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Abstract Corticosteroids (CS), first-line therapeutics for Crohn’s disease (CD) with moderate or severe disease activity, were found to restore intestinal permeability in CD patients, whereas the underlying molecular events are still largely unknown. This study aimed to investigate the effect and mechanisms of CS prednisolone on epithelial barrier using CD patient-derived intestinal organoids. 3D intestinal organoids were generated from colon biopsies of inactive CD patients. To mimic the inflammatory microenvironment, a mixture of cytokines containing TNF-α, IFN-γ, and IL-1β were added to the organoid culture with or without pre-incubation of prednisolone or mifepristone. Epithelial permeability of the organoids was assessed by FITC-D4 flux from the basal to luminal compartment using confocal microscopy. Expression of junctional components were analyzed by qRT-PCR, immunofluorescence staining, and western blot. Activity of signaling pathways were analyzed using western blot. Exposure of the cytokines significantly disrupted epithelial barrier of the intestinal organoids, which was partially restored by prednisolone. On the molecular level, the cytokine mixture resulted in a significant reduction in E-cadherin and ILDR-1, an increase in CLDN-2, MLCK, and STAT1 phosphorylation, whereas prednisolone ameliorated the abovementioned effects induced by the cytokine mixture. This study demonstrates that prednisolone confers a direct effect in tightening the epithelial barrier, identifies novel junctional targets regulated by prednisolone, and underscores intestinal barrier restoration as a potential mechanism that contributes to the clinical efficacy of prednisolone in CD patients. Key messages Prednisolone confers a direct preventive effect against cytokine-induced barrier dysfunction. Prednisolone regulates the expression of CLDN-2, E-cadherin, and ILDR-1. The effect of prednisolone is GR-, MLCK-, and STAT1-dependent.

Background & aims: Ageing is associated with an increased risk of frailty, intestinal microbiota perturbations, immunosenescence and oxidative stress. Prebiotics such as galacto-oligosaccharides (GOS) may ameliorate these ageing-related alterations. We aimed to compare the faecal microbiota composition, metabolite production, immune and oxidative stress markers in prefrail elderly and younger adults, and investigate the effects of GOS supplementation in both groups. Methods: In a randomised controlled cross-over study, 20 prefrail elderly and 24 healthy adults received 21.6 g/day BiotisTM GOS (containing 15.0 g/day GOS) or placebo. Faecal 16S rRNA gene-based microbiota and short-chain fatty acids were analysed at 0,1 and 4 weeks of intervention.Volatile organic compounds were analysed in breath, and stimulated cytokine production, CRP, malondialdehyde, trolox equivalent antioxidant capacity (TEAC) and uric acid (UA) in blood at 0 and 4 weeks. Results: Principle coordinate analysis showed differences in microbial composition between elderly and adults (P

Hesperidin and naringin are citrus flavonoids with known anti-oxidative and anti-inflammatory properties. Evidence from previous studies indicates that both these compounds and the metabolites that are formed during intestinal metabolism are able to exert beneficial effects on intestinal barrier function and inflammation. However, so far, studies investigating the relative contributions of the various compounds are lacking. Therefore, we assessed the effect of citrus flavonoids and their intestinal metabolites on immune-mediated barrier disruption in an

Intestinal epithelial barrier is affected by multiple factors, such as tumour necrosis factor-α (TNF-α). Plasma concentration of TNF-α is higher in patients with Crohn’s disease (CD) than healthy controls (HC) and correlates positively with disease activity. This study aimed to determine the effect of plasma from active, inactive CD patients on intestinal barrier function and to investigate the underlying mechanism. Plasma samples were collected from CD patients and HC. 3D Caco-2 cysts were treated with plasma or TNF-α, with or without pre-incubation of adalimumab (a monoclonal antibody that antagonizes TNF-α) or JNK inhibitor SP600125. The results demonstrated that exposure of the cysts to plasma from CD patients resulted in enhanced paracellular permeability in a disease activity-dependent manner. Compared to HC, active CD plasma decreased ZO-1 and OCCLUDIN expression on mRNA and protein levels, and led to an increased JNK phosphorylation. Pre-incubation with adalimumab or SP600125 ameliorated TJ disruption and barrier dysfunction induced by plasma from CD patients. These results indicate that plasma from CD patients is able to induce epithelial barrier disruption, in part through TNF-α induced TJs modulation. The data also demonstrate an involvement of MAPK pathway, in particular the JNK isoform, in CD patient plasma-induced barrier dysfunction.

