prevalence of intestinal parasites, particularly of Strongyloides stercoralis and intestinal coccidia in patients with the Human Immunodeficiency Virus (HIV)/ Acquired Immunodeficiency Syndrome (AIDS) who were treated at the Specialized Assistance Service (SAE) of Jataí, State of Goiás, Brazil, and analyzed its correlation with clinical, laboratory, and socio-epidemiological parameters. A total of 270 stool samples were analyzed by the Lutz technique, Rugai's method, Agar Plate Culture, Ritchie's method and specific staining, Ziehl-Neelsen modified technique, Kinyoun's method and the rapid safranin method. The prevalence of intestinal parasites was 28.88% including 3.8% of S. stercoralis, Cryptosporidium sp. and Cystoisospora belli. There was a significant positive correlation between intestinal parasites and the clinical status and the use of antiretroviral therapy (ART), smoking, CD4+ lymphocyte counts and sexual orientation. In conclusion, the widespread use of antiretroviral therapy and health assistance contributed to the low prevalence of S. stercoralis and coccidiosis in patients with HIV/ AIDS who were followed up at the SAE.

The growth pattern of the human intestine and its mesentery.

PubMed

Soffers, Jelly H M; Hikspoors, Jill P J M; Mekonen, Hayelom K; Koehler, S Eleonore; Lamers, Wouter H

2015-08-22

It remains unclear to what extent midgut rotation determines human intestinal topography and pathology. We reinvestigated the midgut during its looping and herniation phases of development, using novel 3D visualization techniques. We distinguished 3 generations of midgut loops. The topography of primary and secondary loops was constant, but that of tertiary loops not. The orientation of the primary loop changed from sagittal to transverse due to the descent of ventral structures in a body with a still helical body axis. The 1st secondary loop (duodenum, proximal jejunum) developed intraabdominally towards a left-sided position. The 2nd secondary loop (distal jejunum) assumed a left-sided position inside the hernia before returning, while the 3rd and 4th secondary loops retained near-midline positions. Intestinal return into the abdomen resembled a backward sliding movement. Only after return, the 4th secondary loop (distal ileum, cecum) rapidly "slid" into the right lower abdomen. The seemingly random position of the tertiary small-intestinal loops may have a biomechanical origin. The interpretation of "intestinal rotation" as a mechanistic rather than a descriptive concept underlies much of the confusion accompanying the physiological herniation. We argue, instead, that the concept of "en-bloc rotation" of the developing midgut is a fallacy of schematic drawings. Primary, secondary and tertiary loops arise in a hierarchical fashion. The predictable position and growth of secondary loops is pre-patterned and determines adult intestinal topography. We hypothesize based on published accounts that malrotations result from stunted development of secondary loops.