Intestinal RNA Research Articles

Analyses of clinical, pathological and virological features of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice

Experimental studies of human rotavirus infections in mice are limited and there is lack of information on the quantitative assessment of rotaviral replication and its relationship with histological changes. In the present study, consequences of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice were analyzed for the occurrence of clinical symptoms, histopathology and virological events. The infected animals developed diarrhea and dehydration and showed accumulation of vacuolated enterocytes with lodging of the rotavirus antigens and shortening of villi in the intestine over a period of 5 days. The ileum was identified as the most susceptible and supportive part of small intestine for perpetuation of rotavirus infection in mice. Rotaviral antigen/RNA in stool and RNA in intestine were detected throughout the clinical disease period. At 48–72 h post inoculation, diarrhea was at the peak (90–95%) in the infected animals with increased load of viral RNA and intense pathological lesions suggesting it as the critical time point in the course of infection. The rising titers of antirotavirus neutralizing antibodies ascertained the replication of human rotavirus strain, YO in mice. These data may contribute to the understanding of pathophysiological, immunological and virological characteristics of rotavirus infections in mice.

Developmental control of the Nlrp6 inflammasome and a substrate, IL-18, in mammalian intestine

The inflammasome is a multiprotein complex whose formation is triggered when a NOD-like receptor binds a pathogen ligand, resulting in activated caspase-1, which converts certain interleukins (IL-1β, IL-18, and IL-33) to their active forms. There is currently no information on regulation of this system around the time of birth. We employed transcript profiling of fetal rat intestinal and lung RNA at embryonic days 16 (E16) and 20 (E20) with out-of-sample validation using quantitative RT-PCR. Transcript profiling and quantitative RT-PCR demonstrated that transcripts of core components of the NOD-like receptor Nlrp6 inflammasome (Nlrp6, Pycard, Caspase-1) and one of its substrates, IL-18, were increased at E20 compared with E16 in fetal intestine and not lung. Immunohistochemistry demonstrated increased Pycard in intestinal epithelium. Western blotting demonstrated that IL-18 was undetectable at E16, clearly detectable at E20 in its inactive form, and detectable postnatally in both its inactive and active form. Dramatic upregulation of IL-18 was also observed in the fetal sheep jejunum in late gestation (P = 0.006). Transcription factor binding analysis of the rat array data revealed an overrepresentation of nuclear transcription factor binding sites peroxisome proliferator-activated receptor γ (PPAR-γ) and retinoid X receptor-α and chicken ovalbumin upstream promoter transcription factor 1 in the region 1,000 bp upstream of the transcription start site. Rosiglitazone, a PPAR-γ agonist, more than doubled levels of NLRP6 mRNA in human intestinal epithelial (Caco2) cells. These observations provide the first evidence, to our knowledge, linking activity of PPAR-γ to expression of a NOD-like receptor and adds to a growing body of evidence linking pattern recognition receptors of the innate immune system and intestinal colonization.

Endogenous fraction and urinary recovery of purine derivatives obtained by different methods in Nellore cattle1

Two experiments were conducted to assess the endogenous fraction of purine derivative (PD) excretion, urinary recovery, and intestinal digestibility of purines in Nellore heifers. For both experiments, 8 Nellore heifers fitted with ruminal and abomasal cannulas were allocated to two 4 × 4 Latin squares. The diets were based on corn silage and concentrate (60 and 40% DM basis, respectively); feces and urine samples were obtained by total collection, and abomasal DM flow was estimated using indigestible NDF as an internal marker. In Exp. I, 4 of the 8 heifers (BW 258 ± 20 kg) were also fitted with ileal cannula. The planned treatments were 4 different DMI: 1.2, 1.6, 2.0, and 2.4% of BW (DM basis). The endogenous losses and purine recovery as urinary PD were estimated using linear regression between daily urinary PD excretion (Y) and daily abomasal flow of purine bases (X), expressed in millimoles per kilogram of BW(0.75). In Exp. II, the same 8 Nellore heifers (BW of 296 ± 15 kg) were fed at 1.37% BW (DM basis). The treatments were the infusion of purines (RNA from torula yeast, type VI, Sigma) into the abomasum in increasing amounts (0, 33, 66, and 100 mmol/d). All statistical analyses were performed using the PROC MIXED procedure in SAS. In Exp. I, the DMI range was 1.16 to 1.84% of BW and did not affect (P > 0.05) the apparent RNA digestibility in the small intestine, which had a mean of 75.6%, and a true digestibility of 93.0%. The mean ratio of the N-RNA to the total-N in the ruminal bacteria was 0.137. The daily urinary PD excretion (Y, mmol/kg of BW(0.75)) was a function of RNA flow in the abomasum (X, mmol/kg of BW(0.75)): Y = 0.860X + 0.460, where 0.860 and 0.460 were the PD recovery of purines and the endogenous fraction (in mmol/kg of BW(0.75)), respectively. In Exp. II, the daily urinary PD excretion was a function of RNA flow in the abomasum: Y = 0.741X + 0.301, where 0.741 and 0.301 were the recovery of PD in urine of infused purines and the endogenous losses (in mmol/kg of BW(0.75)), respectively. In conclusion, our data suggest that in Nellore heifers the respective values of endogenous PD excretion (mmol/kg of BW(0.75)), urinary recovery of the purines absorbed in the abomasum, and true digestibility of RNA in the small intestine were 0.30, 0.80, and 0.93.