Intestinal barrier dysfunction is a pathogenic hallmark in Crohn?s disease (CD). Identifying key players that regulate intestinal barrier may provide novel leads for therapeutic intervention. Interleukin-28A (IL-28A) is a newly identified IL-10/interferon cytokine family member, with its most implicated function being antiviral and anti-proliferative properties. However, the role and underlying mechanisms of IL-28A in the regulation of epithelial barrier in CD remain so far unexplored. IL-28A levels were measured in the plasma and biopsies of CD patients and healthy subjects. CD patient-derived intestinal organoids were characterized by differentiation gene markers and then exposed to TNF-a, IFN-c, IL-113 or LPS, or IL-28A with or without GLPG0634 (filgotinib). Epithelial permeability was assessed by FITC-D4 flux. Expression of junctional components was analyzed by qRT-PCR, immunofluorescence staining, or Western blotting. JAK-STAT activity was analyzed by Western blotting. IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IL-28A and its receptor complex IL-28AR/IL-10R2 were detected in CD patient-derived intestinal organoids and showed a selective response to IFN-c exposure. IL-28A triggered epithelial barrier disruption and accompanied by reduced ZO-1 and E-cadherin expression. This effect was mediated by JAK-STAT1 pathway. Pre-incubation with the JAK1 inhibitor filgotinib ameliorated the barrier dysfunction induced by IL-28A. These results identified IL-28A as a novel regulator of epithelial barrier function and could be a putative target for CD treatment. We provide novel basic evidence that restoring intestinal barrier is a potential mechanism that contributes to the clinical benefits of JAK1 inhibitor in patients with CD.NEW & NOTEWORTHY IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IFN-c exposure stimulated IL-28A expression in intestinal organoids. Partially mimicking the effect of IFN-c, IL-28A impaired epithelial barrier function and disrupted junctional components through the activation of JAKSTAT1 signaling, whereas JAK1 inhibitor ameliorated the above-mentioned effects of IL-28A. These findings highlight the newly identified cytokine IL-28A as a novel contributor to CD pathogenesis and could be a putative target for CD treatment. We also provide new evidence for potential applications of JAK inhibition in CD therapy.

Brown adipose tissue (BAT) might be a beneficial mediator in the development and treatment of nonalcoholic steatohepatitis (NASH). We aim to evaluate the gene expression of BAT activity-related genes during the development and the dietary and surgical treatment of NASH. BAT was collected from male C57BL/6J mice that received a high fat-high sucrose diet (HF-HSD) or a normal chow diet (NCD) for 4 and 20 weeks (n=8–9 per dietary group and timepoint) and from mice that underwent dietary intervention (return to NCD) (n=8), roux-en-y gastric bypass (RYGB) (n=6), or sham procedure (n=6) after 12 weeks HF-HSD. Expression of BAT genes involved in lipid metabolism (Cd36 and Cpt1b; p

Intestinal barrier function is suggested to decrease with aging and may be improved by pectin intake. The aim of this study was to investigate the effects of four weeks pectin supplementation on gastrointestinal barrier function in vivo and ex vivo in different age groups. In a randomized, double-blind, placebo-controlled, parallel study, 52 healthy young adults (18&ndash, 40 years) and 48 healthy elderly (65&ndash, 75 years) received 15 g/day pectin or placebo for four weeks. Pre- and post-intervention, in vivo gastrointestinal permeability by a multisugar test, and defense capacity in mucosal samples were assessed. Sigmoid biopsies were collected post-intervention from subgroups for Ussing chamber experiments and gene transcription of barrier-related genes. Pectin intervention did not affect in vivo gastroduodenal, small intestinal, colonic, and whole gut permeability in young adults nor in elderly (p &ge, 0.130). Salivary and fecal sIgA and serum IgA were not significantly different between pectin versus placebo in both age groups (p &ge, 0.128). In both young adults and elderly, no differences in transepithelial electrical resistance and fluorescein flux (p &ge, 0.164) and relative expression of genes analyzed (p &ge, 0.222) were found between pectin versus placebo. In conclusion, intestinal barrier function was not affected by four weeks pectin supplementation neither in healthy young adults nor in healthy elderly.

