Duodenal Biopsy Samples Research Articles

Serotonergic reinforcement of intestinal barrier function is impaired in irritable bowel syndrome.

Alterations in serotonergic (5-HT) metabolism and/or intestinal integrity have been associated with irritable bowel syndrome (IBS). To assess the effects of the precursor of 5-HT, 5-hydroxytryptophan (5-HTP), on mucosal 5-HT availability and intestinal integrity, and to assess potential differences between healthy controls and IBS patients. Fifteen IBS patients and 15 healthy volunteers participated in this randomised double-blind placebo-controlled study. Intestinal integrity was assessed by dual-sugar test and by determining the mucosal expression of tight junction proteins after ingestion of an oral bolus of 100 mg 5-HTP or placebo. Mucosal serotonergic metabolism was assessed in duodenal biopsy samples. 5-HTP administration significantly increased mucosal levels of 5-HIAA, the main metabolite of 5-HT, in both healthy controls (7.1 ± 1.7 vs. 2.5 ± 0.7 pmol/mg, 5-HTP vs. placebo, P = 0.02) and IBS patients (20.0 ± 4.8 vs. 8.1 ± 1.3 pmol/mg, 5-HTP vs. placebo, P = 0.02), with the latter group showing a significantly larger increase. Lactulose/L-rhamnose ratios were significantly lower after administration of 5-HTP (P < 0.05) in healthy controls and were accompanied by redistribution of zonula occludens-1 (ZO-1), pointing to reinforcement of the barrier. In IBS, expression of the tight junction proteins was significantly lower compared to healthy controls, and 5-HTP resulted in a further decrease in occludin expression. Oral 5-HTP induced alterations in mucosal 5-HT metabolism. In healthy controls, a reinforcement of the intestinal barrier was seen whereas such reaction was absent in IBS patients. This could indicate the presence of a serotonin-mediated mechanism aimed to reinforce intestinal barrier function, which seems to dysfunction in IBS patients.

Immunohistochemical and Morphometric Analysis of Intestinal Full-thickness Biopsy Samples from Cats with Lymphoplasmacytic Inflammatory Bowel Disease

The distribution and numbers of CD3(+) T lymphocytes, immunoglobulin(+) plasma cells and calprotectin (L1)(+) macrophages was analyzed in full-thickness, formalin-fixed biopsy samples from the small intestine (duodenum, jejunum and ileum) and from the colon from nine cats with clinical signs of inflammatory bowel disease (IBD). All animals had lymphoplasmacytic enteritis or lymphoplasmacytic enterocolitis. Equivalent samples from the same intestinal regions from 12 healthy pet cats served as controls. Labelled cells in the lamina propria were counted by computer-aided morphometry. The different cell types were similarly distributed in both groups, but there were differences in their numbers. There were more CD3(+) T cells in the duodenum and jejunum of cats with IBD; however, the difference was only significant for the duodenum. There were significantly more IgA(+) cells in the duodenal crypt region. There were significantly more IgG(+) cells in the lower jejunal crypt region. Plasma cells expressing IgM were decreased in cats with IBD, but the difference was not significant. L1(+) macrophages were significantly decreased in the lower crypt area of the colon in cats with IBD and there was a trend to decreased L1(+) cells in the upper crypt area of the duodenum and jejunum. Comparison of the results of this study with previous findings on endoscopically-obtained duodenal biopsy samples from cats with IBD revealed some differences. These discrepancies might relate to differences between control cat populations, types of biopsy samples, methodological factors such as different counting techniques and the activity of the disease at the time of sampling.

Dietary medium-chain triglyceride supplementation has no effect on apolipoprotein B-48 and apolipoprotein B-100 kinetics in insulin-resistant men