The effect of glucagon-like peptide 2 injection on performance, small intestinal morphology, and nutrient transporter expression of stressed broiler chickens

An experiment was conducted to determine the effect of injecting glucagon-like peptide 2 (GLP-2) on the small intestinal weight, morphology, and nutrient transporter expression in pharmacologically stressed broiler chickens. A total of 144 seven-day-old birds were fed either a basal diet (CTRL) or a basal diet plus 30 mg of corticosterone (CORT)/kg of diet for a total of 14 d. Half of the birds from each group were injected daily with GLP-2 (6.7 nmol/kg of BW) or saline for 14 d. The average final BW, ADG, ADFI, and the ratio of feed intake to weight gain (F:G) was recorded over 21 d for the 4 groups of 36 birds, namely CTRL + saline, CTRL + GLP-2, CORT + saline, and CORT + GLP-2. In addition, the absolute and relative small intestinal weight, villus height (VH), and crypt depth (CD) of the duodenum and jejunum, as well as the abundance of sodium and glucose co-transporter 1 (SGLT-1), vitamin D-dependent calcium-binding protein-28,000 molecular weight (CaBP-D28k), and peptide transporter 1 (PepT-1) mRNA in the duodenum and of liver fatty acid-binding protein (L-FABP) mRNA in the jejunum. The total DNA, RNA, and protein content in small intestinal mucosa were also determined. The results showed that CORT administration significantly lowered average final BW, ADG, ADFI, absolute small intestinal weight, VH, and CD of duodenum and jejunum (P < 0.05) while increasing the relative small intestinal weight, F:G, relative abundance of SGLT-1, CaBP-D28k, PepT-1, and L-FABP mRNA (P < 0.05). Glucagon-like peptide 2 injection increased the average final BW, ADG, VH, and CD in duodenum and jejunum and relative abundance of SGLT-1, CaBP-28k, PepT1, and PepT1 mRNA of broiler chickens, respectively (P < 0.05), and decreased F:G (P < 0.05). In chickens fed basal diet plus CORT, injecting GLP-2 decreased F:G (P < 0.05); increased VH and CD of duodenum and CD of jejunum; and increased relative abundance of SGLT-1, CaBP-D28k, PepT-1, and L-FABP mRNA, RNA, and total protein content in small intestine compared with the injection of saline (P < 0.05). In birds fed the basal diet, GLP-2 injection decreased F:G (P < 0.05) and increased final BW, ADG, small bowel weight, CD of jejunum, and relative abundance of CaBP-D28k and PepT-1 mRNA compared with injecting saline (P < 0.05). In conclusion, GLP-2 injection reversed the negative effect of stress on the weight and morphology and the absorptive function of small bowel of broiler chickens. Glucagon-like peptide 2 injection also had a positive effect on the growth performance of healthy broiler chickens.

Morphology, proliferation, and ribonucleic acid and fractional protein syntheses in the small intestinal mucosa of young goats fed soy protein-based diets with or without amino acid supplementation