Background: Liver cirrhosis is associated with intestinal epithelial barrier dysfunction, which may be affected by oxidative stress. Studies in cirrhotic rats provided evidence for intestinal oxidative stress, but studies in cirrhotic patients are scarce. We have shown intestinal barrier dysfunction in patients with compensated cirrhosis. AIM: The present study aimed to investigate whether oxidative stress occurs in the intestinal mucosa of compensated cirrhotic patients and may contribute to barrier dysfunction. Material and methods: Oxidative stress was studied in duodenal and sigmoid biopsies from 15 cirrhotic patients and 22 controls by analyzing transcription of genes involved in glutathione and uric acid metabolism using quantitative real-time polymerase chain reaction. Protein levels of glutathione and glutathione disulphide were measured and the glutathione/glutathione disulphide ratio was calculated as marker of oxidative stress. In addition, intestinal myeloperoxidase and fecal calprotectin were determined. Result: Gene transcription of glutathione synthetase and glutathione reductase were significantly different in duodenal and sigmoid biopsies of cirrhotic patients vs. controls, but no alterations were found for other genes nor for glutathione, glutathione disulphide, glutathione/glutathione disulphide ratio and intestinal myeloperoxidase and fecal calprotectin concentrations. Conclusion: This study did not find indications for oxidative stress and low-grade inflammation in the small and large intestine of stable compensated cirrhotic patients. Although these preliminary findings need further validation, we found intestinal oxidative stress not to be a major mechanism contributing to epithelial barrier dysfunction in patients with compensated cirrhosis.

In the absence of visible mucosal damage, it is hypothesized that the esophageal mucosal barrier is functionally impaired in patients with non-erosive reflux disease (NERD). The aim of the present study was to perform an exploratory analysis of the mucosal barrier in NERD compared to erosive esophagitis (EE) and controls. A second aim was to explore TRPV1 gene transcription in relation to the mucosal barrier function and heartburn symptoms. In this prospective study, 10 NERD patients, 11 patients with active erosive esophagitis and 10 healthy volunteers were included. Biopsies from non-eroded mucosa were obtained for (1) ex vivo analyses (Ussing chamber) of transepithelial electrical resistance (TEER) and permeability (2) gene transcription of tight-junction proteins and transient receptor potential vanilloid subfamily member 1 (TRPV1). No differences in TEER or permeability were found between NERD and healthy volunteers, whereas TEER was lower in patients with erosive esophagitis. TRPV1 gene transcription was not significantly different between EE, NERD and controls. Conclusions: esophageal mucosal barrier function and TRPV1 transcription is not significantly altered in NERD patients. Future research is needed to explore other potential mechanisms that may account for the high symptom burden in these patients.

With great interest, we have read the article by Dotti et al ,1 who analysed the transcriptional signature of epithelial organoid cultures (EpOCs) generated from patients with UC and non-IBD controls. They identified a set of genes that were differentially expressed between UC and control EpOCs, among which several were associated with epithelial secretory and antimicrobial defense. The authors further stated that the organoid culture system could serve as a model to explore the intestinal epithelium of patients with IBD. We hereby provide new support that intestinal organoid culture is also a valuable model to study epithelial barrier function, which has emerged as hallmark for IBD pathogenesis. Although expression of genes and proteins related to epithelial junctional complex has been investigated in biopsies of patients with IBD, functional analyses of the intestinal barrier remains challenging. To this end, we established an EpOCs library from …

OBJECTIVE: To investigate the expression of eNOS and iNOS mRNA and protein in adipose tissue from subcutaneous (s.c.) and omental adipose tissue of obese subjects. DESIGN: Subcutaneous and omental adipose tissue was obtained from subjects undergoing weight reduction surgery. Messenger RNA and protein levels were measured in tissue extracts and related to basal lipolysis, which was measured in isolated adipocytes from the same subjects. SUBJECTS: Eight overweight but otherwise healthy male subjects (age 43.4±10.3 y, BMI 39±3.5 kg/m2, mean±s.e.m.). MEASURESMENTS: For mRNA detection a competitive reverse transcription polymerase chain reaction method was used while protein was detected by Western blot. Glycerol release was determined in isolated adipocytes using a standard luminometric assay. RESULTS: Tissue mRNA levels for eNOS in s.c. tissue were 6098±1969 amol/mg RNA and in omental tissue 6987±2914 amol/mg RNA (mean±s.e.m., P=0.75). iNOS mRNA levels were substantially lower; in s.c. tissue 227±127 amol/mg RNA and in omental tissue 245±162 amol/mg RNA (P=0.8). In Western blot, eNOS protein levels in s.c. and omental tissue were 1.88±2.0 and 7.47±4.11 (OD/mm2 100 µg total protein, P=0.0063), respectively. iNOS protein was expressed at significantly lower levels and barely detectable in both s.c. and omental tissue. Basal rate of lipolysis was two times higher in s.c. compared to omental fat cells (P=0.028). CONCLUSIONS: eNOS protein is markedly increased in omental compared to s.c. adipose tissue in human obese subjects, probably due to post-transcriptional mechanisms. Since basal lipolysis is much lower in omental vs s.c. adipose tissue it is possible that regionally increased NO production, primarily by eNOS, may be involved in the site difference of basal lipolysis in obese subjects.

