Duodenal acidification induces gastric relaxation and alters epithelial barrier function by a mast cell independent mechanism.

Duodenal hyperpermeability and low-grade inflammation in functional dyspepsia is potentially related to duodenal acid exposure. We aimed to evaluate in healthy volunteers the involvement of mast cell activation on the duodenogastric reflex and epithelial integrity during duodenal acidification. This study consisted of 2 parts: (1) Duodenal infusion of acid or saline during thirty minutes in a randomized, double-blind cross-over manner with measurement of intragastric pressure (IGP) using high resolution manometry and collection of duodenal biopsies to measure epithelial barrier function and the expression of cell-to-cell adhesion proteins. Mast cells and eosinophils were counted and activation and degranulation status were assessed. (2) Oral treatment with placebo or mast cell stabilizer disodiumcromoglycate (DSCG) prior to duodenal perfusion with acid, followed by the procedures described above. Compared with saline, acidification resulted in lower IGP (P < 0.01), increased duodenal permeability (P < 0.01) and lower protein expression of claudin-3 (P < 0.001). Protein expression of tryptase (P < 0.001) was increased after acid perfusion. Nevertheless, an ultrastructural examination did not reveal degranulation of mast cells. DSCG did not modify the drop in IGP and barrier dysfunction induced by acid. Duodenal acidification activates an inhibitory duodenogastric motor reflex and, impairs epithelial integrity in healthy volunteers. However, these acid mediated effects occur independently from mast cell activation.

Relationship between bile salts, bacterial translocation, and duodenal mucosal integrity in functional dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a complex disorder, in which multiple mechanisms underlie symptom generation, including impaired duodenal barrier function. Moreover, an altered duodenal bile salt pool was recently discovered in patients with FD. We aimed to evaluate the relationship between bile salts, bacterial translocation, and duodenal mucosal permeability in FD. METHODS: Duodenal biopsies from patients with FD and healthy volunteers (HV) were mounted in Ussing chambers to measure mucosal resistance and bacterial passage in the absence and presence of fluorescein-conjugated Escherichia coli and glyco-ursodeoxycholic acid (GUDCA) exposure. In parallel, duodenal fluid aspirates were collected from patients and bile salts were analyzed. KEY RESULTS: The transepithelial electrical resistance of duodenal biopsies from patients was lower compared with HV (21.4 ± 1.3 Ω.cm2 vs. 24.4 ± 1.2 Ω.cm2 ; P = .02; N = 21). The ratio of glyco-cholic and glyco-chenodeoxycholic acid (GCDCA) to tauro- and GUDCA correlated positively with transepithelial electrical resistance in patients. Glyco-ursodeoxycholic acid slightly altered the mucosal resistance, resulting in similar values between patient and healthy biopsies (22.1 ± 1.0 Ω.cm2 vs. 23.0 ± 1.0 Ω.cm2 ; P = .5). Bacterial passage after 120 minutes was lower for patient than for healthy biopsies (0.0 [0.0-681.8] vs. 1684.0 [0.0-4773.0] E coli units; P = .02). Glyco-ursodeoxycholic acid increased bacterial passage in patient biopsies (102.1 [0.0-733.0] vs. 638.9 [280.6-2124.0] E coli units; P = .009). No correlation was found between mucosal resistance and bacterial passage. CONCLUSIONS & INFERENCES: Patients with FD displayed decreased duodenal mucosal resistance associated with bile salts, however, not associated with bacterial passage in vitro. In addition, the hydrophilic bile salt glyco-ursodeoxycholic acid abolished differences in mucosal resistance and bacterial passage between patient and control group.

Altered duodenal bile salt concentration and receptor expression in functional dyspepsia.

