Many Patients With Irritable Bowel Syndrome Have Atypical Food Allergies Not Associated With Immunoglobulin E.

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Persistent Increased Enteric Glial Expression of S100ß is Associated With Low-grade Inflammation in Patients With Diverticular Disease.

BACKGROUND: Diverticular disease (DD) is a common gastrointestinal inflammatory disorder associated with an enteric neuropathy. Although enteric glial cells (EGCs) are essential regulators of intestinal inflammation and motility functions, their contribution to the pathophysiology of DD remains unclear. Therefore, we analyzed the expression of specific EGC markers in patients with DD. MATERIALS AND METHODS: Expression of the glial markers S100ß, GFAP, Sox10, and Connexin 43 was analyzed by real-time quantitative PCR in colonic specimens of patients with DD and in that of controls. Protein expression levels of S100ß, GFAP, and Connexin 43 were further analyzed using immunohistochemistry in the submucosal and myenteric plexus of patients with DD and in that of controls. Expression of the inflammatory cytokines tumor necrosis factor-α and interleukin-6 was quantified using qPCR, and infiltration of CD3+ lymphocytes was determined using immunohistochemistry. RESULTS: Expression of S100ß was increased in the submucosal and myenteric plexus of patients with DD compared with that in controls, whereas expression of other glial factors remained unchanged. This increased expression of S100ß was correlated to CD3+ lymphocytic infiltrates in patients with DD, whereas no correlation was observed in controls. CONCLUSIONS: DD is associated with limited but significant alterations of the enteric glial network. The increased expression of S100ß is associated with a persistent low-grade inflammation reported in patients with DD, further emphasizing the role of EGCs in intestinal inflammation.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Confocal endomicroscopy shows food-associated changes in the intestinal mucosa of patients with irritable bowel syndrome.

BACKGROUND & AIMS: We investigated suspected food intolerances in patients with irritable bowel syndrome (IBS) using confocal laser endomicroscopy (CLE) for real-time visualization of structural/functional changes in the intestinal mucosa after food challenge. Patients with functional changes after food challenge (CLE+) were placed on personalized exclusion diets and followed up for long-term symptom relief. METHODS: Thirty-six IBS patients with suspected food intolerance and 10 patients with Barrett's esophagus (controls) without IBS symptoms were examined by CLE at University Hospital Schleswig-Holstein (Kiel, Germany). Diluted food antigens were administered directly to the duodenal mucosa through the working channel of the endoscope. Epithelial breaks, intervillous spaces, and the number of intraepithelial lymphocytes (IEL) were measured before and after the food challenge. CLE+ patients were placed on exclusion diets, given symptom score questionnaires, and followed up for 1 year; controls resumed their previous diet. RESULTS: CLE showed a real-time response to food antigens in 22 of 36 patients; no responses were observed in 14 of 36 patients (CLE-) or any of the controls. Baseline IELs were significantly higher in CLE+ than CLE- subjects (P = .004); numbers increased significantly after food challenge (P = .0008). Within 5 minutes of exposure of CLE+ patients to food antigens, IELs increased, epithelial leaks/gaps formed, and intervillous spaces widened. Epithelial leaks and intervillous spaces also increased significantly in CLE+ vs baseline (both P < .001). The concordance of IELs measured by CLE and conventional histology was 70.6%; they did not correlate (P = .89; r(2) = 0.027). Symptom scores improved more than 50% in CLE+ patients after a 4-week exclusion diet and increased to 74% at 12 months; symptoms continued in CLE- patients. CONCLUSIONS: Based on CLE analysis of IBS patients with a suspected food intolerance, exposure to candidate food antigens caused immediate breaks, increased intervillous spaces, and increased IELs in the intestinal mucosa. These changes are associated with patient responses to exclusion diets. Registered at clinicaltrials.gov (registration number: NCT01692613).

Expression and regulation of reelin and its receptors in the enteric nervous system.

