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1.
Nutr Clin Pract ; 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31642109

RESUMO

BACKGROUND: Manganese toxicity can occur as a complication of home parenteral nutrition (HPN). Patients can present with Parkinson disease-like symptoms. Preparations of trace elements (TEs) in parenteral nutrition (PN) generally provide amounts in excess of requirements. Our previous review observed 60% of adult HPN patients had high whole-blood manganese levels. Multi-TE (MTE) solutions were subsequently removed from all HPN formulations in January 2015. The aim of this evaluation was to determine whole-blood concentrations of manganese in adult patients receiving HPN to establish whether levels are now maintained within the normal reference range. METHODS: A retrospective review of whole-blood manganese levels in all patients receiving HPN between January 2018 and January 2019 from 1 hospital site was carried out. RESULTS: 100 patients were included in the review (59 female and 41 male). Normal whole-blood manganese levels (73-219 nmol/L) were observed in 70% of patients and elevated levels (>219 nmol/L) in 30% of patients. In the patients with elevated levels, 57% had not received manganese supplementation for at least 1 year prior to manganese being measured. Markers of cholestasis were similar between the 2 groups. CONCLUSIONS: Incidence of elevated whole-blood manganese concentrations in patients receiving HPN decreased from 60% to 30% upon discontinued use of an MTE solution. Elevated levels remain a concern despite patients being prescribed "manganese-free" PN. Patients receive this TE in amounts adequate to meet requirements through contamination and dietary intake alone, suggesting additional parenteral supplementation of manganese is not required.

3.
BMC Microbiol ; 19(1): 52, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832576

RESUMO

BACKGROUND: Necrotising enterocolitis (NEC) is a common cause of death in preterm infants and is closely linked to the gut microbiota. Spontaneous intestinal perforation (SIP) also occurs in preterm neonates, but results in lower mortality and less adverse neonatal outcomes than NEC. Existing studies are largely limited to non-invasive stool samples, which may not be reflective of the anatomical site of disease. Therefore, we analysed historical formalin-fixed paraffin-embedded (FFPE) tissue from NEC and SIP preterm infants. A total of 13 NEC and 16 SIP infants were included. Extracted DNA from FFPE tissue blocks underwent 16S rRNA gene sequencing. For a subset of infants, diseased tissue and marginal healthy tissue from the same infant were compared. RESULTS: Xylene provided a cost and time effective means of deparaffinization. Tissue from the site of disease was highly comparable to adjacent healthier tissue. Comparing only diseased tissue from all infants showed significantly lower Shannon diversity in NEC (P = 0.026). The overall bacterial communities were also significantly different in NEC samples compared to SIP (P = 0.038), and large variability within NEC infants was observed. While no single OTU or genus was significantly associated with NEC or SIP, at the phylum level Proteobacteria (P = 0.045) and Bacteroidetes (P = 0.024) were significantly higher in NEC and SIP infants, respectively. CONCLUSIONS: Existing banks of intestinal FFPE blocks provide a robust and specific sample for profiling the microbiota at the site of disease. We showed preterm infants with NEC have lower diversity and different bacterial communities when compared to SIP controls.

4.
Sci Rep ; 9(1): 4710, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886164

RESUMO

Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitination.

6.
J Crohns Colitis ; 12(10): 1191-1199, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29912405

RESUMO

Background: Recent findings suggest that αE expression is enriched on effector T cells and that intestinal αE+ T cells have increased expression of inflammatory cytokines. αE integrin expression is a potential predictive biomarker for response to etrolizumab, a monoclonal antibody against ß7 integrin that targets both α4ß7 and αEß7. We evaluated the prevalence and localization of αE+ cells as well as total αE gene expression in healthy and inflammatory bowel disease patients. Methods: αE+ cells were identified in ileal and colonic biopsies by immunohistochemistry and counted using an automated algorithm. Gene expression was assessed by quantitative reverse-transcriptase polymerase chain reaction. Results: In both healthy and inflammatory bowel disease patients, significantly more αE+ cells were present in the epithelium and lamina propria of ileal compared with colonic biopsies. αE gene expression levels were also significantly higher in ileal compared with colonic biopsies. Paired biopsies from the same patient showed moderate correlation of αE expression between the ileum and colon. Inflammation did not affect αE expression, and neither endoscopy nor histology scores correlated with αE gene expression. αE expression was not different between patients based on concomitant medication use except 5-aminosalicylic acid. Conclusion: αE+ cells, which have been shown to have inflammatory potential, are increased in the ileum in comparison with the colon in both Crohn's disease and ulcerative colitis, as well as in healthy subjects. In inflammatory bowel disease patients, αE levels are stable, regardless of inflammatory status or most concomitant medications, which could support its use as a biomarker for etrolizumab.