BACKGROUND: Functional dyspepsia is a common functional gastrointestinal disorder in which a variety of pathophysiological mechanisms such as increased intestinal permeability and low-grade inflammation are involved. The factor causing these alterations, however, has not been identified. OBJECTIVE: We aimed to evaluate the luminal bile salt content and receptor expression in patients with functional dyspepsia and healthy volunteers. METHODS: Gastroduodenoscopy was performed to obtain duodenal biopsies from 25 healthy volunteers and 25 patients with functional dyspepsia (Rome III) to measure duodenal bile salt receptor expression with Western blot. Duodenal fluid aspirates were collected at fixed time points during fasted and fed state conditions and bile salt composition analysis was performed by liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: Patients (N = 17) displayed decreased fasted bile salt concentrations compared to healthy volunteers (N = 20) over time (1.8 ± 0.3 mM vs 3.6 ± 0.5 mM; p = 0.03). In addition, an increased expression of duodenal bile salt sensor vitamin D receptor was found in patients (3.7 ± 1.0-fold; p < 0.0005; N = 24 for both groups). CONCLUSION: Patients with functional dyspepsia are characterized by a decreased duodenal bile salt concentration in fasted state and an increased duodenal vitamin D receptor expression.

Effect of motilin receptor activation on food intake and food timing.

Background: Motilin plasma concentrations are positively correlated with hunger ratings during the fasting state. Moreover, the motilin agonist erythromycin stimulates meal requests. Objectives: The first aim of the study was to evaluate the effect of erythromycin on ad libitum food intake. The second aim was to study the involvement of endogenous motilin and octanoylated ghrelin on voluntary meal initiations. Design: Study 1: Fourteen healthy participants were studied twice after an overnight fast. Intravenous administration of placebo (saline) or erythromycin (40 mg) was given in a double-blind randomized order. Participants had the opportunity to eat ad libitum from an excess free-choice buffet (2330 kcal) for the duration of 1 h. The primary outcome was total caloric intake. Study 2: Thirteen healthy participants were studied after an overnight fast. Baseline blood samples were collected before a breakfast (245 kcal). After a rest period of 90 min, blood samples were collected every 15 min for a duration of 5 h. During this period, volunteers could request small meal portions (164 kcal/serving) at time points of their choosing and unlimited in numbers. The primary outcome was the determination of plasma concentrations before postbreakfast spontaneous meal requests. Results: Ad libitum food intake did not differ between placebo and erythromycin groups (difference compared with placebo: 79 kcal; 95% CI: -245.9, 403.97 kcal; P = 0.3). Octanoylated ghrelin concentrations before spontaneous meal requests were, on average, 36% (95% CI: 5.8%, 65.7%; P = 0.02) higher than values before breakfast, whereas motilin concentrations did not increase (6% increase: 95% CI: -5.9%, 17.2% increase; P = 0.3). Conclusions: Motilin receptor stimulation during the fasting state does not affect total caloric intake nor does endogenous motilin stimulate meal requests after breakfast in the current study population. This trial was registered at www.clinicaltrials.gov as NCT03024879.

Activation of Eosinophils and Mast Cells in Functional Dyspepsia: an Ultrastructural Evaluation.

We recently identified mucosal mast cell and eosinophil hyperplasia in association with a duodenal impaired barrier function in functional dyspepsia (FD). We aimed to further describe the implication of these immune cells by assessing their activation state at the ultrastructural level and by evaluating the association between impaired epithelial integrity and immune activation. Duodenal biopsies were obtained from 24 FD patients and 37 healthy controls. The ultrastructure of mast cells and eosinophils was analyzed by transmission electron microscopy. Transepithelial electrical resistance and paracellular permeability were measured to evaluate epithelial barrier function. The type of degranulation in eosinophils and mast cells was piecemeal. Eosinophils displayed higher degree of degranulation in FD patients than in controls (p < 0.0001). Quantification revealed a decreased granular density in eosinophils of FD patients (p < 0.0001). The degree of degranulation in mast cells was similar in both groups. However, a more heterogeneous profile was found in the FD group (p < 0.0001). No association between epithelial integrity and the number and activation state of mucosal eosinophils and mast cells was found. We demonstrated ultrastructural changes in degranulation state of eosinophils and mast cells, suggesting that eosinophil and mast cell activation play a role in the pathophysiology of FD.