Studies have shown evidence of production of nitric oxide (NO) in adipose tissue, as well as inhibition of lipolysis by NO. We have analyzed nitric oxide synthase (NOS) expression in subcutaneous adipose tissue from 13 nonobese and 18 obese male subjects. Using a competitive reverse transcription polymerase chain reaction method, endothelial (eNOS) and inducible (iNOS), but not neuronal (nNOS), nitric oxide synthase mRNA expression was detected in isolated fat cells and pieces of adipose tissue. Tissue mRNA levels for eNOS were 3,814 ± 825 and 5,956 ± 476 amol/mg RNA (P = 0.043), and for iNOS 306 ± 38 and 332 ± 48 amol/mg RNA, for nonobese and obese individuals, respectively. Western blotting revealed similar eNOS protein levels in isolated fat cells and adipose tissue pieces. Protein levels for eNOS in nonobese and obese individuals, respectively, were (in optical density [OD] units per mm2 per 100 μg of total protein) 0.11 ± 0.08 and 2.80 ± 1.30 (P = 0.043). iNOS protein was detectable, but not measurable, at low levels in a subset of obese patients (3 of 10). iNOS protein levels could not be detected in nonobese individuals. Hormone-sensitive lipase (HSL), the key regulating enzyme in lipolysis, is reduced in obesity. The expression of HSL protein in subcutaneous adipose tissue was studied in the same subset of patients; in agreement with previous results, HSL levels were reduced in obese subjects: 4.64 ± 1.10 and 1.27 ± 0.35 (P = 0.012) in nonobese and obese subjects, respectively. In conclusion, this study shows that eNOS and iNOS, but not nNOS, are present in human subcutaneous adipose tissue. Gene expression and protein levels of eNOS are increased, whereas HSL protein levels are decreased in obesity. It is speculated that increased NO production, preferably by eNOS, and decreased HSL levels may cause decreased subcutaneous adipose tissue lipolysis in obesity. —Elizalde, M., M. Rydén, V. van Harmelen, P. Eneroth, H. Gyllenhammar, C. Holm, S. Ramel, A. Ölund, P. Arner, and K. Andersson. Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans. J. Lipid Res. 2000. 41: 1244–1251.

OBJECTIVE: To investigate in obese subjects the relationship between angiotensinogen gene expression in the abdominal omental and subcutaneous adipose tissue on the one hand and body fat distribution as measured by waist-to-hip ratio (WHR) on the other hand and to compare angiotensinogen gene expression between the two adipose tissue regions. SUBJECTS: Twenty obese subjects undergoing weight reduction surgery with adjustable gastric banding (12 men, eight women; WHR 0.89–1.09; body mass index (BMI) 29–51 kg/m2, age 26–54 y). MEASUREMENTS: Omental and subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured by competitive quantitative reverse transcriptase–polymerase chain reaction. RESULTS: Angiotensinogen mRNA levels were one-third higher in the omental than in the subcutaneous adipose tissue region (P=0.02). The 18S rRNA levels did not differ significantly between the two adipose tissue regions. WHR correlated positively and significantly with angiotensinogen mRNA in both the subcutaneous and the omental adipose tissue (r=0.5). This relationship was independent of age and BMI. However, WHR did not correlate with 18S rRNA in any of the adipose tissue regions. CONCLUSION: The angiotensinogen gene in adipose tissue might be involved in the development of upper-body obesity.

1. Possible nitric oxide (NO)-mediated effects on lipolysis were investigated in vivo in human subcutaneous adipose tissue using microdialysis, as well as in vitro on isolated fat cells of non-obese, healthy volunteers. NO donors were added to the ingoing dialysate solvents. 2. Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates. 3. It was shown that the NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA), but not the biologically inactive enantiomer N(G)-monomethyl D-arginine (D-NMMA), increased glycerol levels in the microdialysates without causing a change of local blood flow. In addition, L-NMMA increased glycerol levels in the microdialysate when local blood flow was stimulated with hydralazine. 4. Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro. 5. Expression of inducible nitric oxide synthase (iNOS) in human adipose tissue was shown by Western blot analysis. Biologically active NOS was demonstrated by measuring total enzymatic activity. 6. In conclusion, the data demonstrate that inhibition of NO release in subcutaneous adipose tissue results in an increased lipolysis in vivo. These effects, which were also observed in vitro, are independent of local blood flow changes. Furthermore, the demonstration of enzymatic NOS activity and the expression of inducible nitric oxide synthase (iNOS) in adipose tissue indicate that locally synthesized NO may play a role in the physiological control of lipolysis in human adipose tissue.