Medium-chain triglyceride (MCT) supplements are used by clinicians to treat patients with severe hypertriglyceridemia who are at risk of pancreatitis. However, the potential mechanisms underlying the effects of MCT on triglyceride-rich lipoprotein (TRL) metabolism have not yet been thoroughly examined in humans. This double-blind randomized crossover study compared the impact of 4 wk of supplementation with 20 g MCT oil/d or 20 g corn oil/d on the kinetics of apolipoprotein (apo) B-48-containing TRLs and apo B-100-containing very-low-density lipoprotein (VLDL), as well as on the expression of key intestinal genes involved in lipid metabolism in 28 obese, insulin-resistant men. The in vivo kinetics of TRL apo B-48 and VLDL apo B-100 were assessed by using a primed-constant infusion of l-[5,5,5-d3]leucine for 12 h in the fed state. Real-time polymerase chain reaction quantification was performed on duodenal biopsy samples taken at the end of each phase of supplementation. Compared with corn oil, MCT supplements had no significant effect on plasma lipoprotein profile or TRL apo B-48 and VLDL apo B-100 kinetics. Positive correlations were observed between the intestinal expression of several key genes involved in lipoprotein metabolism in a subgroup of participants (n = 16) after MCT supplementation. However, there was no difference between MCT and the corn oil control supplement in the intestinal messenger RNA expression levels of these key genes. These data indicate that short-term supplementation with MCT has a neutral effect on TRL apo B-48 and VLDL apo B-100 kinetics and on the intestinal expression of genes involved in lipid and fatty acid metabolism in men with insulin resistance. This trial was registered at www.clinicaltrials.gov as NCT01806142.

Evaluation of endoscopically obtained duodenal biopsy samples from cats and dogs in an adapter-modified Ussing chamber

This study was conducted to evaluate an adapter-modified Ussing chamber for assessment of transport physiology in endoscopically obtained duodenal biopsies from healthy cats and dogs, as well as dogs with chronic enteropathies. 17 duodenal biopsies from five cats and 51 duodenal biopsies from 13 dogs were obtained. Samples were transferred into an adapter-modified Ussing chamber and sequentially exposed to various absorbagogues and secretagogues. Overall, 78.6% of duodenal samples obtained from cats responded to at least one compound. In duodenal biopsies obtained from dogs, the rate of overall response ranged from 87.5% (healthy individuals; n = 8), to 63.6% (animals exhibiting clinical signs of gastrointestinal disease and histopathological unremarkable duodenum; n = 15), and 32.1% (animals exhibiting clinical signs of gastrointestinal diseases and moderate to severe histopathological lesions; n = 28). Detailed information regarding the magnitude and duration of the response are provided. The adapter-modified Ussing chamber enables investigation of the absorptive and secretory capacity of endoscopically obtained duodenal biopsies from cats and dogs and has the potential to become a valuable research tool. The response of samples was correlated with histopathological findings.

Intestinal expression of metal transporters in Wilson’s disease

In Wilson’s disease (WND), biallelic ATP7B gene mutation is responsible for pathological copper accumulation in the liver, brain and other organs. It has been proposed that copper transporter 1 (CTR1) and the divalent metal transporter 1 (DMT1) translocate copper across the human intestinal epithelium, while Cu-ATPases: ATP7A and ATP7B serve as copper efflux pumps. In this study, we investigated the expression of CTR1, DMT1 and ATP7A in the intestines of both WND patients and healthy controls to examine whether any adaptive mechanisms to systemic copper overload function in the enterocytes. Duodenal biopsy samples were taken from 108 patients with Wilson’s disease and from 90 controls. CTR1, DMT1, ATP7A and ATP7B expression was assessed by polymerase chain reaction and Western blot. Duodenal CTR1 mRNA and protein expression was decreased in WND patients in comparison to control subjects, while ATP7A mRNA and protein production was increased. The variable expression of copper transporters may serve as a defense mechanism against systemic copper overload resulting from functional impairment of ATP7B.

Eicosapentaenoic and docosahexaenoic acid supplementation and inflammatory gene expression in the duodenum of obese patients with type 2 diabetes

BackgroundThe extent to which long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) from fish oil such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert their anti-inflammatory effects by down-regulating intestinal inflammation in humans is unknown. We investigated the impact of LCn-3PUFA supplementation on inflammatory gene expression in the duodenum of obese patients with type 2 diabetes.FindingsThis placebo-controlled randomized crossover study included 12 men with type 2 diabetes. After a 4-week run-in period, patients received in a random sequence 5 g/d of fish oil (providing 3 g of EPA + DHA) and a placebo (corn and soybean oil) for 8 weeks each. The two treatment phases were separated by a 12-week washout period. Gene expression was assessed by real-time polymerase chain reaction in duodenal biopsy samples obtained in the fasted state at the end of each treatment phase. Intestinal mRNA expression levels of interleukin (IL)-6 and tumor-necrosis factor (TNF)-α were hardly detectable after either treatment (<100 copies/105 copies of the reference gene ATP5o). Intestinal mRNA expression of IL-18 and of the transcription factor signal transducer and activator of transcription 3 (STAT3) was higher (>5000 copies/105 copies ATP5o) but still relatively low. EPA + DHA supplementation had no impact on any of these levels (all P ≥ 0.73).ConclusionsThese data suggest that duodenal cells gene expression of pro-inflammatory cytokines is low in patients with type 2 diabetes and not affected by EPA + DHA supplementation. Further studies are warranted to determine if inflammatory gene expression in other tissues surrounding the intestine is modulated by EPA + DHA supplementation.Trial registrationClinicalTrials.gov ID: NCT01449773

