GDNF protects enteric glia from apoptosis: evidence for an autocrine loop.

BACKGROUND: Enteric glia cells (EGC) play an important role in the maintenance of intestinal mucosa integrity. During the course of acute Crohn's disease (CD), mucosal EGC progressively undergo apoptosis, though the mechanisms are largely unknown. We investigated the role of Glial-derived neurotrophic factor (GDNF) in the regulation of EGC apoptosis. METHODS: GDNF expression and EGC apoptosis were determined by immunofluorescence using specimen from CD patients. In primary rat EGC cultures, GDNF receptors were assessed by western blot and indirect immunofluorescence microscopy. Apoptosis in cultured EGC was induced by TNF-α and IFN-γ, and the influence of GDNF on apoptosis was measured upon addition of GDNF or neutralizing anti-GDNF antibody. RESULTS: Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls. Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis. CONCLUSIONS: This study proposes the existence of an autocrine anti-apoptotic loop in EGC cells which is operative in Crohn's disease and dependent of GDNF. Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.

Brain derived neurotrophic factor inhibits apoptosis in enteric glia during gut inflammation.

BACKGROUND: Enteric glia cells (EGCs) are essential for the integrity of the bowel. A loss of EGCs leads to a severe inflammation of the intestines. As a diminished EGC network is postulated in Crohn's disease (CD), we aimed to investigate if EGCs could be a target of apoptosis during inflammation in CD, which can be influenced by Brain derived neurotrophic factor (BDNF). MATERIAL/METHODS: GFAP, BDNF and cCaspase-3 were detected in the gut of patients with CD. Primary EGC cultures were established and cultivated. Tyrosine receptor kinase (TrkB) receptors on these cells were investigated by western blot and immunofluorescence. Rate of apoptosis was induced by tumor necrosis factor (TNF-alpha) and interferon (IFN-gamma). Apoptosis was determined by a fluorometric caspase 3/7 activation assay after preincubation of these cells with BDNF or neutralizing anti-BDNF antibodies. RESULTS: Mucosal GFAP-positive EGCs undergo apoptosis revealed by cCaspase-3 in the gut of patients with CD expressing BDNF highly. The combination of TNF-alpha and IFN-gamma was able to induce apoptosis in primary EGCs, whereas these factors alone did not. Brain derived neurotrophic factor (BDNF) attenuate glia cell apoptosis to a small extent, but neutralizing antibodies against BDNF dramatically increased apoptosis. CONCLUSIONS: Mucosal EGC apoptosis is an important finding in the gut of patients with CD. Proinflammatory cytokines, which are highly increased in CD, induce EGC apoptosis, whereas the neurotrophin BDNF might be protective for EGC. Since EGCs are implicated in the maintenance of the enteric mucosal integrity, EGC apoptosis may contribute to the pathophysiological changes in CD.

Distribution of enteric glia and GDNF during gut inflammation.

BACKGROUND: The enteric glia network may be involved in the pathogenesis of inflammatory bowel disease (IBD). Enteric glia cells (EGCs) are the major source of glial-derived neurotrophic factor (GDNF), which regulates apoptosis of enterocytes. The aim of the study was to determine the distribution of EGCs and GDNF during gut inflammation and to elucidate a possible diminished enteric glia network in IBD. METHODS: The expression of glial fibrillary acidic protein (GFAP) in colonic biopsies of patients with IBD, controls and patients with infectious colitis was detected by immunohistochemistry and Western blot. Tissue GDNF levels were measured by ELISA. RESULTS: The expression of GFAP and GDNF in the mucosal plexus is highly increased in the inflamed colon of patients with ulcerative colitis (UC) and infectious colitis. Although the GDNF and GFAP content are increased in Crohn's disease (CD), it is significantly less. Additionally the non-inflamed colon of CD patients showed a reduced GFAP and no GDNF expression compared to controls and the non-inflamed colon of UC patients. CONCLUSIONS: GFAP and GDNF as signs of activated EGCs are increased in the inflamed mucosa of patients with UC and infectious colitis, which underline an unspecific role of EGC in the regulation of intestinal inflammation. The reduced GFAP and GDNF content in the colon of CD patients suggest a diminished EGC network in this disease. This might be a part of the pathophysiological puzzle of CD.

The endothelin axis influences enteric glia cell functions.