The study was designed to examine whether feeding soy protein isolate as partial replacement of casein (CN) affects jejunal protein synthesis and whether effects may be ameliorated by supplementation of those AA known to be at lower concentrations in soy protein isolate than in CN. Goat kids (14 d) were fed comparable milk protein diets, in which 50% of the crude protein was CN (CAS), soy protein isolate (SPI), or soy protein isolate supplemented with AA (SPIA) for 43 d (n=8 per group). On d 42, plasma concentrations of protein, urea, and AA were measured before and after morning feeding. In the morning of d 43, [15N]RNA from yeast [13mg/kg of body weight (BW)] was given with the diet to measure the reutilization of dietary RNA precursors for mucosal RNA biosynthesis. Four hours later, an oral dose of l-[1-13C]leucine (180mg/kg of BW) was administered and blood samples were collected between −15 and +45min relative to tracer administration for analysis of plasma 13C α-ketoisocaproic acid and 13C recovery in blood CO2. Kids were killed 60min after the tracer application, and jejunal tissue was collected to determine mucosal morphology, cell proliferation, enzyme activities, RNA synthesis, and fractional protein synthesis rate. Plasma protein concentrations were higher in CAS than in SPI and SPIA. Plasma concentrations of Thr were higher in CAS than in SPI and SPIA, and those of Met were lower in SPI than in CAS and SPIA. In mid-jejunum, villus circumferences were higher in CAS than in SPI and SPIA, and villus height and villus height:crypt depth ratio were higher in CAS than in SPI. In mid-jejunum, mucosal protein concentrations were higher in CAS than in SPI and SPIA and mucosal activities of aminopeptidase N tended to be higher in CAS than in SPI, whereas activities of dipeptidyl peptidase IV tended to be lower in SPI than in SPIA. Activities of 5′ nucleotidase and xanthine oxidase were lower in CAS than in SPI. The 13C recovery in blood CO2 tended to be higher in SPI than in CAS. In mid-jejunum, 15N enrichment of RNA tended to be higher in CAS than in SPI, and 13C enrichment of protein-bound Leu was higher in SPI than in CAS. In mid-jejunum, the fractional protein synthesis rate tended to be higher in SPI than in CAS. Our results revealed changes in intestinal growth after soy protein feeding that were associated with effects on intestinal RNA and protein synthesis but that were not ameliorated by AA supplementation.

Modulation of Brain Dead Induced Inflammation by Vagus Nerve Stimulation

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

Molecular Cloning, Tissue Distribution and Expression of Porcine y+L Amino Acid Transporter-1

In this study, we cloned, sequenced and characterized porcine y+L Amino Acid Transporter-1 (y+LAT1). By screening a translated EST database with the protein sequence of the human y + LAT1 and by using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine y + LAT1 was isolated from porcine intestine RNA. It was 2,111 bp long, encoding a 511 amino acid trans-membrane glycoprotein composed of 12 transmembrane domains. The predicted amino acid sequence was found to be 91%, 90%, 87% and 87% identical to those of cattle, human, mouse and rat y + LAT1 respectively. Real-time RT-PCR results indicated that the small intestine had the highest y + LAT1 mRNA abundance and the lung had the lowest y + LAT1 mRNA abundance. Baby hamster kidney (BHK) cells transfected with green fluorescent protein (GFP) tagged porcine y + LAT1 cDNA indicated that the cellular localization of the gene product in BHK was on the plasma membrane.

Identification of Two Common Variants Contributing to Serum Apolipoprotein B Levels in Mexicans

Although the Mexican population has a high predisposition to dyslipidemias and premature coronary artery disease, this population is underinvestigated for the genetic factors conferring the high susceptibility. This study attempted to determine these genetic factors. First, we investigated apolipoprotein B (apoB) levels in Mexican extended families with familial combined hyperlipidemia using a two-step testing strategy. In the screening step, we screened 5721 single-nucleotide polymorphisms (SNPs) for linkage signals with apoB. In the test step, we analyzed the 130 SNPs residing in regions of suggestive linkage signals for association with apoB. We identified significant associations with two SNPs (ie, rs1424032 [P=6.07x10(-6)] and rs1349411 [P=2.72x10(-4)]) that surpassed the significance level for the number of tests performed in the test step (P<3.84x10(-4)). Second, these SNPs were tested for replication in Mexican hyperlipidemic case-control samples. The same risk alleles as in the families with familial combined hyperlipidemia were significantly associated (P<0.05) with apoB in the case-control samples. The rs1349411 resides near the apoB messenger RNA editing enzyme (APOBEC1) involved in the processing of APOB messenger RNA in the small intestine. The rs1424032 resides in a highly conserved noncoding region predicted to function as a regulatory element. We identified two novel variants, rs1349411 and rs1424032, for serum apoB levels in Mexicans.

N-3 Polyunsaturated fatty acids modulate carcinogen-directed non-coding microRNA signatures in rat colon.