Gliadin Does Not Induce Mucosal Inflammation or Basophil Activation in Patients With Nonceliac Gluten Sensitivity

Nonceliac gluten-sensitive (NCGS) patients report intestinal and extra-intestinal symptoms shortly after ingesting gluten; these symptoms disappear on gluten-free diets, although these patients have no serologic markers of celiac disease or intestinal damage. In fact, there is no evidence for mucosal or serologic modifications in those individuals. We investigated immunologic responses of duodenal mucosa samples and peripheral blood basophils, isolated from NCGS patients, after exposure to gliadin. Participants underwent a complete clinical evaluation to exclude celiac disease while on a gluten-containing diet, a skin prick test to exclude wheat allergy, and upper endoscopy (n= 119) at 2 tertiary medical centers in Italy. Patients were considered to have NCGS based on their symptoms and the current definition of the disorder. Subjects were assigned to the following groups: patients with celiac disease on gluten-free diets (n= 34), untreated patients with celiac disease (n= 35), patients with NCGS (n= 16), or controls (n= 34). Duodenal biopsy samples collected during endoscopy were incubated with gliadin peptides, and levels of inflammatory markers were assessed. Peripheral blood basophils were extracted and incubated with gliadin peptides or a mix of wheat proteins; activation was assessed based on levels of CD203c, CD63, and CD45. Duodenal mucosa samples collected from 69 patients with celiac disease showed markers of inflammation after incubation with gliadin. Some, but not all, markers of inflammation were detected weakly in biopsy samples from 3 controls and 3 NCGS patients (P= .00 for all markers). There were no significant increases in the levels of CD63 and CD203c in NCGS patients. Unlike the duodenal mucosa from patients with celiac disease, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin. The invitro gliadin challenge therefore should not beused to diagnose NCGS.

The Duodenal Microbiota Composition of Adult Celiac Disease Patients Is Associated with the Clinical Manifestation of the Disease

Celiac disease is classically manifested in the gastrointestinal (GI) tract but extraintestinal symptoms, such as dermatitis herpetiformis (DH), are also common. Besides several well-known shared genetic risk factors and an environmental trigger, gliadin, factors determining the clinical outcome of the disease are not known. In this study, the role of duodenal microbiota in the celiac disease outcome was studied by analyzing mucosa-associated microbiota in celiac disease patients with a variety of intestinal and extraintestinal symptoms. Microbiota in duodenal biopsy samples obtained from 33 patients with celiac disease with GI, DH, anemia, or mixed symptoms, as well as screen-detected asymptomatic celiac disease and 18 control subjects were analyzed using PCR denaturing gradient gel electrophoresis and a subset of samples additionally by the 16S ribosomal RNA gene sequencing. The composition and diversity of mucosal microbiota was associated with the manifestation of celiac disease when analyzed using PCR denaturing gradient gel electrophoresis and the 16S ribosomal RNA gene sequencing. The patients with celiac disease with GI symptoms or anemia had lower microbial diversity than those with DH. Moreover, the patients with GI symptoms had different intestinal microbiota composition and structure, dominated by Proteobacteria, in comparison to those with DH or control subjects (patients with dyspepsia). The relatively similar intestinal microbiota composition in the control subjects and those with DH was characterized by the high abundance of Firmicutes. The two common outcomes of celiac disease, classical GI and extraintestinal manifestations, had marked differences on the diversity and composition of intestinal microbiota. This association suggested that intestinal microbiota may have a role in the manifestation of the disease.