BACKGROUND: The biologic effects of endothelin-1 (ET-1) are not limited to its vasoconstricting activity. A new and highly interesting role of the endothelin axis is its involvement in immune functions. As ET-1 is highly increased during gut inflammation, the aim of this study was to see if the endothelin axis influences enteric glia cell (EGC) functions, and through them, the immune response, during gut inflammation. MATERIAL/METHODS: Cultured EGCs were treated with interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNFalpha), IL-4, interferon-gamma, and ET-1. Secretion of ET-1 was detected by ELISA. Cultured EGCs were labeled with anti-glial fibrillary acidic protein (GFAP), endothelin-A (ETA), and endothelin-B (ETB), antibodies. The expression of ETA and ETB receptors was evaluated using reverse transcription PCR. Glial fibrillary acidic protein expression was determined by Western blot. RESULTS: ET-1 secretion of EGCs could be stimulated by IL-1 beta and TNFalpha in a time and dose-dependent manner, whereas IL-4 and interferon-gamma showed no effect on ET-1 production. Cultured EGCs expressed ETA and ETB-receptors. Endothelin B mRNA expression was increased after incubation with IL-1 beta. Incubation of cells with IL-1 beta, TNFalpha, and ET-1 led to a significant increase of GFAP in EGCs. CONCLUSIONS: Enteric glia cells express functional ETA and ETB receptors and produce huge amounts of ET-1 during gut inflammation, which increase GFAP expression in EGCs. These ET-1/ET receptors autocrine or paracrine loops might provide a new means to modulate EGC function, such as change in gut motility, cytokine production, and regulating gut homeostasis.

Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn's disease.

OBJECTIVES: Imbalanced apoptosis of enterocytes is likely to be 1 of the mechanisms underlying Crohn's disease (CD). Apoptosis of enterocytes is regulated by glial-derived neurotrophic factor (GDNF), which is increased in CD. The cellular source of GDNF during gut inflammation is unclear. The aim of the study was to identify the source of GDNF in CD during gut inflammation. MATERIALS AND METHODS: Glial fibrillary acidic protein (GFAP), GDNF, and smooth muscle actin (SMA) was detected in the gut from patients with CD by immunohistochemistry. Cultured enteric glia cells (EGC) were labeled with anti-GFAP, anti-GDNF, and antibodies and a Golgi marker (anti-58K antibodies) after blocking Golgi export with monensin. Cultured EGCs were treated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and lipopolysaccharides. Secretion of neurotrophic factors was detected by enzyme-linked immunosorbent assay. RESULTS: Mucosal GFAP-positive EGCs are increased in the colon of patients with CD. This type of glia but not subepithelial myofibroblasts expresses significant amounts of GDNF. In vitro GDNF is continuously secreted from cultured EGCs. The neurotrophic factor secretion could be stimulated by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharides in a time- and dose-dependent manner. The increased GDNF secretion by EGCs sustained for>12 hours after withdrawal of the proinflammatory cytokines. CONCLUSIONS: A mucosal GFAP expressing EGC population is dramatically increased in CD. This population is a major cellular source of the upregulated GDNF in the inflamed gut. Therefore, mucosal EGC may play a key role in protecting the gut epithelium and may contribute to reestablish the integrity of the injured epithelium.

Benefit of Surveillance for Pancreatic Cancer in High-Risk Individuals: Outcome of Long-Term Prospective Follow-Up Studies From Three European Expert Centers.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Hereditary factors play a role in the development of PDAC in 3% to 5% of all patients. Surveillance of high-risk groups, may facilitate detection of PDAC at an early stage. The aim of this study was to assess whether surveillance aids detection of early-stage PDAC or precursor lesions (PRLs) and improves the prognosis. PATIENTS AND METHODS: Screening outcomes were collected from three European centers that conduct prospective screening in high-risk groups including families with clustering of PDAC (familial pancreatic cancer [FPC]) or families with a gene defect that predisposes to PDAC. The surveillance program consisted of annual magnetic resonance imaging, magnetic resonance cholangiopancreatography, and/or endoscopic ultrasound. RESULTS: Four hundred eleven asymptomatic individuals participated in the surveillance programs, including 178 CDKN2A mutation carriers, 214 individuals with FPC, and 19 BRCA1/2 or PALB2 mutation carriers. PDAC was detected in 13 (7.3%) of 178 CDKN2A mutation carriers. The resection rate was 75%, and the 5-year survival rate was 24%. Two CDKN2A mutation carriers (1%) underwent surgical resection for low-risk PRL. Two individuals (0.9%) in the FPC cohort had a pancreatic tumor, including one advanced PDAC and one early grade 2 neuroendocrine tumor. Thirteen individuals with FPC (6.1%) underwent surgical resection for a suspected PRL, but only four (1.9%) had high-risk lesions (ie, high-grade intraductal papillary mucinous neoplasms or grade 3 pancreatic intraepithelial neoplasms). One BRCA2 mutation carrier was found to have PDAC, and another BRCA2 mutation carrier and a PALB2 mutation carrier underwent surgery and were found to have low-risk PRL. No serious complications occurred as consequence of the program. CONCLUSION: Surveillance of CDNK2A mutation carriers is relatively successful, detecting most PDACs at a resectable stage. The benefit of surveillance in families with FPC is less evident.

Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC).

Intestinal epithelial cells (IEC) form the largest surface of the human body and are of pivotal importance to digest and absorb nutrients. Furthermore these cells play a critical role shielding the organism against microorganisms and toxins present in the intestinal lumen. It is therefore not surprising that a large group of researchers take great interest in the study of these cells. However, to date it is a challenge to purify viable primary human intestinal epithelial cells and it has been even more fastidious to maintain IEC in culture ex-vivo as IEC undergo apoptosis within hours due to loss of cell anchorage ('anoikis') following the isolation process. Over recent years the authors aimed to continuously improve the isolation technique for primary IEC, allowing a simple, effective and rapid isolation of highly purified non-apoptotic human IEC. In this study the newly improved method is presented and applied to establish ex-vivo cultures of highly purified, fully viable primary IEC displaying important functional properties, making these cells amenable for ex-vivo research on primary human intestinal epithelial cells.

Role of neurotrophins in inflammation of the gut.

Until now neurotrophins like nerve growth factor (NGF), brain-derived neurotrophic factor (BDNA), neurotrophin (NT)-3 and neurotrophic factors like glial-derived neurotrophic factor (GDNF) have been almost exclusively investigated concerning their role in differentiation, growth and survival of specific neurons in the peripheral and central nervous system. However, in the last decade several non-neuronal functions of neurotrophins and neurotrophic factors have been characterized. In the gastrointestinal tract, neurotrophins and neurotrophic factors regulate neuropeptide expression, interact with immunoregulatory cells and epithelial cells and regulate motility during inflammation. This highlights this new and complex regulatory system as important and may lead to new options in the treatment of acute and chronic inflammation of the gut.

Enteric nervous plasticity and development: dependence on neurotrophic factors.

The enteric nervous system in the mammalian gut is histologically and to some extent functionally similar to the central nervous system. Thus, structural and functional similarities between these systems are evident. As shown for the central nervous system, differentiation of neural crest-derived precursor cells of the enteric nervous system also depends essentially on different neurotrophic factors. Moreover, recent studies have revealed that these trophic factors also play a critical role throughout life by regulating neurotransmitter and neuropeptide synthesis, and by influencing neuronal morphology and synaptic functions. Consequently, our understanding of these complex interactions of the enteric nervous system and neurotrophic factors requires synergistic efforts from neurophysiology, biochemistry, and pharmacology in order to understand the complex phenomena of enteric nervous development and plasticity in the gut. Knowledge of these mechanisms might help to develop strategies for therapy of neuronal abnormalities, which cause different gastrointestinal diseases.

Quantitative ultrasound of the proximal phalanges and dual-energy X-ray absorptiometry in Crohn's disease patients with osteopenia.

BACKGROUND: Osteopenia and osteoporosis are frequent complications in Crohn's disease, and these features are associated with an increased risk of vertebral and appendicular fractures. Bone mineral density (BMD) measurements are widely accepted to assess the fracture risk in postmenopausal osteoporosis. In recent years, quantitative ultrasound (QUS) has become attractive for the diagnosis of osteopenia as a nonionizing method. The aim of the present study was to investigate QUS and BMD measurements in osteopenic patients with Crohn's disease. METHODS: BMD of the lumbar spine and femoral neck and QUS of proximal phalanges II-V (DBM Sonic 1200; IGEA) were performed prospectively in 171 patients with Crohn's disease. The amplitude-dependent sound-of-speed (AD-SoS) and the ultrasound bone profile score (UBPS) were calculated using the WinSonic PRO 1.1 software program. X-ray examination of the spine was performed in 131 patients. Vertebral deformity was morphometrically defined according to the published methods of McCloskey and Eastell. RESULTS: BMD of the lumbar spine and femoral neck correlated significantly (r = 0.62), but no correlation between BMD and QUS could be demonstrated. Vertebral deformities (VD) were detected in 28/131 (21.4%) patients. Two patients had a history of femoral fracture (FF). Lumbar BMD was lower in patients with either VD or FF than in those patients with no preexisting fractures (T-score: -2.46 vs -2.04; P = 0.0233). QUS parameters correlated negatively to patients' age but could not be used to discriminate between patients with and without VD/FF. CONCLUSIONS: Osteoporosis-related fractures are associated with a low lumbar bone density in Crohn's disease patients. QUS of the proximal phalanges cannot detect manifest osteoporosis in Crohn's disease patients and is therefore not valuable as a screening tool for these patients.

The role of enteric glia in gut inflammation.

