M. Steinkamp

Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia

Background: Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn’s disease (CD) and necrotising enterocolitis (NEC) show pathological changes in enteric glia. Therefore, factors controlling GFAP+ enteric glia are of great interest. The aim of the present study was to characterise enteric glia and determine the effect of interleukin 1β (IL-1β), interleukin 4 (IL-4), tumour necrosis factor α (TNF-α), and lipopolysaccharides (LPS) on cultured enteric glia. Methods: Dissected rat colon and cultured enteric glia cells were double labelled with anti-GFAP and anti-S-100 antibodies. For regulatory studies, enteric glia cells were treated with cytokines and LPS. Proliferation was assayed using bromodeoxyuridine (BrdU) and mitosis of enteric glia was blocked by demecolcine. Results: We were able to distinguish GFAP negative (−) from GFAP+ glia subtypes in situ and in primary cultures. Incubation of cells with IL-1β, TNF-α, and LPS led to a significant increase in GFAP+ enteric glia while IL-4 had no effect on GFAP expression. After incubation with IL-1β, total intracellular GFAP of enteric glia cells was increased. Upregulation of GFAP+ enteric glia could also be observed after stimulation with IL-1β on blocking mitosis. BrdU uptake in stimulated enteric glia showed no increased proliferation rate. Conclusions: Two different types of enteric glia based on GFAP expression exist in the gut. Proinflammatory cytokines and LPS cause a dramatic increase in GFAP+ enteric glia. This suggests that cytokines play an important role in controlling GFAP+ enteric glia which might in turn be involved in modulating the integrity of the bowel during inflammation.

Distribution of enteric glia and GDNF during gut inflammation

BackgroundThe 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.MethodsThe 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.ResultsThe 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 Crohns 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.ConclusionsGFAP 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.

Refinement of screening for familial pancreatic cancer

Objective Surveillance programmes are recommended for individuals at risk (IAR) of familial pancreatic cancer (FPC) to detect early pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC). However, the age to begin screening and the optimal screening protocol remain to be determined. Methods IAR from non-CDKN2A FPC families underwent annual screening by MRI with endoscopic ultrasonography (EUS) in board-approved prospective screening programmes at three tertiary referral centres. The diagnostic yield according to age and different screening protocols was analysed. Results 253 IAR with a median age of 48 (25–81) years underwent screening with a median of 3 (1–11) screening visits during a median follow-up of 28 (1–152) months. 134 (53%) IAR revealed pancreatic lesions on imaging, mostly cystic (94%), on baseline or follow-up screening. Lesions were significantly more often identified in IAR above the age of 45 years (p<0.0001). In 21 IAR who underwent surgery, no significant lesions (PDAC, pancreatic intraepithelial neoplasia (PanIN) 3 lesions, high-grade intraductal papillary mucinous neoplasia (IPMN)) were detected before the age of 50 years. Potentially relevant lesions (multifocal PanIN2 lesions, low/moderate-grade branch-duct IPMNs) occurred also significantly more often after the age of 50 years (13 vs 2, p<0.0004). The diagnostic yield of potentially relevant lesions was not different between screening protocols using annual MRI with EUS (n=98) or annual MRI with EUS every 3rd year (n=198) and between IAR screened at intervals of 12 months (n=180) or IAR that decided to be screened at ≥24 months intervals (n=30). Conclusions It appears safe to start screening for PDAC in IAR of non-CDKN2a FPC families at the age of 50 years. MRI-based screening supplemented by EUS at baseline and every 3rd year or when changes in MRI occur appears to be efficient.

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

Summary 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-α) and interferon (IFN-γ). 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-α and IFN-γ 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.

Increased Duodeno-Gastro-Esophageal Reflux (DGER) in symptomatic GERD patients with a history of cholecystectomy.

BACKGROUND Duodenal-Gastro-Esophageal Reflux (DGER) represents an independent risk factor for the development of complicated Gastro-esophageal-reflux-disease (GERD) and Barretts esophagus. Clinical and epidemiological data suggest a potential association between cholecystectomy (CCE) and augmented bile reflux. METHODS 132 patients (67 women, 65 men, median age 55) with typical symptoms of GERD were enrolled in the study and divided in cholecystectomized (CCE-group: n = 107) and non- cholecystectomized (nCCE-group: n = 25) patients. Standardized clinical work-up of patients included combined esophageal 24 h pH-measurement and Bilitec 2000 esophageal manometry and upper endoscopy. RESULTS In the statistical analysis no differences between the cholecystectomized group (CCE-group, n = 25) and the patients without cholecystectomy (nCCE-group, n = 107) could be observed in quantity or quality of reflux symptoms. Furthermore, neither acid reflux nor severity of inflammation and frequency of Barretts esophagus significantly differed between the nCCE and CCE-group. However, the percentage of patients with pathological DGER were significantly higher in the CCE-group as compared to the nCCE-group (76 vs. 55 %, p < 0.01). Moreover, the CCE-group revealed significant higher levels of pathological DGER compared to the nCCE-group (15.5 % +/- 14.1 vs. 8.6 % +/- 15.4; p < 0.05). CONCLUSION To conclude, our data provide first evidence of elevated DGER after CCE in patients with typical clinical symptoms of GERD using the Bilitec device. Both the frequency and the extent of DGER was significantly increased in the CCE-group. Prospective studies are urgently needed to elucidate the impact of CCE on DGER in patients with clinical symptoms of a reflux disease.