Subacute stress and chronic stress interact to decrease intestinal barrier function in rats.

Psychological stress increases intestinal permeability, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders. We assessed the effect of subacute, chronic and combined stress on intestinal barrier function and mast cell density. Male Wistar rats were allocated to four experimental groups (n = 8/group): 1/sham; 2/subacute stress (isolation and limited movement for 24 h); 3/chronic crowding stress for 14 days and 4/combined subacute and chronic stress. Jejunum and colon were collected to measure: transepithelial electrical resistance (TEER; a measure of epithelial barrier function); gene expression of tight junction molecules; mast cell density. Plasma corticosterone concentration was increased in all three stress conditions versus sham, with highest concentrations in the combined stress condition. TEER in the jejunum was decreased in all stress conditions, but was significantly lower in the combined stress condition than in the other groups. TEER in the jejunum correlated negatively with corticosterone concentration. Increased expression of claudin 1, 5 and 8, occludin and zonula occludens 1 mRNAs was detected after subacute stress in the jejunum. In contrast, colonic TEER was decreased only after combined stress, and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the chronic and combined stress condition in the colon only. In conclusion, our data show that chronic stress sensitizes the gastrointestinal tract to the effects of subacute stress on intestinal barrier function; different underlying cellular and molecular alterations are indicated in the small intestine versus the colon.

Impaired barrier function in patients with house dust mite-induced allergic rhinitis is accompanied by decreased occludin and zonula occludens-1 expression.

BACKGROUND: Tight junction (TJ) defects have recently been associated with asthma and chronic rhinosinusitis. The expression, function, and regulation of nasal epithelial TJs remain unknown in patients with allergic rhinitis (AR). OBJECTIVE: We investigated the expression, function, and regulation of TJs in the nasal epithelium of patients with house dust mite (HDM)-induced AR and in an HDM-induced murine model of allergic airway disease. METHODS: Air-liquid interface cultures of primary nasal epithelial cells of control subjects and patients with HDM-induced AR were used for measuring transepithelial resistance and passage to fluorescein isothiocyanate-dextran 4 kDa (FD4). Ex vivo transtissue resistance and FD4 permeability of nasal mucosal explants were measured. TJ expression was evaluated by using real-time quantitative PCR and immunofluorescence. In addition, the effects of IL-4, IFN-γ, and fluticasone propionate (FP) on nasal epithelial cells were investigated in vitro. An HDM murine model was used to study the effects of allergic inflammation and FP treatment on transmucosal passage of FD4 in vivo. RESULTS: A decreased resistance in vitro and ex vivo was found in patients with HDM-induced AR, with increased FD4 permeability and reduced occludin and zonula occludens-1 expression. AR symptoms correlated inversely with resistance in patients with HDM-induced AR. In vitro IL-4 decreased transepithelial resistance and increased FD4 permeability, whereas IFN-γ had no effect. FP prevented IL-4-induced barrier dysfunction in vitro. In an HDM murine model FP prevented the allergen-induced increased mucosal permeability. CONCLUSION: We found impaired nasal epithelial barrier function in patients with HDM-induced AR, with lower occludin and zonula occludens-1 expression. IL-4 disrupted epithelial integrity in vitro, and FP restored barrier function. Better understanding of nasal barrier regulation might lead to a better understanding and treatment of AR.

A weakly acidic solution containing deoxycholic acid induces esophageal epithelial apoptosis and impairs integrity in an in vivo perfusion rabbit model.