BACKGROUND & AIMS: In the central nervous system (CNS), reelin coordinates migration and lamination of neurons and regulates synaptic plasticity, whereas its role in the enteric nervous system (ENS) remains enigmatic. Thus we determined the expression pattern and localization of reelin in the human ENS and monitored the time course of mRNA expression of the reelin signaling system in the rat intestine as well as in GDNF treated ENS cultures. RESULTS: Reelin, its receptors and Dab1 were expressed in all intestinal layers as well as in isolated myenteric ganglia. Enteric ganglia and nerve fibers were immunoreactive for reelin which co-localized with PGP 9.5 and synaptophysin. In the rat small intestine, highest expression levels of reelin were detected at early postnatal stages. Enteric nerve cell cultures treated with GDNF showed marked up-regulation of reelin and its receptors. CONCLUSIONS: Reelin and its receptors are strongly expressed in the human ENS. Reelin is specifically localized in enteric neurons with highest expression levels during early postnatal life as well as in neuronal network forming enteric nerve cell cultures pointing to putative functions in the differentiation and maintenance of the ENS. EXPERIMENTAL METHODS: Gene expression of reelin, its receptors and Dab1 were analyzed in the human colon and isolated enteric ganglia. Co-localization of reelin with the pan-neuronal marker PGP 9.5 and the synaptic vesicle marker synaptophysin was studied by dual-label-immunocytochemistry. The time course of reelin expression was monitored in an ontogenetic study of rat intestines as well as in GDNF-treated cultures of enteric neurons.

The enteric serotonergic system is altered in patients with diverticular disease.

OBJECTIVE: Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (5HT-4R) were thus studied in patients with DD. DESIGN: Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of 5HT-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of 5HT-4R was assessed within myenteric ganglia and muscle layers. Correlation of 5HT-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined. RESULTS: 5HT-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. 5HT-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. 5HT-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD. CONCLUSIONS: The serotonergic system is compromised in DD. Altered expression of 5HT-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

Oral glutamine supplementation improves intestinal permeability dysfunction in a murine acute graft-vs.-host disease model.

Although a profound barrier dysfunction has been reported, little is known about the pathophysiological mechanism evoking gastrointestinal graft-vs.-host disease (GI-GvHD) and apparent therapeutic options. The aim of this study was to evaluate the influence of oral glutamine on the course of GI-GvHD in an acute semiallogenic graft-vs.-host disease (GvHD) in irradiated B6D2F1 mice. An acute semiallogenic GvHD was induced by intraperitoneal injection of lymphocytes from C57BL/6 mice to irradiated B6D2F1 mice. Half of the GvHD animals received oral glutamine supplementation for 6 days started at the time of lymphocyte transfer. Six days after induction of the semiallogenic GvHD, jejunum specimens were prepared. The expression of the proinflammatory cytokine TNF-α and the tight junction protein occludin was investigated by PCR. Histological changes along with the apoptotic response were evaluated and intestinal permeability was assessed. Animals with GvHD showed a strong increase in paracellular permeability as a sign of the disturbed barrier function. TNF-α expression was significantly increased and the expression of the tight junction protein occludin decreased. GvHD led to mucosal atrophy, crypt hyperplasia, crypt apoptosis, and a disintegration of the tight junctions. Glutamine-treated mice showed reduced expression of TNF-α, increased occludin expression, fewer histological changes in the jejunum, smaller number of apoptotic cells in the crypt, and reduced gastrointestinal permeability. In conclusion, oral glutamine seems to have beneficial effects on the severity of inflammatory changes in the course of GvHD and might be a therapeutic option.

A novel endoscopic prototype device for gastric full-thickness biopsy for the histopathologic diagnosis of GI neuromuscular pathology: in vivo porcine long-term survival study (with videos).