7.
J Crohns Colitis ; 12(suppl_2): S653-S668, 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-29767705

RESUMO

Integrins are cell surface receptors with bidirectional signalling capabilities that can bind to adhesion molecules in order to mediate homing of leukocytes to peripheral tissues. Gut-selective leukocyte homing is facilitated by interactions between α4ß7 and its ligand, mucosal addressin cellular adhesion molecule-1 [MAdCAM-1], while retention of lymphocytes in mucosal tissues is mediated by αEß7 binding to its ligand E-cadherin. Therapies targeting gut-selective trafficking have shown efficacy in inflammatory bowel disease [IBD], confirming the importance of leukocyte trafficking in disease pathobiology. This review will provide an overview of integrin structure, function and signalling, and highlight the role that these molecules play in leukocyte homing and retention. Anti-integrin therapeutics, including gut-selective antibodies against the ß7 integrin subunit [etrolizumab] and the α4ß7 integrin heterodimer [vedolizumab and abrilumab], and the non-gut selective anti-α4 integrin [natalizumab], will be discussed, as well as novel targeting approaches using small molecules.

8.
EMBO Rep ; 19(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29437695

RESUMO

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18-induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin-2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1- and WIPI2-positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin-2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.

9.
Inflamm Bowel Dis ; 24(3): 583-592, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29462388

RESUMO

Background/Aims: Current models of Crohn's disease (CD) describe an inappropriate immune response to gut microbiota in genetically susceptible individuals. NOD2 variants are strongly associated with development of CD, and NOD2 is part of the innate immune response to bacteria. This study aimed to identify differences in fecal microbiota in CD patients and non-IBD controls stratified by NOD2 genotype. Methods: Patients with CD and non-IBD controls of known NOD2 genotype were identified from patients in previous UK IBD genetics studies and the Cambridge bioresource (genotyped/phenotyped volunteers). Individuals with known CD-associated NOD2 mutations were matched to those with wild-type genotype. We obtained fecal samples from patients in clinical remission with low fecal calprotectin (<250 µg/g) and controls without gastrointestinal disease. After extracting DNA, the V1-2 region of 16S rRNA genes were polymerase chain reaction (PCR)-amplified and sequenced. Analysis was undertaken using the mothur package. Volatile organic compounds (VOC) were also measured. Results: Ninety-one individuals were in the primary analysis (37 CD, 30 bioresource controls, and 24 household controls). Comparing CD with nonIBD controls, there were reductions in bacterial diversity, Ruminococcaceae, Rikenellaceae, and Christensenellaceae and an increase in Enterobacteriaceae. No significant differences could be identified in microbiota by NOD2 genotype, but fecal butanoic acid was higher in Crohn's patients carrying NOD2 mutations. Conclusions: In this well-controlled study of NOD2 genotype and fecal microbiota, we identified no significant genotype-microbiota associations. This suggests that the changes associated with NOD2 genotype might only be seen at the mucosal level, or that environmental factors and prior inflammation are the predominant determinant of the observed dysbiosis in gut microbiota.