A neuro-glia interaction is part of gut inflammation and essential for the integrity of the bowel. A loss of enteric glia cells (EGCs) led to a fatal haemorrhagic jejuno-ileitis and death in a few days. Although a diminished EGC network is postulated in inflammatory bowel disease and enteric glia pathology is described in Chagas' disease the role of EGCs in the onset of these disease complexes is not definitely clear. Several lines of evidence implicate that the secretion of different factors by enteric glia may be the key for modulating gut homeostasis. As mucosal integrity might be important for remission in Crohn's disease and inflammation of the enteric nervous system is part of the pathology in Chagas' disease, the role of EGCs during gut inflammation could be part of the key to understand these diseases.

Simultaneous onset of ulcerative colitis and disseminated pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is an immune-mediated inflammatory skin condition representing one of the most distinct extraintestinal manifestations of inflammatory bowel disease (IBD). PG occurs independently from intestinal disease activity in about 1-2% of patients suffering from ulcerative colitis or Crohn's disease and is characterized by chronic deep skin ulcers whose exact pathogenesis is still unknown. So far, patients with ulcerative colitis have only been reported to develop PG during the course of IBD but not at the initial manifestation of bowel symptoms. This is the first report demonstrating the simultaneous onset of ulcerative colitis and severe multifocal PG. In addition, we provide first evidence that infliximab may have a particularly powerful effect in early disseminated PG compared to late-onset PG, advocating an early application of this drug.

Glutamate receptor subunit expression in primary enteric glia cultures.

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.

Lactose intolerance in active Crohn's disease: clinical value of duodenal lactase analysis.

BACKGROUND: Patients with active Crohn's disease (CD) often report having abdominal symptoms after ingestion of milk products, but the pathomechanism for lactose malabsorption seems to be complex. GOALS: To investigate the prevalence of clinical milk intolerance and to objectify symptoms with hydrogen (H 2 ) breath testing, analysis of lactase protein, and enzyme activity in the duodenal mucosa in patients with CD and in healthy controls. STUDY: In 49 patients with CD and 24 controls, H 2 breath testing was performed. All individuals underwent endoscopy of the upper gastrointestinal tract, in which multiple pinch samples were taken from the distal duodenum. Lactase activity was measured using the method of Dahlquist. The lactase protein expression was analyzed by gel electrophoresis using the monoclonal antibody mlac 10 and by immunochemistry using the monoclonal antibody mlac 4. RESULTS: Prevalence of milk intolerance in healthy controls was 16.6% versus 46.9% in patients with CD, with a high frequency (83.3%) in patients with active disease (CD activity index >150). Milk intolerance was correlated to the duration of inflammatory bowel disease ( p = 0.023) but not to the location or previous bowel resection. Hydrogen breath testing had a moderate sensitivity in detecting lactose maldigestion (70.4%) and a high specificity (95.6%). Duodenal lactase levels were also correlated to disease activity, whereas correlations to clinical symptoms remained poor. Patients with milk intolerance had a significantly reduced bone density at the lumbar spine (z-score, -1.33 +/- 0.92 vs. -0.19 +/- 0.95 [mean +/- SD]; p = 0.002) CONCLUSIONS: Milk intolerance is a frequent problem in active CD, which can be objectified accurately by H 2 lactose breath testing. Decreased lactase levels in the duodenal mucosa may be found during an acute flare but are not the predominant cause of milk intolerance in CD.

Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells.

BACKGROUND & AIMS: Ablation of the enteric glia leads to a fulminant hemorrhagic jejunoileitis. We hypothesized that glial-derived neurotrophic factor (GDNF) may be involved in mucosal protection of the gut. Therefore, we examined the regulation of GDNF and its receptor (GFR-alpha1) in colonic inflammation and its effects on colonic epithelial cell apoptosis. METHODS: The expression of GDNF and GFR-alpha1 was investigated in experimental colitis of rats and in human inflammatory bowel disease (IBD). GDNF-induced activation of Akt (protein kinase B [PKB]) and mitogen-activated protein kinase (MAPK) in the colonic epithelial cell lines HT-29 and SW480 was studied. Furthermore, the antiapoptotic potency of GDNF in SW480 cells was evaluated. RESULTS: GDNF was specifically up-regulated in experimental rat colitis and in IBD. In contrast, GFR-alpha1 was constitutively expressed in rat and human colonic epithelium. GDNF potently activated MAPK and Akt (PKB) in colonic epithelial cells. Moreover, GDNF strongly prevented apoptosis in SW480 cells. Our data show that GDNF-mediated protection against apoptosis depends on activation of the MAPK and phosphatidylinositol 3-kinase/Akt (PKB) pathways. CONCLUSIONS: GDNF is up-regulated in IBD and has strong antiapoptotic properties in colonic epithelial cells. This points to a novel role of the neurotrophic factor GDNF for mucosal protection and regeneration in IBD.