Background: Esophageal epithelial integrity is considered an important factor in the prevention of tissue damage by gastric refluxate.We hypothesized that in patients with gastroesophageal reflux disease (GERD) the esophageal epithelial barrier function is impaired and less resistant in response to acid exposure. We therefore investigated esophageal epithelial integrity in GERD patients and in healthy controls both in the basal state and in response to acid exposure Methods: 14 patients with chronic GERD (8 with erosive esophagitis, 6 with non-erosive reflux disease) and 10 healthy controls (HC) were enrolled. Before endoscopy, GERD patients discontinued PPI therapy for 7 days. Six esophageal biopsies frommacroscopically normal mucosa were obtained approximately 5 cm above the gastroesophageal junction and directly transferred to a mini-Ussing chamber system. After an equilibration period, baseline TEER was assessed. Half of the biopsies were then exposed at their luminal side to an acidic solution (pH1) for 30 minutes. During exposure and after removal of the acidic solution, changes in TEER were analyzed relative to baseline TEER. Permeation to the paracellular permeation marker fluorescein (375 DA 1 mg/ml) was assessed in all biopsies (previous acid-exposed and non-exposed) for 120 minutes. Only subjects with at least two adequate biopsies (one for acid exposure, one as a control) were included. Results: Esophageal epithelium of GERD patients showed lower baseline TEER (127.7±13.3 Ω vs. 174.3±17.6 Ω, p=0.04) and a trend toward higher transmucosal permeation of fluorescein in the nonexposed biopsies when compared to healthy controls (serosal concentration (pmol/ml) after 120 min: 48.2 (7.6-66.7) vs. 6.8 (3.6-20.2), p=0.09 and AUC: 79.8 (12.9-135.6) vs. 9.0 (5.5-31.3), p=0.07). Acid exposure provoked a fall in TEER that was equal for the biopsies of GERD patients and healthy controls (-52.1±2.5% vs. -50.0±4.4% of baseline TEER). After removal of the acidic solution, TEER recovered also to a similar extent in GERD patients and healthy controls (89.6±3.8% vs. 93.8±3.0% of baseline TEER). However, maximum TEER was reached earlier in biopsies of GERD patients (54±9 min vs. 83±6 min (HC), p= 0.02) and at the end of the experiment, TEER relative to baseline was lower in biopsies of GERD patients (73.7±5.8% vs. 89.7±3.3% (HC) of baseline TEER, p

To study the effects of neonatal asphyxia on gene expression of the dopaminergic systems, we determined quantitatively the mRNA levels of tyrosine hydroxylase, dopamine transporter, dopamine D(1) and D(2) receptors in substantia nigra/ventral tegmental area, striatum and limbic area. The mRNA levels were determined at one and 4 weeks after asphyxia by a quantitative reverse transcription polymerase chain reaction method. Spontaneously and Caesarean section born rats showed similar mRNA levels with the exception of an increase of tyrosine hydroxylase mRNA levels in the limbic area of 4-week-old animals. Five min of asphyxia did not change the mRNA levels in any region compared to that in the spontaneously born rats. Fifteen and twenty min of asphyxia induced region-specific alterations in mRNA levels. In SN/VTA an increase of tyrosine hydroxylase mRNA levels in the 1-week-old rats and in striatum an increase of D(1) and D(2) dopamine receptor mRNA levels in the 4-week-old rats were observed. Fifteen min of asphyxia induced a selective increase of D(1) and D(2) dopamine receptor mRNA levels in the limbic area of 4-week-old rats. These observations indicate that neonatal asphyxia triggers a cascade of gene expressions for tyrosine hydroxylase and D(1) and D(2) dopamine receptors. In 1-week-old rats, the gene expression of tyrosine hydroxylase increased in the cell body region substantia nigra/ventral tegmental area. This change may increase the D(1) and D(2) dopamine receptor expression in the target regions striatum and limbic area during further development.

The influence of an acute dose of lead nitrate or cadmium chloride on the activity of glutathione-S-transferase (GST) was investigated in rats. CdCl2 (10 mumol/kg) did not influence the enzyme activity in either organ. In contrast Pb(NO3)2 (100 mumol/kg) caused a significant increase of GST activity in both organs. SDS polyacrylamide gel electrophoresis (SDS-PAGE) showed that in liver the activity enhancement is due mainly to the induction of the isoenzyme GST 7-7, while in the kidneys the activity of all the isoenzymes is increased.