Impaired esophageal mucosal integrity may be an important contributor in the pathophysiology of gastroesophageal reflux disease (GERD). Nevertheless, the effect of potentially harmful agents on epithelial integrity is mainly evaluated in vitro for a short period of time and the possible induction of epithelial apoptosis has been neglected. Our objective was to assess the effect of an acidic and weakly acidic solution containing deoxycholic acid (DCA) on the esophageal epithelium in an in vivo rabbit model of esophageal perfusion and to evaluate the role of the epithelial apoptosis. The esophagus of 55 anesthetized rabbits was perfused for 30 min with different solutions at pH 7.2, pH 5.0, pH 1.0, and pH 5.0 containing 200 and 500 µM DCA. Thereafter, animals were euthanized immediately or at 24 or 48 h after the perfusion. Transepithelial electrical resistance, epithelial dilated intercellular spaces, and apoptosis were assessed in Ussing chambers, by transmission electron microscopy, and by TUNEL staining, respectively. No macroscopic or major microscopic alterations were observed after the esophageal perfusions. The acidic and weakly acidic solution containing DCA induced similar long-lasting functional impairment of the epithelial integrity but different ultrastructural morphological changes. Only the solution containing DCA induced epithelial apoptosis in vivo and in vitro in rabbit and human tissue. In contrast to acid, a weakly acidic solution containing DCA induces epithelial apoptosis and a long-lasting impaired mucosal integrity. The presence of apoptotic cells in the esophageal epithelium may be used as a marker of impaired integrity and/or bile reflux exposure.

Efficacy of Mirtazapine in Patients With Functional Dyspepsia and Weight Loss.

BACKGROUND & AIMS: A subset of patients with functional dyspepsia (FD) present with early satiation and weight loss, for which there are no established therapeutic options. We investigated the efficacy of mirtazapine (an antidepressant and antagonist of the histamine receptor H1, the α2 adrenergic receptor, and the serotonin receptors 5-HT2C and 5-HT-3) in patients with FD and weight loss. METHODS: We conducted a randomized, placebo-controlled pilot trial that studied 34 patients with FD (29 women; mean age, 35.9 ± 2.3 years) with weight loss >10% of original body weight (mean loss, 12.4 ± 2.3 kg) without depression or anxiety. After a run-in period, patients were randomly assigned to groups given placebo (n = 17) or mirtazapine 15 mg each day for 8 weeks (n = 17) in a double-blind manner. Subjects were evaluated during a 2-week baseline and 8-week treatment for dyspepsia symptom severity, quality of life (on the basis of the Nepean Dyspepsia Index), and gastrointestinal-specific anxiety; they were given a nutrient challenge test and weighed. Data were analyzed by using linear mixed models, followed by planned contrasts with adaptive step-down Bonferroni multiple testing correction. RESULTS: Two patients in each group dropped out. At weeks 4 and 8, mirtazapine significantly reduced mean dyspepsia symptom severity scores compared with week 0 (P = .003 and P = .017, respectively); there was no significant reduction in the placebo group (P > .37 for weeks 4 and 8). The difference in change from week 0 between mirtazapine and placebo showed a trend with a large effect size at week 4 (P = .059) that was not significant at week 8 (P = .55). However, improvements from week 0 to weeks 4 and 8 were significantly larger in the mirtazapine group than placebo group for early satiation, quality of life, gastrointestinal-specific anxiety, weight, and nutrient tolerance (mostly with large effect sizes). CONCLUSIONS: In a randomized, placebo-controlled trial, mirtazapine significantly improved early satiation, quality of life, gastrointestinal-specific anxiety, nutrient tolerance, and weight loss in patients with FD. ClinicalTrials.gov number: NCT01240096.

The normoglycaemic biobreeding rat: a spontaneous model for impaired gastric accommodation.

OBJECTIVE: Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function. DESIGN: Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220 days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation. RESULTS: No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30 days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220 days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220 days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats. CONCLUSIONS: BB-DP rats of 220 days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.

Evidence for neuronal and structural changes in submucous ganglia of patients with functional dyspepsia.