We have hypothesized that dietary modulation of intestinal non-coding RNA [microRNA (miRNA)] expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). To fully understand the effects of these agents on the expression of miRNAs, Sprague-Dawley rats were fed diets containing corn oil or fish oil with pectin or cellulose and injected with azoxymethane (AOM, a colon-specific carcinogen) or saline (control). Real-time polymerase chain reaction using miRNA-specific primers and Taq Man probes was carried out to quantify effects on miRNA expression in colonic mucosa. From 368 mature miRNAs assayed, at an early stage of cancer progression (10 week post AOM injection), let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were significantly (P < 0.05) affected by diet x carcinogen interactions. Overall, fish oil fed animals exhibited the smallest number of differentially expressed miRNAs (AOM versus saline treatment). With respect to the tumor stage (34 week post AOM injection), 46 miRNAs were dysregulated in adenocarcinomas compared with normal mucosa from saline-injected animals. Of the 27 miRNAs expressed at higher (P < 0.05) levels in tumors, miR-34a, 132, 223 and 224 were overexpressed at >10-fold. In contrast, the expression levels of miR-192, 194, 215 and 375 were dramatically reduced (< or = 0.32-fold) in adenocarcinomas. These results demonstrate for the first time the utility of the rat AOM model and the novel role of fish oil in protecting the colon from carcinogen-induced miRNA dysregulation.

Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctionalization of duplicated genes

BackgroundIn the Duplication-Degeneration-Complementation (DDC) model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps) genes by dietary fatty acids (FAs) in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid), sunflower oil (12% lipid, rich in linoleic acid), linseed oil (12% lipid, rich in linolenic acid), or low fat (4% lipid, low fat diet) for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined.ResultFA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level of fabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a given fabp gene. As such, up-regulation of the steady-state level of fabp mRNAs by FAs occurred at the level of initiation of transcription. None of the sister duplicates of these fabp genes exhibited an increase in their steady-state transcript levels in a specific tissue following feeding zebrafish any of the four experimental diets.ConclusionDifferential induction of only one of the sister pair of duplicated fabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.

Reduced NHE3-mediated Na+ absorption increases survival and decreases the incidence of intestinal obstructions in cystic fibrosis mice

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 Na+/H+ exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target.

Early Administration of Probiotics Alters Bacterial Colonization and Limits Diet-Induced Gut Dysfunction and Severity of Necrotizing Enterocolitis in Preterm Pigs 3

Following preterm birth, bacterial colonization and enteral formula feeding predispose neonates to gut dysfunction and necrotizing enterocolitis (NEC), a serious gastrointestinal inflammatory disease. We hypothesized that administration of probiotics would beneficially influence early bacterial colonization, thereby reducing the susceptibility to formula-induced gut atrophy, dysfunction, and NEC. Caesarean-delivered preterm pigs were provided total parenteral nutrition (1.5 d) followed by enteral feeding (2 d) with porcine colostrum (COLOS; n = 5), formula (FORM; n = 9), or formula with probiotics (FORM-P; Bifidobacterium animalis and Lactobacillus: L. acidophilus,L. casei,L. pentosus,L. plantarum; n = 13). Clinical NEC scores were reduced (P < 0.05) in FORM-P (2.0 ± 0.2) and COLOS groups (1.7 ± 0.5) compared with FORM pigs (3.4 ± 0.6). Lower NEC scores were associated with elevated intestinal weight, mucosa proportion, villus height, RNA integrity, and brush border aminopeptidase A and N activities, and lower gastric organic acid concentration in the FORM-P and COLOS groups (P < 0.05). Diversity of the mucosa-associated bacteria in the distal small intestine was similar among formula-fed pigs, yet the abundance of specific bacterial groups differed between FORM-P and FORM pigs. FORM-P pigs had lower colonization density of a potential pathogen, Clostridium perfringens, and had commensal Lactobacillus bacteria more closely associated with enterocytes along the villus-crypt axis relative to FORM pigs. These results suggest that probiotic administration immediately after birth promotes the colonization of a beneficial commensal microbiota capable of limiting the formula-induced mucosal atrophy, dysfunction, and pathogen load in preterm neonates, thereby reducing the incidence and severity of NEC.