Impact of Pantoprazole on Duodeno-Gastro-Esophageal Reflux (DGER)

BACKGROUND Duodeno-gastro-esophageal reflux (DGER) is considered as an independent risk factor for complicated reflux disease (GERD). Patients with Barretts esophagus have significantly higher levels of DGER than patients with uncomplicated GERD. However, the clinical response to conventional high-dose PPI therapy in patients with uncomplicated GERD and DGER is largely unknown. METHODS 30 patients with uncomplicated GERD and combined pathological reflux (acid and bile) were enrolled in the study. Clinical work-up included evaluation of clinical symptoms, esophageal manometry and upper endoscopy. After 6 - 8 weeks of treatment with Pantoprazole 80 mg/d pH measurement and Bilitec 2000 were repeated, and the pattern of symptoms was re-evaluated. RESULTS Under treatment with Pantoprazole 80 mg/d acid reflux was normalised in 28 patients (93 %). Similarly the mean percentage of DGER (time with an absorption greater than 0.14) was significantly reduced from 19.6 % (+/- 13.7) to 5.7 % (+/- 7.7, p < 0.05). In 15 patients (50 %) an elevated DGER persisted under treatment with Pantoprazole (DGER-NR group) whereas in 15 cases (50 %) a normalisation could be achieved (DGER-R group). The DGER-NR group had significantly higher levels of bile reflux before (and under) treatment compared to the DGER-R group: 22.9 % (9.98 %) vs. 15.6 % (0.72 %), respectively. Overall, the median quality of life index (QLI) improved from 4.78 (+/- 0.86) before to 8.04 +/- 1.84) under therapy. The clinical response under treatment was marikedly reduced in the DGER-NR group compared to the DGER-R group: QLI 7.3 vs. 8.9. Particularly heartburn and nocturnal coughing persisted. CONCLUSIONS Our data confirm that high-dose pantoprazole therapy effectively exerts acid suppression in GERD patients with combined pathological reflux. However, DGER could only normalised in 50 % of patients. High levels of DGER at diagnosis enhance the risk of persistent DGER under PPI therapy and are associated with a reduced clinical outcome.

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.

Endoskopische Möglichkeiten der Behandlung von Nahtinsuffizienzen im Rektum

Hintergrund: Anastomoseninsuffizienzen stellen eine gefürchtete Komplikation der kolorektalen Chirurgie dar. Endoskopische Techniken haben sich in der Therapie der Leckagen zunehmend etabliert. In dieser Übersicht möchten wir einen Überblick der wichtigsten endoskopischen Verfahren geben. Methode: Systematische Recherche der vorhandenen Literatur. Ergebnisse: Die bedeutendsten endoskopischen Verfahren zur Behandlung der kolorektalen Anastomoseninsuffizienzen stellen die Fibrininjektion, die Vakuumschwammtherapie (Endo-Sponge), spezielle Clip-Systeme (OTSC) sowie die Stentimplantation dar. Der breiten klinischen Anwendung dieser Verfahren steht jedoch eine unzureichende Studienlage gegenüber. Schlussfolgerung: Die Einschätzung der Wertigkeit der einzelnen endoskopischen Verfahren untereinander und in speziellen therapeutischen Situationen hängt im Wesentlichen von der Erfahrung der Untersucher ab. Es bedarf der Durchführung randomisierter, prospektiver Studien, um diese Erfahrungen zu objektivieren.

Glatiramer Acetate: A Novel Therapeutic Approach in Crohn's Disease?