Usefulness of the organ culture system when villous height/crypt depth ratio, intraepithelial lymphocyte count, or serum antibody tests are not diagnostic for celiac disease

The existence of mild forms of celiac disease (CD) can make the histology-based diagnosis difficult to reach. Since anti-endomysium (EMA) and anti-tissue transglutaminase (anti-tTG) are detectable in culture supernatants of duodenal biopsies from CD patients, our aim was to assess if this system can support the histology in the diagnostic work-up. A total of 559 suspected CD patients underwent serum EMA/anti-tTG detection, upper endoscopy with duodenal biopsy sampling, histologic analysis, and organ culture to detect EMA/anti-tTG in supernatants. A subgroup of 30 patients with organ culture positive results were put on a gluten-free diet (GFD). Their gluten-dependency was evaluated by the psychological general well-being and beck depression inventory indexes. Statistical analysis was performed by Cohen k inter-test, Friedman test, and Dunn multiple comparison. Two hundred forty-one out of 559 (43.1%) patients showed intestinal villous atrophy, whereas serum and organ culture EMA/anti-tTG were positive in 293/559 (52.4%) and 334/559 (59.7%) patients, respectively. The strength of agreement resulted good for serology vs histology (k = 0.730), good for organ culture vs histology (k = 0.662), and very good for serology vs organ culture (k = 0.852). After 12 months of GFD, psychological general well-being index significantly increased, and beck depression inventory index significantly decreased (P < 0.001 for each one). Data highlight the organ culture system as a useful tool to assist the histology in diagnosing CD, mainly in cases without villous atrophy or in seronegative patients. The marked improvement in quality of life after a GFD further supports the reliability of this system in diagnosing CD.

An enteral leucine supply modulates human duodenal mucosal proteome and decreases the expression of enzymes involved in fatty acid beta-oxidation

Leucine is well known to regulate protein metabolism in muscle. We recently reported that enteral leucine infusion decreased proteasome activity in human duodenal mucosa and enhanced intestinal cell proliferation, but its effects on gut proteome remain unknown. Therefore, we aimed to assess the effects of an enteral leucine infusion on the whole proteome of duodenal mucosa. In this work, 5 healthy volunteers received for 5h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g kg(-1) h(-1)) or maltodextrins supplemented with leucine (0.035 g kg(-1) h(-1)). At the end of infusion, endoscopic duodenal biopsy samples were collected and analyzed by 2D-PAGE. Eleven protein spots were differentially and significantly (P<0.05) expressed in response to the leucine-supplemented maltodextrins compared with maltodextrins alone. Forty percent of identified proteins by mass spectrometry were located in mitochondria. Four proteins were involved in lipid metabolism: HADHA, ACADVL and CPT2 expressions were reduced, whereas FABP1 expression was increased. In addition, the expression of DHA kinase involved in glycerol metabolism was also downregulated. Finally, leucine supplementation altered the duodenal mucosal proteome by regulating the expression of several enzymes mainly involved in lipid metabolism. These results suggest that leucine supplementation may slowdown fatty acid beta-oxidation in human duodenal mucosa.

The Rise of Tropheryma whipplei: a 12-Year Retrospective Study of PCR Diagnoses in Our Reference Center

Tropheryma whipplei is the causative agent of classic Whipple's disease (WD) and other clinical entities, such as localized infection. Asymptomatic carriers have also been reported, mainly based on the testing of fecal samples. Our objective was to undertake a retrospective analysis of molecular biology usage for the diagnosis of WD over a 12-year period in our reference center. We tested 27,923 samples from 15,473 patients. The number of patients tested and the number of patients with a positive PCR result for T. whipplei have increased significantly over the last 12 years (P < 0.0001). Overall, T. whipplei was more frequently recovered from stools (43%), saliva (15%), duodenal biopsy samples (12.5%), blood (5%), and cerebrospinal fluid (CSF) (6%) and less commonly from cardiac valves (3%), urine (0.5%), skin biopsy samples (1%), lymph nodes (2.5%), aqueous humor (0.5%), and intra-articular fluid (1%). Among all the positive samples, we observed that stool samples and skin biopsy samples exhibited a higher prevalence of positivity by real-time quantitative PCR (qPCR) at 10.07% and 15.4%, respectively. The number of patients with a positive PCR result for T. whipplei has increased significantly over the last 12 years, although the positive ratio has not changed. Improvements in diagnostic tools have contributed greatly toward greater knowledge of WD and, consequently, the interest of physicians in this condition. In addition, we propose here an update of the diagnostic strategy for WD when qPCR is being used.