Toll-like receptor 2 is protective of ischemia–reperfusion-mediated small-bowel injury in a murine model

In a murine model of intestinal injury, we hypothesized that Toll-like receptor 2 (TLR2), a recognition molecule for commensal bacteria, plays an important role in the development of mucosal immunity and is protective against ischemia/reperfusion injury via the modulation of both innate and acquired immunity. Interventional laboratory study. Academic medical research center. Four-week-old C57BL/6 wild-type (n = 12) and C57BL/6 TLR2-deficient mice (TLR2-/-) (n = 12). Twenty-four mice underwent laparotomy only or laparotomy plus superior mesenteric artery occlusion (n = 6/group) for 60 mins, followed by 90 mins of recovery. Mid-jejunal sections were taken for histopathology and messenger RNA expression (reverse transcriptase-polymerase chain reaction, normalized to 18s and laparotomy-only controls). Intestinal injury was scored from 0 (no injury) to 4 (transmural necrosis). Statistical analyses were performed using Mann-Whitney U test and Student's t-test (p < .05 significant). TLR2-/- mice had elevated intestinal injury scores (mean +/- SEM) after ischemia/reperfusion vs. wild-type (2.17 +/- 0.40 vs. 0.67 +/- 0.33, p < .05). Intestinal cytokine messenger RNA (mean fold change +/- SEM) of interferon-gamma (0.29 +/- 0.12 vs. 3313 +/- 1710), interleukin-4 (0.25 +/- 0.13 vs. 2.70 +/- 1.08), and interleukin-6 (250.63 +/- 69.60 vs. 320,300 +/- 215,964) in TLR2-/- was significantly decreased (p < .05) after ischemia/reperfusion vs. wild-type. Tumor necrosis factor-alpha messenger RNA levels were unchanged. TLR2-/- mice have a dysregulated mucosal innate immune response and fail to mount a protective response after ischemia-reperfusion compared with wild-type mice. This murine model of intestinal injury may correlate with the early postnatal course of premature infants who may have decreased TLR2 expression and/or decreased luminal commensal bacteria secondary to antibiotic therapy, thus decreasing TLR2-mediated signaling.

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-γ RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as α toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-γ expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

Immunological and gene expression responses to aSalmonellainfection in the chicken intestine

Besides infection in humans, Salmonella enteritidis can also cause serious illness in young chickens. However, the genetic and immunological parameters important for the disease in chickens are not well characterized. In this study, processes in the chicken intestine in response to a Salmonella infection were investigated in two different chicken lines. One-day-old chickens were orally infected with Salmonella. T-cell subpopulations, phagocytic properties of intestinal mononuclear cells and RNA expression levels of the jejunum were investigated. The two chicken lines differed in the amount of cfu in the liver and growth retardation after the infection. Differences in phagocytic activity of intestinal mononuclear cells were found between control and Salmonella infected chickens. The number of CD4+ T-cells of the intestine decreased after the Salmonella infection in one chicken line, while the number of CD8+ T-cells increased in both chicken lines, but the time post infection of this increase differed between the lines. In one chicken line the expression levels of the genes carboxypeptidase M and similar to ORF2 decreased after the Salmonella infection, which might be related to a decrease in the amount of macrophages. With the microarray, ten genes were found that were regulated in only one of the chicken lines, while we found six genes regulated in response to the infection in both chicken lines. So differences in genetic background of the chickens influence the intestinal host response of the Salmonella infection as observed by phagocytic activity, gene expression and changes in the number of T-cell subpopulations and macrophages.

Risk and risk reduction involving arginine intake and meat consumption in colorectal tumorigenesis and survival

Elevated polyamine and nitric oxide levels (both derived from arginine) promote tumorigenesis, whereas non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal cancer (CRC) incidence in experimental and epidemiologic studies. We investigated dietary arginine-induced intestinal tumorigenesis and NSAID-inhibitory effects in Apc(Min/+) mice differentially expressing nitric oxide synthase-2 (Nos2). We also studied effects of estimated arginine exposures through meat consumption on tumor characteristics and survival in human CRC cases. Dietary arginine increased high-grade colon adenoma incidence in Apc(Min/+)Nos2(+/+) mice, but not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N(1)-acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (p = 0.04) and decreased tumor number in the small intestines of Apc(Min/+)Nos2(+/+) mice (p = 0.0003). Five hundred and eleven cases from our NCI-supported CRC gene-environment study were analyzed based on self-reported meat (as a surrogate for arginine) consumption. Familial CRC cases (n = 144) in the highest meat consumption quartile (Q4) had no statistically significant differences in tumor grade compared to cases in Q1-Q3 (p = 0.32); however, they were observed to have decreased overall survival (OS) (10-year OS = 42% vs. 65%; p = 0.017), and increased risk of death in an adjusted analysis (hazards ratio [HR] = 2.24; p = 0.007). No differences in tumor grade, OS or adjusted HR were observed for sporadic CRC cases (n = 367) based on meat consumption. Our results suggest important roles for arginine and meat consumption in CRC pathogenesis, and have implications for CRC prevention.