BACKGROUND: Many GI motility disorders are associated with underlying GI neuromuscular pathology, which requires full-thickness biopsies (FTB) for histopathologic diagnosis. Currently, none of the endoscopy-based attempts to obtain FTB specimens have proven suitable for routine use. This study evaluated a novel endoscopic prototype device (ED) for this purpose. OBJECTIVE: To determine (1) the ability of the ED to obtain suitable FTB specimens, (2) associated complications, (3) feasibility of reliable defect closure, and (4) ability to evaluate intramural neuromuscular components. DESIGN: Preclinical proof-of-concept study in 30 pigs. SETTING: Animal laboratory. INTERVENTION: Gastric FTB specimens were obtained with a circular cutter and anchor. The defect was closed by over-the-scope clips/T-tags. The resection site was inspected via laparoscopy. After 2 to 4 weeks, necropsy was carried out to evaluate late complications. MAIN OUTCOME MEASUREMENTS: Feasibility, safety, and closure rate of the procedure. FTB specimens were assessed by histology/immunohistochemistry to visualize enteric neuromusculature. RESULTS: A total of 29 of 30 procedures were successfully performed; one hemorrhage required endoscopic treatment. A total of 29 of 30 FTB specimens (mean diameter 9.1 mm) were retrieved in 7.1 ± 0.4 minutes (range 3.0-12.5 minutes), displaying optimal tissue quality. Defect closure took 10.8 ± 0.9 minutes (range 7.2-32 minutes). Laparoscopy did not reveal damage to adjacent organs. Necropsy showed well-healed scars at the resection site and no complications, peritonitis, or abscess formation. Histology showed smooth muscle layers and submucosal and myenteric ganglia. LIMITATIONS: Survival animal pilot study, no patients. CONCLUSION: The novel ED enabled safe harvesting of well-preserved FTB specimens. Defect closure proved to be reliable. All neuromuscular structures relevant for histopathologic evaluation of GI neuromuscular pathology were demonstrated. Further studies are needed to verify the efficacy of this prototype device in the entire gut and in humans.

Localization Pattern of Dispatched Homolog 2 (DISP2) in the Central and Enteric Nervous System.

Dispatched homolog (DISP) proteins have been implicated in the regulation of hedgehog signaling during embryologic development. Although DISP2 has recently been associated with neuronal development and control of cognitive functions, its localization pattern in the mammalian central and peripheral nervous system has not yet been investigated. In this study, the Disp2 expression profile was assessed in human tissues from publicly available transcriptomic datasets. The DISP2 localization pattern was further characterized in the human and rat central nervous system (CNS), as well as within the colonic enteric nervous system (ENS) using dual-label immunohistochemistry. Colocalization of DISP2 with neuronal and glial markers was additionally analyzed in murine primary ENS culture. At transcriptomic level, DISP2 expression was predominant in neuronal cell types of the CNS and ENS. DISP2 immunoreactivity was mainly located within PGP9.5-positive neurons rather than in S100-positive glial cells throughout the nervous system. Investigation of human and rat brain tissues, colonic specimens, and murine ENS primary cultures revealed that DISP2 was located in neuronal cell somata, as well as along neuronal processes both in the human and murine CNS and ENS. Our results indicate that DISP2 is prominently localized within neuronal cells of the CNS and ENS and support putative functions of DISP2 in these tissues.

Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.

Expression pattern and localization of alpha-synuclein in the human enteric nervous system.

BACKGROUND: Alpha-synuclein (α-syn) is abundantly expressed in the central nervous system and involved in the regulation of neurotransmission. Insoluble fibrils of phosphorylated α-synuclein (p-α-syn) have been implicated in several neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer's disease). The aim of the study was to determine the gene expression pattern and localization of α-syn/p-α-syn in the human enteric nervous system (ENS). METHODS: Human colonic specimens (n=13, 15-83 years) were processed for α-syn and p-α-syn immunohistochemistry. Colocalization of α-syn was assessed by dual-labeling with pan-neuronal markers (PGP 9.5, HuC/D). For qPCR studies, tissue was obtained from full-thickness sections, tunica muscularis, submucosa, mucosa, and laser-microdissected (LMD) enteric ganglia. RESULTS: Highest α-syn levels were detectable within the tunica muscularis and submucosa. Ganglia isolated by LMD showed high expression of α-syn mRNA. All myenteric and submucosal ganglia and nerve fibers were immunoreactive for α-syn. Dual-labeling revealed colocalization of α-syn with both pan-neuronal markers. p-α-syn immunoreactivity was consistently observed in specimens from adults with increasing age. CONCLUSIONS: α-syn is abundantly expressed in all nerve plexus of the human ENS including both neuronal somata and processes. The presence of p-α-syn within the ENS is a regular finding in adults with increasing age and may not be regarded as pathological correlate. The data provide a basis to unravel the functions of α-syn and to evaluate altered α-syn in enteric neuropathies and α-synucleinopathies of the CNS with gastrointestinal manifestations.