Variable activation of immune response by quinoa (Chenopodium quinoa Willd.) prolamins in celiac disease

Celiac disease is an enteropathy triggered by dietary gluten found in wheat, barley, and rye. The current treatment is a strict gluten-free diet. Quinoa is a highly nutritive plant from the Andes, with low concentrations of prolamins, that has been recommended as part of a gluten-free diet; however, few experimental data support this recommendation. We aimed to determine the amount of celiac-toxic prolamin epitopes in quinoa cultivars from different regions of the Andes and the ability of these epitopes to activate immune responses in patients with celiac disease. The concentration of celiac-toxic epitopes was measured by using murine monoclonal antibodies against gliadin and high-molecular-weight glutenin subunits. Immune response was assessed by proliferation assays of celiac small intestinal T cells/interferon-γ (IFN-γ) and production of IFN-γ/IL-15 after organ culture of celiac duodenal biopsy samples. Fifteen quinoa cultivars were tested: 4 cultivars had quantifiable concentrations of celiac-toxic epitopes, but they were below the maximum permitted for a gluten-free food. Cultivars Ayacuchana and Pasankalla stimulated T cell lines at levels similar to those for gliadin and caused secretion of cytokines from cultured biopsy samples at levels comparable with those for gliadin. Most quinoa cultivars do not possess quantifiable amounts of celiac-toxic epitopes. However, 2 cultivars had celiac-toxic epitopes that could activate the adaptive and innate immune responses in some patients with celiac disease. These findings require further investigation in the form of in vivo studies, because quinoa is an important source of nutrients for patients with celiac disease.

Interactions Among Secretory Immunoglobulin A, CD71, and Transglutaminase-2 Affect Permeability of Intestinal Epithelial Cells to Gliadin Peptides

The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunoglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgA-CD71 complexes and intestinal permeability to the gliadin 3H-p31-49 peptide were analyzed in polarized monolayers of Caco-2 cells. Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgA-CD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p31-49, transport of intact 3H-p31-49 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apical-basal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

Abstract 458: Omega-3 Supplementation Does Not Affect Inflammatory Gene Expression in the Small Intestine of Patients with Type 2 Diabetes

Recent evidence suggests that diet-induced inflammation in the small intestine is linked to obesity and insulin resistance. Long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to have anti-inflammatory effects by down-regulating inflammatory gene expression in adipocytes and mononuclear cells. However, the extent to which EPA and DHA may exert their anti-inflammatory effects by down-regulating inflammation in the gut is unknown. The objective of the study was to investigate the impact of EPA+DHA supplementation on the expression of inflammatory genes in the small intestine of patients with type 2 diabetes. A total of 12 men with type 2 diabetes were recruited in this placebo-controlled randomized crossover study. After a 4-week run-in period, patients received in random sequence 5 g/d of fish oil providing 3 g of EPA+DHA or placebo (corn and soybean oil) for 8 weeks, each separated by a 12-week washout period. Gene expression was assessed by real-time PCR in duodenal biopsy samples obtained in the fasted state at the end of each treatment phase. Intestinal mRNA expression levels for interleukin(IL)-6 and tumor-necrosis factor(TNF)-α were hardly detectable after either treatment (&lt; 100 copies/10^5 copies of the reference gene ATP synthase O subunit, ATP5o). Intestinal mRNA expression of IL-18 and of the transcription factor STAT3 (signal transducer and activator of transcription 3) was higher (&gt; 5000 copies/10^5 copies ATP5o) but still relatively low and EPA+DHA supplementation had no impact on any of these levels (P ≥ 0.73 between treatments). Plasma C-reactive protein (CRP) concentrations after supplementation with EPA+DHA (5.2 ± 4.5 mg/L) were not significantly different than values measured after placebo (8.0 ± 10.8 mg/L, P = 0.2). In conclusion, these data suggest that gene expression of pro-inflammatory cytokines and STAT3 in duodenal cells is low in patients with type 2 diabetes and not affected by EPA+DHA supplementation.

Does acute tryptophan depletion affect peripheral serotonin metabolism in the intestine?