11.
Front Nutr ; 4: 14, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28534028

RESUMO

Large randomized controlled trials (RCTs) in preterm infants offer unique opportunities for mechanistic evaluation of the risk factors leading to serious diseases, as well as the actions of interventions designed to prevent them. Necrotizing enterocolitis (NEC) a serious inflammatory gut condition and late-onset sepsis (LOS) are common feeding and nutrition-related problems that may cause death or serious long-term morbidity and are key outcomes in two current UK National Institutes for Health Research (NIHR) trials. Speed of increasing milk feeds trial (SIFT) randomized preterm infants to different rates of increases in milk feeds with a primary outcome of survival without disability at 2 years corrected age. Enteral lactoferrin in neonates (ELFIN) randomizes infants to supplemental enteral lactoferrin or placebo with a primary outcome of LOS. This is a protocol for the mechanisms affecting the gut of preterm infants in enteral feeding trials (MAGPIE) study and is funded by the UK NIHR Efficacy and Mechanistic Evaluation programme. MAGPIE will recruit ~480 preterm infants who were enrolled in SIFT or ELFIN. Participation in MAGPIE does not change the main trial protocols and uses non-invasive sampling of stool and urine, along with any residual resected gut tissue if infants required surgery. Trial interventions may involve effects on gut microbes, metabolites (e.g., short-chain fatty acids), and aspects of host immune function. Current hypotheses suggest that NEC and/or LOS are due to a dysregulated immune system in the context of gut dysbiosis, but mechanisms have not been systematically studied within large RCTs. Microbiomic analysis will use next-generation sequencing, and metabolites will be assessed by mass spectrometry to detect volatile organic and other compounds produced by microbes or the host. We will explore differences between disease cases and controls, as well as exploring the actions of trial interventions. Impacts of this research are multiple: translation of knowledge of mechanisms promoting gut health may explain outcomes or suggest alternate strategies to improve health. Results may identify new non-invasive diagnostic or monitoring techniques, preventative or treatment strategies for NEC or LOS, or provide data useful for risk stratification in future studies. Mechanistic evaluation might be especially informative where there are not clear effects on the primary outcome (ISRCTN 12554594).

12.
J Crohns Colitis ; 11(5): 610-620, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28453768

RESUMO

Background and Aims: The αEß7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEß7-E-cadherin interactions. Methods: αEß7+ and αEß7- colonic T cell localization, inflammatory cytokine production and expression of regulatory T cell-associated markers were evaluated in cohorts of control subjects and patients with active UC by immunohistochemistry, flow cytometry and real-time PCR of FACS-purified cell populations. Results: CD4+αEß7+ T lymphocytes from both healthy controls and UC patients had lower expression of regulatory T cell-associated genes, including FOXP3, IL-10, CTLA-4 and ICOS in comparison with CD4+αEß7- T lymphocytes. In UC, CD4+αEß7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEß7- lymphocytes. Additionally the CD4+αEß7+ subset was enriched for Th17 cells and the recently described Th17/Th1 subset co-expressing both IL-17A and IFNγ, both of which were found at higher frequencies in UC compared to control. Conclusion: αEß7 integrin expression on human colonic CD4+ T cells was associated with increased production of pro-inflammatory Th1, Th17 and Th17/Th1 cytokines, with reduced expression of regulatory T cell-associated markers. These data suggest colonic CD4+αEß7+ T cells are pro-inflammatory and may play a role in UC pathobiology.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite Ulcerativa/imunologia , Colo/citologia , Integrinas/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Colite Ulcerativa/metabolismo , Colo/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
13.
Nat Genet ; 49(2): 256-261, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067908

RESUMO

Genetic association studies have identified 215 risk loci for inflammatory bowel disease, thereby uncovering fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals and conducted a meta-analysis with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new susceptibility loci, 3 of which contain integrin genes that encode proteins in pathways that have been identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes (ITGA4 and ITGB8) and at previously implicated loci (ITGAL and ICAM1). In all four cases, the expression-increasing allele also increases disease risk. We also identified likely causal missense variants in a gene implicated in primary immune deficiency, PLCG2, and a negative regulator of inflammation, SLAMF8. Our results demonstrate that new associations at common variants continue to identify genes relevant to therapeutic target identification and prioritization.


Assuntos
Predisposição Genética para Doença/genética , Doenças Inflamatórias Intestinais/genética , Integrinas/genética , Alelos , Estudo de Associação Genômica Ampla/métodos , Humanos , Inflamação/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética
14.
Nat Genet ; 49(2): 186-192, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067910

RESUMO

To further resolve the genetic architecture of the inflammatory bowel diseases ulcerative colitis and Crohn's disease, we sequenced the whole genomes of 4,280 patients at low coverage and compared them to 3,652 previously sequenced population controls across 73.5 million variants. We then imputed from these sequences into new and existing genome-wide association study cohorts and tested for association at ∼12 million variants in a total of 16,432 cases and 18,843 controls. We discovered a 0.6% frequency missense variant in ADCY7 that doubles the risk of ulcerative colitis. Despite good statistical power, we did not identify any other new low-frequency risk variants and found that such variants explained little heritability. We detected a burden of very rare, damaging missense variants in known Crohn's disease risk genes, suggesting that more comprehensive sequencing studies will continue to improve understanding of the biology of complex diseases.