OBJECTIVES: An intact and well-functioning enteric nervous system is necessary to efficiently organize gut function. Functional gastrointestinal disorders are pathological entities in which gut function is impaired without a clearly established pathophysiology. On the basis of the relative ease with which intestinal biopsies can be obtained, and taking advantage of a recently developed optical recording technique, we evaluated whether functional neuronal defects exist in enteric nerves of patients with functional dyspepsia (FD). METHODS: The submucous plexus isolated from duodenal biopsies taken from FD patients and control subjects was used to functionally and morphologically examine nerves and ganglionic architecture (neurons and glial cells). In light of previous studies reporting eosinophil and mast cell infiltration in the gut mucosa of FD patients, we also examined whether these cells infiltrated the submucous plexus and whether this correlated with neuronal activity and specific clinical symptoms. RESULTS: We demonstrate that neuronal functioning is impaired in the submucous plexus of FD patients, as shown by decreased calcium responses to depolarization and electrical stimulation. Glial (S100) and neuronal (HuCD) markers show signs of gliosis, altered ganglionic architecture, and neuronal abnormalities in the submucous plexus of FD patients. We found that eosinophils and mast cells infiltrated the submucous layer of FD patients to a much larger extent than in controls. A significant correlation was found between the number of these cells and the calcium transient amplitudes measured in submucous ganglia. CONCLUSIONS: We provide the first direct evidence that FD is characterized by functional and structural abnormalities within the submucous ganglion plexus, which may be of future predictive and diagnostic value in the treatment of FD patients.

From intestinal permeability to dysmotility: the biobreeding rat as a model for functional gastrointestinal disorders.

BACKGROUND: Impaired intestinal barrier function, low-grade inflammation and altered neuronal control are reported in functional gastrointestinal disorders. However, the sequence of and causal relation between these events is unclear, necessitating a spontaneous animal model. The aim of this study was to describe the natural history of intestinal permeability, mucosal and neuromuscular inflammation and nitrergic motor neuron function during the lifetime of the BioBreeding (BB) rat. METHODS: Normoglycemic BB-diabetes prone (DP) and control rats were sacrificed at different ages and jejunum was harvested to characterize intestinal permeability, inflammation and neuromuscular function. RESULTS: Both structural and functional evidence of increased intestinal permeability was found in young BB-DP rats from the age of 50 days. In older animals, starting in the mucosa from 70 days and in half of the animals also in the muscularis propria from 110 days, an inflammatory reaction, characterized by an influx of polymorphonuclear cells and higher myeloperoxidase activity, was observed. Finally, in animals older than 110 days, coinciding with a myenteric ganglionitis, a loss of nitrergic neurons and motor function was demonstrated. CONCLUSION: In the BB-rat, mucosal inflammatory cell infiltration is preceded by intestinal barrier dysfunction and followed by myenteric ganglionitis and loss of nitrergic function. This sequence supports a primary role for impaired barrier function and provides an insightful model for the pathogenesis of functional gastrointestinal disorders.

Impaired duodenal mucosal integrity and low-grade inflammation in functional dyspepsia.

OBJECTIVE: Functional dyspepsia (FD) is an extremely common functional gastrointestinal disorder, the pathophysiology of which is poorly understood. We hypothesised that impaired intestinal barrier function is involved in the onset and persistence of this disorder by inducing low-grade inflammation. Therefore, our aim was to evaluate duodenal mucosal integrity and low-grade inflammation in patients with FD. DESIGN: Duodenal biopsy specimens were obtained from 15 patients with FD fulfilling the Rome III criteria and 15 age- and gender-matched healthy volunteers. Transepithelial electrical resistance (TEER) and paracellular permeability were measured in Ussing chambers. Expression of cell-to-cell adhesion proteins was evaluated by real-time PCR, western blot and/or immunofluorescence. Numbers of mast cells, eosinophils and intraepithelial lymphocytes were assessed by immunohistochemistry. RESULTS: Patients with FD displayed lower TEER and increased paracellular passage compared with healthy controls, which is indicative of impaired mucosal integrity. In addition, abnormal expression of cell-to-cell adhesion proteins at the level of tight junctions, adherens junctions and desmosomes was shown. Furthermore, patients were characterised by the presence of low-grade inflammation, as demonstrated by increased infiltration of mucosal mast cells and eosinophils. A significant association between the expression level of several cell-to-cell adhesion proteins, the extent of increased permeability and the severity of low-grade inflammation was found. CONCLUSIONS: These findings challenge the classical paradigm that patients with FD show no structural changes in the gastrointestinal tract. We suggest that impaired intestinal barrier function is a pathophysiological mechanism in FD. Thus, restoration of intestinal barrier integrity may be a potential therapeutic target for treating patients with FD.