Serotonin (5-hydroxytryptamine; 5-HT), a tryptophan metabolite, plays an important regulatory role in the human central nervous system and in the gastrointestinal tract. Acute tryptophan depletion (ATD) is currently the most widely established method to investigate 5-HT metabolism. The aim of this study was to assess the effect of an acute decrease in the systemic availability of tryptophan on intestinal 5-HT metabolism and permeability. Thirty-three healthy volunteers (17 with ATD, 3 of whom dropped out; 16 placebo) participated in this randomized placebo-controlled study. Plasma and duodenal mucosal concentrations of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and kynurenic acid (KA) were measured by HPLC-mass spectrometry. Intestinal barrier function was assessed with a multisugar plasma test, and analysis of tight junction transcription was performed in duodenal biopsy samples obtained by gastroduodenoscopy. Mucosal 5-HT, 5-HIAA, and KA concentrations remained unaltered by ATD. In contrast, ATD significantly decreased plasma 5-HT (P < 0.05) and 5-HIAA (P < 0.0001) concentrations. After endoscopy, a significant increase in plasma 5-HT concentrations was observed in the placebo group (P = 0.029) compared with the ATD group. Moreover, a significant increase in plasma KA concentrations over time was found in the placebo group (P < 0.05). No changes in intestinal barrier function were observed. An acute decrease in precursor availability does not affect mucosal concentrations of serotonergic metabolites, in contrast with systemic concentrations. ATD alters biochemical responses to acute stress from the endoscopic examination reflected by lower 5-HT concentrations. Changes in 5-HT concentrations were paralleled by alterations in KA concentrations, which suggest competition between the 2 metabolic pathways for the mutual precursor. This trial was registered at clinicaltrials.gov as NCT00731003.

Down regulation of anti-inflammatory cytokines in ileal mucosa associates with active disease in juvenile idiopathic arthritis

Methods We performed gastroduodenoscopy and colonoscopy in 16 children with JIA suffering from gastrointestinal (GI) symptoms. The control children (n=25) had GIsymptoms, but eventually were shown not to have any significant GI disease. We analyzed in duodenal and ileal biopsy samples the mRNA expression of IFNg, TGFb, IL6, IL4, FoxP3, IL17A, CTLA4, ICOS, GITR, IL10, IL18, IL12p40, IL23, TLR2, TLR4, and TLR5 with RT-PCR. We also assessed correlation between the clinical markers of JIA and the mucosal mRNA levels.

Effects of an enteral glucose supply on protein synthesis, proteolytic pathways, and proteome in human duodenal mucosa

Previous studies have shown that the glucose supply reduces postoperative insulin resistance and improves patient outcomes. However, the effects of luminal glucose on intestinal mucosal proteins remain unknown. We aimed to assess the effects of an enteral glucose supply on protein synthesis, proteolytic pathways, and proteome in human duodenal mucosa. Twenty healthy volunteers received a 5-h enteral infusion of either saline or glucose (0.12 g · kg(-1) · h(-1)). Simultaneously, a continuous intravenous infusion of l-[1-(13)C]leucine (12 μmol · kg(-1) · h(-1)) was maintained until endoscopy. The duodenal mucosal protein fractional synthesis rate (FSR) was calculated from leucine enrichments assessed in protein and free amino acid pools by gas chromatography-mass spectrometry. Cathepsin D, calpains, and chymotrypsin-like proteasome mucosal activities were evaluated by using specific fluorogenic substrates. A 2-dimensional PAGE-based comparative proteomics analysis was also performed on additional duodenal mucosal biopsy samples to identify differentially expressed proteins. Duodenal mucosal protein FSR and protease activities were not affected by glucose infusion relative to saline. Nevertheless, the comparative proteomics analysis indicated that 10 protein spots were significantly differentially expressed (ie, at least ±1.5-fold modulated; Student's t test, P < 0.05) in response to the glucose infusion relative to saline. Of the 8 proteins identified by mass spectrometry, α-enolase, cytoplasmic aconitate hydratase, and glutathione S-transferase ω-1 were upregulated, whereas epoxide hydrolase 2 was downregulated. Enteral glucose supply affected neither duodenal mucosal protein FSR nor activities of mucosal proteases but altered the duodenal mucosal proteome by modulating the expression of several enzymes involved mainly in carbohydrate and xenobiotic metabolism. This trial is registered at clinicaltrials.gov as NCT00213551.

Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum

Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 μmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/μg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/β, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/β-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.