Assuntos
Adenilil Ciclases/genética , Predisposição Genética para Doença/genética , Doenças Inflamatórias Intestinais/genética , Colite Ulcerativa/genética , Doença de Crohn/genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética
15.
Small GTPases ; 7(4): 265-269, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27669114

RESUMO

Formation of autophagosomes requires vesicular trafficking from virtually every subcellular compartment to the formation site. This traffic must be tightly regulated but also adaptable as different membrane compartments will contribute varying amounts of membrane, lipids and proteins to the forming autophagosome depending on the stimulus. In mammalian cells, efforts to understand how autophagosomes form have been focused on the role of Rab proteins in autophagy. Rab proteins provide specificity through their interaction with coat proteins, vesicle tethers and SNAREs. Recent data emerging from these studies have defined a subset of Rab proteins and their regulators, the RabGAPS (GTPase activating proteins) in both autophagosome formation and maturation. This review will focus on the role of a set of RabGAPs shown to regulate autophagy, in particular TBC1D14, and its interactors, RAB11 and TRAPPIII. Through our studies on TBC1D14, we have gained an understanding of the contribution of membrane from the recycling endosome, and the role of TRAPPIII in maintaining ATG (Autophagy protein) 9 trafficking in autophagosome formation.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Autofagia , Endossomos/metabolismo , Humanos
16.
Autophagy ; 12(7): 1212-3, 2016 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-27171758

RESUMO

Amino acid withdrawal induces the formation of autophagosomes, which results in dozens of these large double-membrane vesicles appearing in the starved cell within 10-15 min, and the initiation of autophagy. This vesicle-mediated response clearly requires an adequate supply of membrane and a tight molecular regulation creating a substantial challenge for the cell in terms of vesicle trafficking pathways. Several membrane sources, which contribute to autophagosome initiation and formation, have been identified including the ER, Golgi, plasma membrane, mitochondria and recycling endosomes. How contributions from these organelles are regulated is an intensive area of study. Members of several families of membrane traffic regulators, including small GTPases, such as RAB proteins, and their regulators, SNARE proteins and BAR domain-containing proteins, have recently been shown to support autophagosome formation.


Assuntos
Autofagia/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Endossomos/metabolismo , Humanos , Mitocôndrias/metabolismo
17.
Cell Cycle ; 15(14): 1797-8, 2016 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-27105289
18.
Cold Spring Harb Protoc ; 2016(3): pdb.prot083675, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26933243

RESUMO

The dynamic nature of insulin-sensitive glucose transporter isoform 4 (GLUT4) storage vesicles (GSVs) makes their characterization challenging. Fractionation techniques can facilitate isolation of GSVs from insulin-sensitive cells. In this protocol, we describe preparation of a total membrane fraction from 3T3-L1 adipocytes. The resulting pellet contains all membranes and allows for easier identification of membrane proteins, including the insulin-sensitive pool of GLUT4. A method for concentration of the soluble fraction is also included.


Assuntos
Adipócitos/ultraestrutura , Fracionamento Celular/métodos , Membrana Celular , Animais , Linhagem Celular , Camundongos
19.
Cold Spring Harb Protoc ; 2016(3): pdb.prot083683, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26933244

RESUMO

The insulin-sensitive pool of glucose transporter isoform 4 (GLUT4) can be isolated from total cell membranes using the 16K fractionation protocol, described here. This method produces a light membrane-containing supernatant that includes the insulin-sensitive pool of GLUT4 in GLUT4 storage vesicles. The 16K pellet fraction contains the heavy membranes (including the plasma membrane, mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum). The distribution of proteins between the two fractions is determined via immunoblotting. By subjecting insulin-stimulated versus unstimulated cells to this protocol, the mobilization of proteins out of the insulin-sensitive GLUT4 pool can be assessed.


Assuntos
Adipócitos/ultraestrutura , Fracionamento Celular/métodos , Membrana Celular/química , Vesículas Citoplasmáticas/química , Transportador de Glucose Tipo 4/isolamento & purificação , Animais , Linhagem Celular , Camundongos
20.
Sci Rep ; 6: 23326, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26987296

RESUMO

CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival.


Assuntos
Lectinas Tipo C/genética , Lisossomos/fisiologia , Proteínas de Transporte de Monossacarídeos/genética , Doença dos Neurônios Motores/patologia , Mutação , Células de Purkinje/citologia , Animais , Autofagia , Sobrevivência Celular , Células Cultivadas , Complexo de Golgi/patologia , Células HeLa , Humanos , Lectinas Tipo C/metabolismo , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Doença dos Neurônios Motores/genética
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