Psychological stress and corticotropin-releasing hormone increase intestinal permeability in humans by a mast cell-dependent mechanism.

OBJECTIVE: Intestinal permeability and psychological stress have been implicated in the pathophysiology of IBD and IBS. Studies in animals suggest that stress increases permeability via corticotropin-releasing hormone (CRH)-mediated mast cell activation. Our aim was to investigate the effect of stress on intestinal permeability in humans and its underlying mechanisms. DESIGN: Small intestinal permeability was quantified by a 2 h lactulose-mannitol urinary excretion test. In a first study, 23 healthy volunteers were subjected to four different conditions: control; indomethacin; public speech and anticipation of electroshocks. In a second study, five test conditions were investigated in 13 volunteers: control; after pretreatment with disodium cromoglycate (DSCG); administration of CRH; DSCG+CRH and DSCG+public speech. RESULTS: Indomethacin, as a positive comparator (0.071±0.040 vs 0.030±0.022; p<0.0001), and public speech (0.059±0.040; p<0.01), but not the shock protocol increased intestinal permeability. Similarly, salivary cortisol was only increased after public speech. Subgroup analysis demonstrated that the effect of public speech on permeability was only present in subjects with a significant elevation of cortisol. CRH increased the lactulose-mannitol ratio (0.042±0.021 vs 0.028±0.009; p=0.02), which was inhibited by the mast cell stabiliser DSCG. Finally, intestinal permeability was unaltered by public speech with DSCG pretreatment. CONCLUSIONS: Acute psychological stress increases small intestinal permeability in humans. Peripheral CRH reproduces the effect of stress and DSCG blocks the effect of both stress and CRH, suggesting the involvement of mast cells. These findings provide new insight into the complex interplay between the central nervous system and GI function in man.

In functional dyspepsia, hypersensitivity to postprandial distention correlates with meal-related symptom severity.

BACKGROUND & AIMS: Hypersensitivity to gastric distention, an important feature of functional dyspepsia, is assessed by stepwise balloon distention of the proximal stomach in fasting patients. However, symptoms of functional dyspepsia are often worse after a meal, so studies of postprandial balloon distentions might be more relevant. We compared the effects of fasting and postprandial stomach distention in patients with functional dyspepsia. METHODS: Twenty healthy controls and 62 patients with functional dyspepsia participated in a gastric barostat study at Leuven University Hospital with graded isobaric distentions before and after a liquid meal. On a separate day, all patients underwent a gastric emptying breath test with assessment of postprandial severity of 6 different dyspeptic symptoms scored at 15-minute intervals for 4 hours. For each symptom, a meal-related severity score was obtained by adding all scores; the cumulative symptom score (CSS) was obtained by adding individual symptom severity scores. RESULTS: In patients, but not in controls, postprandial sensitivity to balloon distention was significantly greater than fasting sensitivity. The CSS and individual symptom scores did not differ between patients with normal or hypersensitivity to fasting distention, but patients who were hypersensitive to postprandial distention had a significantly higher CSS, along with scores for postprandial fullness, bloating, and nausea (all P < .05). On multivariate analysis, hypersensitivity to postprandial distention was associated with hypersensitivity to fasting distention and with impaired accommodation to a meal. CONCLUSIONS: Postprandial, but not fasting, distention thresholds are related to the severity of meal-related symptoms in patients with functional dyspepsia.