Abnormalities of neuromuscular anatomy in diverticular disease.

The pathogenesis of diverticular disease is still poorly understood and considered to be multifactorial. Whereas classical pathogenetic concepts have focused on risk factors including increasing age, low-fiber diet and connective tissue disorders, novel concepts take into account that patients with diverticular disease exhibit disturbed intestinal motility patterns (that may result in functional obstruction and painful sensations) therefore postulating an underlying enteric neuro-/myopathy. Recent studies including quantitative evaluations of the enteric nervous system (ENS) in diverticular disease yielded hypoganglionic conditions of both myenteric and submucosal plexus as well as a nerve tissue remodeling in chronic diverticular disease. The disturbed neuromuscular communication was proven by demonstrating alterations in several enteric neurotransmitter systems, exemplified for the cholinergic, serotonergic, nitrergic system as well as for vasointestinal peptide, galanin and tachykinins. Novel lines of evidence have added the involvement of neurotrophic factors such as glial cell line-derived neurotrophic factor which is supposed to regulate ENS development and maintenance and which is downregulated in patients with diverticular disease. Consistent with the hypothesis of an enteric myopathy, deficits in smooth muscle integrity and composition such as hypertrophy, fibrotic transformation and gene expression deficits could be delineated. Taken together, the structural and functional findings on alterations of the ENS and the enteric musculature in diverticular disease provide evidence to strengthen the hypothesis that an enteric neuro-/myopathy may contribute to the development of colonic diverticula and the generation of symptoms in the course of the disease.

Behind every smile there's teeth: Cathepsin B's function in health and disease with a kidney view.

Cathepsin B (CatB) is a very abundant lysosomal protease with endo- and carboxydipeptidase activities and even ligase features. In this review, we will provide a general characterization of CatB and describe structure, structure-derived properties and location-dependent proteolytic actions. We depict CatB action within lysosome and its important roles in lysosomal biogenesis, lysosomal homeostasis and autophagy rendering this protease a key player in orchestrating lysosomal functions. Lysosomal leakage and subsequent escape of CatB into the cytosol lead to harmful actions, e.g. the role in activating the NLPR3 inflammasome, affecting immune responses and cell death. The second focus of this review addresses CatB functions in the kidney, i.e. the glomerulus, the proximal tubule and collecting duct with strong emphasis of its role in pathology of the respective segment. Finally, observations regarding CatB functions that need to be considered in cell culture will be discussed. In conclusion, CatB a physiologically important molecule may, upon aberrant expression in different cellular context, become a harmful player effectively showing its teeth behind its smile.

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage and inflammation following ischaemia/reperfusion injury in mouse.

AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.

IL-1ß and ADAM17 are central regulators of ß-defensin expression in Candida esophagitis.

Candida albicans resides on epithelial surfaces as part of the physiological microflora. However, under certain conditions, it may cause life-threatening infections, including Candida sepsis. We have recently shown that human ß-defensins (hBDs) hBD-2 and hBD-3 are upregulated in Candida esophagitis and that this antifungal host response is distinctly regulated by NF-κB and MAPK/activator protein-1 (AP-1) pathways. Here, we show that C. albicans induces hBD-2 through an autocrine IL-1ß loop and that activation of the epidermal growth factor receptor (EGFR) by endogenous transforming growth factor-α (TGF-α) is a crucial event in the induction of hBD-3. To further dissect upstream signaling events, we investigated expression of the central sheddases for EGFR ligands ADAM10 and ADAM17 in the healthy and infected esophagus. Next, we used pharmaceutical inhibitors and small-interfering RNA-mediated knock down of ADAM10 and ADAM17 to reveal that ADAM17-induced shedding of TGF-α is a crucial step in the induction of hBD-3 expression in response to Candida infection. In conclusion, we describe for the first time an autocrine IL-1ß loop responsible for the induction of hBD-2 expression and an ADAM17-TGF-α-EGFR-MAPK/AP-1 pathway leading to hBD-3 upregulation in the course of a Candida infection of the esophagus.

