Temporal development of necrotizing enterocolitis in the preterm pig
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ABSTRACT: Necrotizing enterocolitis (NEC), a serious gastrointestinal disease that afflicts 5-10% of preterm infants, often progresses rapidly from mild food intolerance into extensive haemorrhage, inflammation and necrosis. Events leading to NEC have remained poorly defined. Similar disease characteristics are observed in preterm pigs 24-48 h after feeding formula. Using this model, we aimed to characterize the temporal development of NEC, and describe the functional and immunological response of the preterm intestine preceding NEC. Keywords: time course Pigs from treatment groups TPN (n=5), and 8 h (n=5) and 24 h (n=5-6) FORM and COLOS were randomly selected for microarray analysis. Equal amounts of total distal small intestinal RNA from all pigs was pooled to make the reference sample. Samples and reference pool were labelled with Oyster 550 and 650, respectively. The in-house spotted porcine oligonucleotide microarray version 4 (POM4) is a low density microarray consisting of 384 different oligonucleotide probes representing more than 200 different immune related genes and eight different array control oligonucleotides (ArrayControl; Ambion, Nærum, Denmark). The immunologically relevant 60-70mer oligonucleotide probes represent interferons and interleukins (and receptors), chemokines (and receptors), acute phase proteins, apoptosis-related factors and sequences with relevance to Toll-like receptors and their intracellular signalling pathways.
Project description:Preterm neonates are susceptible to gastrointestinal (GI) disorders such as necrotizing enterocolitis (NEC). Maternal milk, and especially colostrum, protects against NEC via growth promoting, immunomodulatory and antimicrobial factors. The fetal enteral diet, amniotic fluid (AF), contains similar bioactive components and we hypothesized that postnatal AF administration would reduce inflammatory responses and NEC in preterm neonates. Thirty preterm pigs (92% gestation) were delivered by caesarean section and fed total parental nutrition (TPN) for 48 h followed by enteral porcine colostrum (COLOS, n=7), infant formula (FORM, n=13) or formula + porcine AF (AF, n=10). Using a previously validated model of NEC in preterm pigs, we determined the structural, functional, microbiological and immunological responses to AF when administered prior to and after introduction of a suboptimal enteral formula diet. Keywords: Healthy versus inflammed tissues in relation to necrotizing enterocolitis Pigs from each treatment group (COLOS, n=4; FORM, n=6; and AF, n=7) were randomly selected for microarray analysis of frozen distal small intestine samples. The FORM group was further divided into formula-fed healthy pigs (F-HEA, n=3) and formula-fed NEC pigs (F-NEC, n=3) in order to compare sick versus healthy formula fed pigs. Equal amounts of total distal small intestinal RNA from all pigs were pooled to make the reference sample. Samples and reference pool were labelled with Oyster 550 and 650, respectively. The in-house spotted porcine oligonucleotide microarray version 4 (POM4) is a low density microarray consisting of 384 different oligonucleotide probes representing more than 200 different immune related genes.
Project description:Caesarean-delivered preterm pigs were fed 3 d of parenteral nutrition followed by 2 d of enteral formula feeding. Antibiotics (n=11) or control saline (n=13) were given twice daily from birth to tissue collection at d 5. NEC-lesions and intestinal structure, function, microbiology and immunity markers were recorded. We used Affymetrix microarrays to investigate gene expression in intestinal tissues of preterm piglets treated with antibiotics or control saline. Twenty-four preterm piglets were delivered by caesarean section on day 105 of gestation from two healthy sows. All piglets were initially provided with parenteral nutrition via a vascular catheter, combined with small amounts of minimal enteral nutrition. On day three, all parenteral nutrition was stopped and total enteral nutrition was given through an oro-gastric feeding tube. Piglets were allocated into controls ( n=13) and an intervention group receiving oral and systemic broad-spectrum antibiotics ( n=11). To assure high systemic and intra luminal MIC values antibiotics were given both orally and intramuscularly. All antibiotics were given directly after feeding with an oral bolus and control pigs were given corresponding amounts of saline. On day five, all piglets were euthanized, and small intestinal tissue collected.
Project description:Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) but specific biomarkers for diagnosis and effective treatment are lacking. Neutrophil extracellular traps (NETs) are related to sepsis in adults but not investigated in infant conditions. This is the first proteome study to document that circulating NETs are involved in neonatal LOS and NEC. cfDNA and NET proteins may provide new potential diagnostic markers for these diseases.
Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2µg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, Rødovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5µl cDNA from each synthesis, 0.5µl Salmon sperm (10µg/µl) and 13.5µl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29µl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5µl of each Capture reagent and 26µl SDS-Based hybridization buffer was mixed to a final volume of 52µl. 26µl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).
Project description:In this study we investigated how changes in pH and ocean chemistry consistent with the scenarios of the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, long before they affect biomineralization. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrated up-regulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur well before impacts on calcification. We applied a reference microarray design for the experiment outlined in the study, which was a three condition experiment of ocean acidification: control pH 8.0-8.2, medium pH 7.8-7.9 and high pH 7.6-7.7, and across three time points: time zero, day 1 and day 28. Samples from time zero and control treatments were used to generate the reference sample for the microarray hybridization experiments. A total of 27 microarrays were used in the entire experiment, 3 biological replicates per treatment and timepoint. Reference samples in each array was labeled with Cy3, and the actual experimental samples with Cy5.
Project description:This SuperSeries is composed of the following subset Series: GSE10413: Gene expression profiling of caffeic acid phenethyl ester-treated human umbilical vein endothelial cells-1 GSE10429: Gene expression profiling of caffeic acid phenethyl ester -treated human umbilical vein endothelial cells-2 Keywords: SuperSeries Refer to individual Series
Project description:Caffeic acid phenethyl ester (CAPE), derived from various plant sources, has been shown to ameliorate ischemia/reperfusion (I/R) injury in vivo, and this has been attributed to its ability to reduce the oxidative stress. Here we investigated the cytoprotection of CAPE against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) to evaluate potential gene expression involvement. CAPE exhibited dose-dependent cytoprotection of HUVEC that required preincubation. A gene screen with microarrays was performed to identify the potential cytoprotective gene(s) induced by CAPE. Heme oxygenase-1 (HO-1) was highly upregulated by CAPE and this was confirmed with reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Keywords: gene expression in HUVEC, CAPE cytoprotective dose response Confluent HUVEC were incubated with cytoprotective dose of CAPE at 5 µg/ml or 0.1% DMSO as vehicle control for 6 hrs. Both treatments were done in triplicates. Total RNA was isolated at the end of the treatment and applied to microarray experiments in order to identify transcriptional response of HUVEC to CAPE. Microarray experiments were based on a two-color reference design using human universal reference RNA to compare results bwtween CAPE treatment and vehicle control groups.
Project description:To study hepatic gene expression differences, liver samples from infected pigs (n = 10) were compared with liver samples from the non-infected control group (n = 5). Ten microarrays were performed, such that 5 randomly selected cDNA samples from the infected group were labeled with Oyster 550 (Genisphere Inc., Hatfield, PA, USA) and compared directly to 5 control animals labeled with Oyster 650 (Genisphere) analysing a pair of samples (one from an infected animal and one from a randomly chosen non-infected animal) on each microarray slide. The remaining five cDNA preparations from the infected group were labeled with Oyster 650 and compared in the same way to the five controls labeled with Oyster 550 on the remaining 5 microarray slides. This dye-swap design was applied to reduce variation due to dye effects and to provide as much biological replication as possible. Keywords: Disease state analysis Two-condition experiment, Disease vs. Control, Biological replicates: 10 disease replicates and 5 control replicates, 10 Microarrays comparing one disease replicate with one control replicate.
Project description:Caffeic acid phenethyl ester (CAPE), derived from various plant sources, has been shown to ameliorate ischemia/reperfusion (I/R) injury in vivo, and this has been attributed to its ability to reduce the oxidative stress. Here we investigated the cytoprotection of CAPE against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) to evaluate potential gene expression involvement. CAPE exhibited dose-dependent cytoprotection of HUVEC that required preincubation. A gene screen with microarrays was performed to identify the potential cytoprotective gene(s) induced by CAPE. Heme oxygenase-1 (HO-1) was highly upregulated by CAPE and this was confirmed with reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Keywords: Gene expression in HUVEC, CAPE cytoprotective dose response Confluent HUVEC were incubated with cytoprotective dose of CAPE at 5 µg/ml or 0.1% DMSO as vehicle control for 6 hrs. Both treatments were done in triplicates. Total RNA was isolated at the end of the treatment and applied to microarray experiments in order to identify transcriptional response of HUVEC to CAPE. Microarray experiments were based on a two-color reference design using human universal reference RNA to compare results bwtween CAPE treatment and vehicle control groups.