Distribution and abundance of nutrient transporter mRNA in the intestinal tract of the black bear, Ursus americanus

End products of digestion are absorbed by the body through the action of transporter proteins expressed on the apical membrane of intestinal epithelial cells. We investigated the mRNA abundance and distribution of a peptide transporter (PepT1), a glucose transporter (SGLT1), two amino acid transporters (NBAT and b o,+AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears ( Ursus americanus). Intestinal total RNA was isolated from 10 bears and abundance of PepT1, SGLT1, NBAT, b o,+AT, and APN mRNA were determined by Northern blots. Abundance of PepT1 ( P < 0.05), APN ( P < 0.05), and SGLT1 ( P < 0.0001) changed quadratically from the proximal to distal intestine with abundance being greatest in the midregion. Abundance of b o,+AT mRNA increased linearly ( P < 0.05) from the proximal to distal intestine. The number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. PepT1 mRNA was present at 10-fold or greater levels than amino acid transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute a major form in which amino acids are absorbed in the black bear. The abundance of NBAT and b o,+AT mRNA was greater towards the distal intestine, suggesting a role in salvaging unabsorbed amino acids.

The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.

The Essential Role of Insulin-Like Growth Factor-1 in the Intestinal Tropic Effects of Glucagon-Like Peptide-2 in Mice

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that acts through unknown pathways to induce intestinal growth. We investigated the role of the insulin-like growth factors (IGF-1 and IGF-2) as mediators of GLP-2-enhanced growth in the murine intestine. IGF-1 expression and secretion were determined in GLP-2-responsive primary intestinal cultures treated with GLP-2. Parameters of intestinal growth were assessed in wild-type (CD1, Igf1(+/+) and Igf2+), heterozygous (Igf1(+/-)), and null (Igf1(-/-) and Igf2(-P)) mice treated chronically with saline, GLP-2, IGF-1, or R-Spondin1. GLP-2 increased IGF-1 messenger RNA expression and IGF-1 secretion in intestinal cultures and increased expression of IGF-1 messenger RNA in mouse small intestine in vivo. Igf1(+/+) and Igf2+ mice responded to .1 microg/g(-1) per day(-1) GLP-2 with increased intestinal weights, morphometric parameters, and proliferative indices. In contrast, Igf1(-/-) mice were unresponsive to the same dose of GLP-2, failing to demonstrate changes in intestinal weight, morphometry, or proliferation. However, a significant effect of 1 microg/g(-1) per day(-1) GLP-2 was observed in Igf1(-/-) mice, but only in terms of small intestinal weight when normalized for body weight. Furthermore, Igf2(-P) mice demonstrated a partially impaired response in terms of small intestinal growth. Both Igf1(-/-) and Igf2(-P) mice exhibited normal-enhanced intestinal growth in response to IGF-1 and/or R-Spondin1. GLP-2 enhances intestinal IGF-1 expression and secretion, and IGF-1 is required for small and large intestinal growth in response to GLP-2. These findings identify IGF-1 as an essential mediator of the intestinotropic actions of GLP-2.

Expression of Polymeric Immunoglobulin Receptor mRNA and Protein in Human Paneth Cells: Paneth Cells Participate in Acquired Immunity

Paneth cells are important effectors of intestinal innate immunity. It has been generally accepted that Paneth cells do not participate in the synthesis of polymeric immunoglobulin receptor (pIgR) or the secretion of immunoglobulin A (IgA) in the small intestine. However, we have previously shown that pIgR is specifically localized in Paneth cells of the rat small intestine. We therefore investigated the possibility that pIgR is also localized in human Paneth cells. Double-labeled fluorescent immunohistochemistry and double-labeled fluorescent in situ hybridization were used to determine RNA and protein expression in normal human small intestine. Both pIgR mRNA and protein were colocalized with lysozyme in normal human Paneth cells. Furthermore, IgA was colocalized with lysozyme in the secretory granules of human Paneth cells. This is the first study to demonstrate that pIgR and IgA are colocalized in the secretory granules of human Paneth cells. These findings suggest that, in addition to their well-recognized role in innate immunity, Paneth cells are involved in IgA-mediated acquired immunity in the gastrointestinal tract. Furthermore, these results add to accumulating evidence that Paneth cells participate in intestinal inflammation.