Establishment and characterization of porcine colonic epithelial cells grown in primary culture.

BACKGROUND: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. METHODS: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. RESULTS: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. CONCLUSIONS: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.

Morphological alterations of the enteric nervous system in young male patients with rectal prolapse.

OBJECTIVES: The pathogenesis of rectal prolapse (RP) defined by a circumferential, full-thickness invagination of the rectal wall into the anal canal is controversial. RP is normally encountered in elderly women and attributed to several etiological factors (e.g., advanced age, pudendal nerve injury, laxity of supporting ligaments). RP affecting young male patients is unlikely to be explained by these factors and may be due to a rectal motility disorder. Therefore, the enteric nervous system (ENS) as key regulator of intestinal motility was evaluated by a systematic morphometric analysis. PATIENTS AND METHODS: Full-thickness rectosigmoid specimens obtained from young male patients with symptomatic RP (n = 5) and male controls (n = 15) were processed for conventional histology and immunohistochemistry using anti-HuC/D as pan-neuronal marker. Enteric ganglia, nerve and glial cells were quantified separately in the myenteric (MP) and submucosal plexus (SMP). RESULTS: Compared to controls, patients with RP showed significantly (p < 0.05) increased mean ganglionic area both in MP and SMP, increased mean neuronal content of submucosal ganglia, and nearly threefold higher frequency of submucosal ganglia containing ≥7 neurons. CONCLUSION: The morphometric analysis reveals distinct quantitative alterations of the ENS in young male patients with RP mainly characterized by submucosal hyperganglionosis similar to histopathological features described in intestinal neuronal dysplasia. The data give evidence that RP in this unusual subgroup is associated with morphological changes of enteric ganglia which may contribute to the development of RP and complement established etiological concepts.

Expression Profiling of Rectal Biopsies Suggests Altered Enteric Neuropathological Traits in Parkinson's Disease Patients.

Still little is known about the nature of the gastrointestinal pathological alterations occurring in Parkinson's disease (PD). Here, we used multiplexed mRNA profiling to measure the expression of a panel of 770 genes related to neuropathological processes in deep submucosal rectal biopsies of PD patients and healthy controls. Altered enteric neuropathological traits based on the expression of 22 genes related to neuroglial and mitochondrial functions, vesicle trafficking and inflammation was observed in 9 out of 12 PD patients in comparison to healthy controls. These results provide new evidences that intestinal neuropathological alterations may occur in a large proportion of PD patients.

Alpha Synuclein Connects the Gut-Brain Axis in Parkinson's Disease Patients - A View on Clinical Aspects, Cellular Pathology and Analytical Methodology.

Parkinson's disease (PD) is marked by different kinds of pathological features, one hallmark is the aggregation of α-synuclein (aSyn). The development of aSyn pathology in the substantia nigra is associated to the manifestation of motor deficits at the time of diagnosis. However, most of the patients suffer additionally from non-motor symptoms, which may occur already in the prodromal phase of the disease years before PD is diagnosed. Many of these symptoms manifest in the gastrointestinal system (GIT) and some data suggest a potential link to the occurrence of pathological aSyn forms within the GIT. These clinical and pathological findings lead to the idea of a gut-brain route of aSyn pathology in PD. The identification of pathological aSyn in the intestinal system, e.g., by GIT biopsies, is therefore of highest interest for early diagnosis and early intervention in the phase of formation and propagation of aSyn. However, reliable methods to discriminate between physiological and pathological forms of enteral aSyn on the cellular and biochemical level are still missing. Moreover, a better understanding of the physiological function of aSyn within the GIT as well as its structure and pathological aggregation pathways are crucial to understand its role within the enteric nervous system and its spreading from the gut to the brain. In this review, we summarize clinical manifestations of PD in the GIT, and discuss biochemical findings from enteral biopsies. The relevance of pathological aSyn forms, their connection to the gut-brain axis and new developments to identify pathologic forms of aSyn by structural features are critically reviewed.