To the Editor: In the early 1960s, a research team at the biophysics department of the Weizman Institute of Science, Rehovot, Israel, was determined to develop polypeptides able to elicit an immune response when injected into animals. To this end, they designed an immunogenic polypeptide of approximately 4000 Da (glatiramer acetate, copolymer 1, Copaxone) consisting of only 4 amino acids (alanine, glutamic acid, lysine, and tyrosine) and resembling the protein composition of myelin basic protein (MBP). MBP is the main protein component of the myelin sheath in the spinal cord and of white matter in the brain and represents one of several putative autoantigens in multiple sclerosis (MS). Several mammalian animal models have demonstrated that crude injections of myelincontaining tissue causes experimental allergic encephalitis (EAE), which closely mimics MS. Surprisingly, injection of glatiramer acetate (GA) suppressed rather than induced EAE in mammalians.1,2 A first prospective, double-blind, placebo-controlled clinical trial in MS patients conducted by Bornstein et al provided the first evidence of therapeutic effects of GA. Daily subcutaneous injections of 20 mg of GA for 2 years significantly reduced disease progression and frequency of relapse in MS patients.3 Large multicenter trials confirmed the initial observations made by Bornstein et al.4 In 1996, GA was eventually approved by the Food and Drug Administration (FDA) for the treatment of the relapsing-remitting form of MS. Today, Copaxone constitutes an important therapeutic tool in the treatment of MS and is characterized by a high safety profile and only minimal side effects such as irritation at injections sites and, rarely, transient vasomotor response. Extensive studies on the mechanisms of action of GA demonstrated that GA exerts its therapeutic effect by modulating the immune response at different levels of specificity. GA binds promiscuously and with high affinity to various class II major histocompatibility (MHC) molecules of murine and human origin, displacing antigens from the MHC antigenbinding site. This leads to the inhibition of various pathological effector functions.5 Furthermore, GA acts as a potent inducer of Th2/3 cells that secrete high amounts of regulatory substances such as interleukin-10 (IL-10) and transforming growth factor(TGF), but not Th1 inflammatory cytokines, thus deviating the immune response from a Th1to a Th2-biased cytokine profile.6 The Th1-mediated immunopathological nature of inflammatory bowel disease (IBD), in particular, Crohn’s disease, prompted researchers to examine the effects of GA in IBD. Indeed, previous studies convincingly demonstrated that GA treatment ameliorates the various pathological manifestations of several IBD animal models including trinitrobenzene sulfonic acid–induced colitis and dextran sulfate sodium (DSS)–induced colitis, as well as a spontaneous model of colitis in C3H/HeJBir IL-10deficient mice.7,8 In these mouse models amelioration of clinical symptoms such as weight loss, intestinal bleeding and diarrhea was accompanied by reduced expression of proinflammatory cytokines such as tumor necrosis factor– and interferonand enhanced levels of regulatory anti-inflammatory TGFand IL-10. Recently, the finding of a direct association between the therapeutic effects of GA in IBD and its immunomodulatory effect in the injured colon was unveiled.9 In this study, naive mice were immunized by GA, leading to the generation of a Th2/3-subtype T-cell population that drastically suppressed disease manifestation on their adoptive transfer to mice with DSS-induced colitis. In contrast, adoptive transfer of control lysozyme-specific cells did not result in any beneficial effect on the disease. In addition, GA-specific shortterm T-cell lines were in the inner layer of the colon after intraperitoneal injection in colitis-induced mice and secreted the regulatory cytokine TGFin situ.9 Here, we report a 33-year-old male white patient who was diagnosed with the relapsing-remitting form of MS 7 years ago at age 26. One year later, in April 2001, Crohn’s disease with mild to moderate ileocoecal involvement was diagnosed, and treatment with steroids and regular follow-up visits in our IBD outpatient clinic were initiated. In September 2001, daily injections of 20 mg of Copaxone were started for treatment of MS. Retrospectively, bowel symptoms such as abdominal pain, stool frequency, and intestinal bleeding were drastically reduced a few weeks after commencement of Copaxone treatment, and the patient achieved full remission of Crohn’s disease without the need of any concomitant IBD treatment. In 2006, the Copaxone treatment was discontinued because of remittent exacerbations of the MS, and methotrexat treatment (10 mg/week) was started. Soon after the termination of Copaxone, bowel symptoms flared up again, and the patient complained of abdominal pain and increasing stool frequency with the addition of blood. Furthermore, ultrasonography revealed markedly enhanced disease activity in the terminal ileum (Fig. 1). After consultation with the neurologist in charge, we decided to reinitiate Copaxone treatment. Soon after, bowel symptoms alleviated, and the patient again achieved full remission of Thomas M. Gress and Martin Steinkamp shared last authorship. Copyright