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function.

BACKGROUND & AIMS: Bacterial infections commonly occur in decompensated cirrhosis resulting from bacterial translocation from the intestine. We studied the role of intestinal macrophages and the epithelial barrier in cirrhosis. METHODS: Forty-four patients with NASH/ASH cirrhosis (decompensated n=29, compensated n=15) and nineteen controls undergoing endoscopy were recruited. Serum was obtained and LPS and LBP levels determined. Intestinal macrophages were characterized by flow cytometry, immunohistochemistry, and nitric oxide (NO) production measured in supernatant of cultured duodenal samples. Quantitative RT-PCR was performed on duodenal biopsies assessing 84 inflammatory genes. Protein levels of cytokines/chemokines were assessed in serum and supernatant. The duodenal wall was assessed by electron microscopy, tight junction protein expression determined by RT-PCR, immunohistochemistry, and Western blot and, functional analysis performed by transepithelial resistance measurement and permeability studies. RESULTS: Increased plasma LPS, LBP levels and higher numbers of duodenal CD33(+)/CD14(+)/Trem-1(+) macrophages, synthesizing iNOS and secreting NO were present in decompensated cirrhosis. Upregulation of IL-8, CCL2, CCL13 at the transcriptional level, and increased IL-8, and IL-6 were detected in supernatant and serum in cirrhosis. IL-6 and IL-8 co-localised with iNOS(+) and CD68(+), but not with CD11c(+) cells. Electron microscopy demonstrated an intact epithelial barrier. Increased Claudin-2 was detected by Western blot and immunohistochemistry, while decreased transepithelial resistance and increased duodenal permeability were detected in decompensated cirrhosis. CONCLUSIONS: Our study shows the presence of activated CD14(+)Trem-1(+)iNOS(+) intestinal macrophages, releasing IL-6, NO, and increased intestinal permeability in patients with cirrhosis, suggesting that these cells may produce factors capable of enhancing permeability to bacterial products.

Changes in gastrointestinal tract function and structure in functional dyspepsia.

Functional dyspepsia is an extremely common disorder of gastrointestinal function. The disorder is thought to be heterogeneous, with different pathophysiological mechanisms underlying varied symptom patterns. A diversity of changes in gastrointestinal tract function and structure has been described in functional dyspepsia. These involve alterations in the stomach, such as impaired accommodation, delayed gastric emptying and hypersensitivity, and alterations in the duodenum, such as increased sensitivity to duodenal acid and/or lipids and low-grade inflammation. In this Review, we summarize all these abnormalities in an attempt to provide an integrated overview of the pathophysiological mechanisms in functional dyspepsia.

Gene markers in dendritic cells unravel pieces of the skin sensitization puzzle.

The underlying events of how dendritic cells (DC) are capable of evoking an antigen-specific skin sensitization response are not yet understood. Recently, we revealed a set of genes in human cord blood CD34(+) DC (CD34-DC) that show a discriminating behaviour after skin sensitizing exposure. Based on their differential expression, an in vitro assay was developed to identify chemicals as sensitizing or not. This study was designed to investigate the genes' involvement in the DC response to skin sensitizers and as such gain insights in the sensitization cascade. Functional connection of the marker genes was inquired by constructing a molecular network using Ingenuity software. By real-time RT-qPCR, we established the effective expression of 3 additional gene transcripts in the generated network in CD34-DC, of which CREB1 and TNF-alpha were significantly altered in expression by sensitizing versus non-sensitizing exposure. Next, it was tested whether the discriminating response of CCR2 and COX2 marker genes was translated at the protein level in CD34-DC exposed to 3 sensitizers versus 3 non-sensitizers. Significantly differential protein expression of CCR2 and COX2 was confirmed using flow cytometry. Our results indicate that the marker genes may be functionally relevant in DC mediated skin sensitization.

Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells.

The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.