Expression of GH/IGF-1 Axis Compounds in Moderate-Severe Crohn's Disease: Role of Mucosal Inflammation and Anti TNF-α Treatment

AIM: Growth failure in children and adolescents, weight loss and catabolism in adults, are well known features of active Crohn's disease (CD). Increasing evidence shows that these features may be due to growth hormone (GH) resistance caused by persistent chronic inflammation. Aim of this study was to evaluate, in CD patients, a possible peripheral GHresistance (intestinal mucosa), and eventual modifications following anti TNF-α treatment. MATERIALS AND METHODS: 9 patients (5 F, median age 42.2 years, range 19-67) with moderate-severe active CD (CDAI>220), consecutively scheduled to receive three infliximab infusions at a dose of 5 mg/kg for induction of remission, were studied. Biopsy specimens from normal appearing duodenal mucosa were collected, in the 2 weeks prior to the first and after the third infusion. Confocal and real time PCR analysis of insulin-like growth factor 1 (IGF-1), activated signal transducer and activator of transcription protein 5 (phosphoSTAT5) and suppressor of cytokine signalling protein 3 (SOCS-3) were carried out and normalised to GAP, 18S and H3 referring genes. Four dyspeptic patients (2 F, median age 39 years, range 24-57), who underwent upper endoscopy with duodenal biopsy sampling, represented our control group. Differences between CD patients and controls were analysed by one-way ANOVA and REST-coupled ANOVA analysis. RESULTS: In CD patients, a significant inverse correlation was found when comparing phospho-STAT5 levels with CDAI (r=-0.71; p=0.003). Confocal analysis showed a significant decrease in phospho-STAT5 immunoreactivity in sections from CD patients, compared to controls (p<0.001). After treatment, phospho-STAT5 levels significantly increased (p<0.001). Molecular data for phospho-STAT5 were in keeping with biochemical findings. At baseline, compared with controls, in CD patients, the PCR analysis of IGF-1, showed a trend towards a decrease, while SOCS3 showed a trend towards an increase. Following treatment, SOCS-3 and IGF-1 target genes showed a trend toward a decrease and an increase, respectively. Nevertheless, following infliximab induction treatment, IGF-1, phospho-STAT5 and SOCS-3 levels did not reach those of the control group. CONCLUSION: The present study underline the effect of inflammation on the growth hormone axis not only at the level of hepatic metabolism, but also in the intestinal mucosa, since biologic agents able to inhibit pro-inflammatory cytokines, in particular TNF-α, the main inflammatory mediator, reverse growth hormone resistance. These therapeutic approaches could prevent the onset of those clinical conditions with a negative impact on child growth and quality of life of adults.

IL-15 Interferes With Suppressive Activity of Intestinal Regulatory T Cells Expanded in Celiac Disease

Celiac disease (CD) is a condition in which the regulation of the mucosal immune response to dietary gliadin might be altered. The transcription factor forkhead box P3 (Foxp3) has been identified as a marker of a subset of regulatory T cells (Treg). In this study, we have investigated the presence and the suppressive function of Treg cells in the celiac small intestinal mucosa, their correlation with the disease state, and the inducibility by gliadin in an organ culture system; moreover, we tried to define whether interleukin 15 (IL-15), overexpressed in CD, could influence the regulatory activity of such cells. The expression of Foxp3, CD3, CD4, and CD8 were analyzed by immunohistochemistry and flow cytometry in duodenal biopsies taken from patients with untreated CD, treated CD, and from non-CD controls, as well as in vitro cultured biopsy samples from treated CD patients, upon challenge with gliadin. Furthermore, we analyzed the suppressive function of CD4+CD25+ T cells, isolated from untreated CD biopsy samples, on autologous responder CD4+CD25- T cells, in the presence of a polyclonal stimulus, with or without IL-15. Higher density of CD4+CD25+Foxp3+ T cells was seen in duodenal biopsy samples from active CD patients in comparison with treated CD and non-CD controls. In coculture, CD4+CD25+ T cells were functionally suppressive, but their activity was impaired by IL-15. Cells from CD subjects showed increased sensitivity to the IL-15 action, likely due to enhanced expression of IL-15 receptor. Finally, we demonstrated an expansion of Foxp3 in treated CD mucosa following in vitro challenge with gliadin. These data suggest that CD4+CD25+Foxp3+ T cells are induced in situ by gliadin. However, their suppressor capacity might be impaired in vivo by IL-15; this phenomenon contributes to maintain and expand the local inflammatory response in CD.