Periportal Cuffing in Inflammatory Bowel Diseases: Mystery of Stars and Stripes

To the Editor: Primary sclerosing cholangitis (PSC), pericholangitis, gallstones, and chronic hepatitis are common hepatobiliary disorders noted in association with inflammatory bowel disease (IBD). Echo-rich periportal cuffing (ErPC) constitutes a rare ultrasonographic phenomenon characterized by periportal encasement of the liver, also referred to as ‘‘fixed star heaven phenomenon’’ due to its distinctive appearance resembling ‘‘stars’’ and ‘‘stripes.’’ Cases of ErPC have been described in a variety of conditions such as hepatitis, IBD, and liver transplants. Previously, we evaluated 10,500 ultrasound examinations for periportal cuffing and assessed the associated clinical diagnoses. Strikingly, ErPC occurred most frequently in patients with Crohn’s disease (CD) and ulcerative colitis (UC). Surprisingly, the etiopathogenesis of ErPC is entirely unknown in IBD and the sonographic phenomenon has not been analyzed by other imaging modalities before. A current hypothesis is that ErPC occurs due to the relative increase in echo reflectors caused by lymphatic fluid obstructing the periportal areolar tissue, as observed in liver transplants or severe hepatitis; however, ErPC may also be the result of extraintestinal liver inflammation in IBD resulting in periportal zones of increased echogenicity. Figure 1 displays the typical sonographic appearance of ErPC in IBD. Here we systematically analyzed for the first time 4 patients with IBD (2 CD, 2 UC, 2 male, 2 female; mean age: 41 years) and ErPC by magnetic resonance imaging (MRI) / magnetic resonance cholangiopancreatography (MRCP), contrast-enhanced ultrasonography (CEUS), and serological tests in order to shed light on this rare sonographic phenomenon. Initially, ErPC was discovered by routine abdominal ultrasonography in our IBD outpatient clinic. In all patients, liver functions tests showed normal results and hepatitis A, B, and C, autoimmune hepatitis, and primary biliary cirrhosis (PBC) were ruled out. At the time of abdominal imaging ErPC was not associated with acute flares of intestinal inflammation as evidenced by low and medium Crohn’s Disease Activity Index (CDAI) scores (mean: 140). Following sonographic detection of ErPC, patients were evaluated by MRI/ MRCP. For PSC, typical morphological findings on MRCP include multiple segmental luminal irregularities, with strictures alternating with dilatations (‘‘beading’’) and peripheral bile duct attenuations (‘‘pruning’’). In contrast, low signal intensity periportal lesions (‘‘periportal halo sign’’) on T2weighted MRI/MRCP are caused by periportal edema or periportal lymph fluid accumulation and have been recently reported in patients with PBC and other hepatobiliary conditions. Interestingly, MRI/MRCP findings of all 4 patients with confirmed ErPC on ultrasonography showed no pathological features of the intrahepatic and extrahepatic biliary tree, thus ruling out active PSC as well as periportal fluid accumulation. CEUS with microbubble contrast medium (Sonovue, Bracco, Milan, Italy) showed an unspecific hypoechoic contrast enhancement in both the arterial (5–30 seconds) and the parenchymal phase (3–5 minutes), further supporting the absence of periportal fluid collection. Strikingly, perinuclear antineutrophil cytoplasmatic antibodies (p-ANCA) but not cytoplasmic ANCA (c-ANCA) were markedly elevated in all 4 patients (titers ranging from 1:40–1:600), suggesting a possible role of ErPC in the development of autoimmune/immune-mediated inflammatory liver disorders. Intriguingly, sonographic follow-up examinations in our 4 patients revealed that ErPC completely disappeared after a few weeks only to reappear after several weeks. This ‘‘on-and-off’’ nature of ErPC, positive p-ANCA titers, and the fact that periportal edema and lymph fluid obstruction could be excluded by MRI/ MRCP and CEUS prompted us to hypothesize that ErPC results from aberrant trafficking of mucosal lymphocytes to the portal tract of the liver via the enterohepatic circulation. Although there is still a paucity of experimental data, the role of such an enterohepatic lymphocyte pathway is hotly debated in the pathogenesis of extraintestinal complications in IBD. For PSC, which is associated with IBD in up to 85%, the hypothesis has been formulated that the destruction of bile ducts is mediated by long-lived memory T cells originally activated in the gut and recruited to the liver due to inflammation-induced expression of chemokines and adhesion molecules. The first evidence for such a paradigm is provided by recent data showing that the gut-homing mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) and CCL25 are detected in liver endothelium of PSC, whereas under normal circumstances expression of these molecules is restricted to the gut. Furthermore, several mouse models of IBD such as the SAMP-1/ Yit mouse and the IL-2Ra (CD25)-deficient mouse have been recently shown to develop not only colitis but also biliary inflammation closely resembling histological features of primary biliary cirrhosis. To conclude, ErPC is a rare sonographic phenomenon that is frequently associated with IBD. Here we systematically analyzed for the first time 4 patients with ErPC and IBD CopyrightVC 2009 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.21165 Published online 21 December 2009 inWiley InterScience (www.interscience.wiley.com).