Contribution of CD3+CD8- and CD3+CD8+ T Cells to TNF-α Overexpression in Crohn Disease-Associated Perianal Fistulas and Induction of Epithelial-Mesenchymal Transition in HT-29 Cells.

BACKGROUND: Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta, and interleukin (IL)-13 are known to trigger epithelial-mesenchymal transition (EMT), promoting fistula formation. Here, we investigated the role of T-lymphocytes (T cells) in fistula pathogenesis. METHODS: CD3+CD8-, CD3+CD8+, or CD45+CD3- cells from healthy volunteers, patients with CD, and patients with CD with perianal fistula were co-cultured with HT-29 cells. The EMT, cytokine production, and mRNA expression were analyzed. Perianal CD fistula specimens were immunohistochemically stained for cytokines and their receptors. The effect of cytokines on EMT induction was investigated using an EMT spheroid model. RESULTS: Patients with CD with fistula revealed more CD3+CD8- and less CD3+CD8+ T cells in blood than healthy control patients and patients with CD without fistula. In perianal fistula specimens, CD4+ cells-and to a lesser extent CD8+ cells-were highly present around fistula tracts. When co-cultured with HT-29 cells, both cell subsets promoted EMT-related gene expression and TNF-α production in a time-dependent manner. The CD3+CD8- T cells from patients with CD with fistula also produced higher amounts of IL-13 than cells from healthy control patients or patients with CD without a fistula. We found that IL-22 and IL-22Rα1 were highly expressed in perianal CD fistula specimens and that IL-22 cotreatment potentiated TNF-α-induced EMT in HT-29 spheroids. CONCLUSIONS: Our data indicate that both CD3+CD8- and CD3+CD8+ T cells play an important role in the pathogenesis of perianal CD fistulas by the secretion of TNF-α. Our data support clinical evidence indicating that anti-TNF-α therapy is effective in fistula treatment and identify IL-13 and IL-22 as possible novel therapeutic targets for fistula therapy.

Hallmarks of epithelial to mesenchymal transition are detectable in Crohn's disease associated intestinal fibrosis.

BACKGROUND: Intestinal fibrosis and subsequent stricture formation represent frequent complications of Crohn's disease (CD). In many organs, fibrosis develops as a result of epithelial to mesenchymal transition (EMT). Recent studies suggested that EMT could be involved in intestinal fibrosis as a result of chronic inflammation. Here, we investigated whether EMT might be involved in stricture formation in CD patients. METHODS: Human colonic tissue specimens from fibrotic areas of 18 CD and 10 non-IBD control patients were studied. Immunohistochemical staining of CD68 (marker for monocytes/macrophages), transforming growth factor-ß1 (TGFß1), ß-catenin, SLUG, E-.cadherin, α-smooth muscle actin and fibroblast activation protein (FAP) were performed using standard techniques. RESULTS: In fibrotic areas in the intestine of CD patients, a large number of CD68-positive mononuclear cells was detectable suggesting an inflammatory character of the fibrosis. We found stronger expression of TGFß1, the most powerful driving force for EMT, in and around the fibrotic lesions of CD patients than in non-IBD control patients. In CD patients membrane staining of ß-catenin was generally weaker than in control patients and more cells featured nuclear staining indicating transcriptionally active ß-catenin, in fibrotic areas. In these regions we also detected nuclear localisation of the transcription factor, SLUG, which has also been implicated in EMT pathogenesis. Adjacent to the fibrotic tissue regions, we observed high levels of FAP, a marker of reactive fibroblasts. CONCLUSIONS: We demonstrate the presence of EMT-associated molecules in fibrotic lesions of CD patients. These findings support the hypothesis that EMT might play a role for the development of CD-associated intestinal fibrosis.

Quality of life after proctocolectomy and ileal pouch-anal anastomosis in patients with ulcerative colitis.

PURPOSE: While ileal pouch-anal anastomosis is performed in many patients with ulcerative colitis, conflicting data exist about its effects on quality of life. We aimed to determine quality of life and to identify risk factors for impaired quality of life in these patients. METHODS: Forty-eight of 82 patients (59%; median follow-up 57 months [range 21-93 months]) after ileal pouch-anal anastomosis for ulcerative colitis were compared to 48 matched healthy controls. Generic, health-, and disease-related, as well as symptom-specific quality of life was analyzed using five well-established quality of life instruments. RESULTS: Although generic quality of life was comparable between groups, health-related quality of life was impaired after ileal pouch-anal anastomosis. While high stool frequency was associated with impaired health-related and disease-specific quality of life, fecal incontinence and history of pouchitis also caused a deterioration of generic and symptom-related quality of life. Seventy-seven percent of patients reported their quality of life to be better compared to the situation before surgery and 88% would undergo ileal pouch-anal anastomosis again. CONCLUSIONS: Overall quality of life after ileal pouch-anal anastomosis is good. However, high stool frequency, fecal incontinence, and pouchitis are associated with impaired quality of life and should be prevented or treated to the best possible extent.

A role for tumor necrosis factor and bacterial antigens in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: Intestinal fistulae represent a severe complication of Crohn's disease (CD). The authors have demonstrated that epithelial-to-mesenchymal transition plays a pivotal role in their pathogenesis. High levels of interleukin-13 and tumor necrosis factor (TNF) are detected in myofibroblast-like transitional cells covering the fistula tracts. Here, a functional role was investigated for the transcription factor Ets-1, TNF, and the bacterial wall component (muramyl dipeptide [MDP]) in the pathogenesis of CD-associated fistulae. METHODS: Perianal fistulae from CD patients were analyzed by immunohistochemistry. Primary colonic lamina propria fibroblasts (CLPFs) were isolated from CD patients with or without fistulizing disease. Messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction in CLPF or HT29 intestinal epithelial cells (IECs) grown as monolayers or spheroids. RESULTS: Strong expression of Ets-1 transcription factor was demonstrated in transitional cell covering the fistula tracts by immunohistochemistry. TNF induced mRNA expression of ETS-1 and ß6-integrin in HT29 IEC and in CLPF from fistulizing CD patients. These effects could be fully blocked by administration of anti-TNF antibodies. In HT29 cells, TNF further induced mRNA levels of TNF and transforming growth factor beta by treatment for 24 hours. In fistula CLPF derived from CD patients, TNF induced expression of ß6-integrin, TNF, and transforming growth factor beta. Of note, the bacterial wall component, MDP, induced mRNA levels of ETS-1, transforming growth factor beta, interleukin-13, SNAIL1, and ß6-integrin in HT29 IEC monolayers and fistula CLPF by treatment for 24 hours. CONCLUSIONS: TNF and MDP induce the expression of factors associated with epithelial-to-mesenchymal transition and invasion in IEC and fistula CLPF. Our findings indicate that TNF and MDP might synergize in the pathogenesis of CD-associated fistulae.

The role for dickkopf-homolog-1 in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: One of the most challenging conditions in Crohn's disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae. METHODS: Dkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-ß or IL-13. Knock-down of DKK-1 or ß-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR. RESULTS: Dkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-ß induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-ß-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of ß-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-ß. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF. DISCUSSION: We demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.

Interleukin-13 and transforming growth factor ß synergise in the pathogenesis of human intestinal fistulae.

OBJECTIVE: Epithelial to mesenchymal transition (EMT) seems to play an important role in the pathogenesis of fistulae, a common clinical complication of Crohn's disease (CD). TGFß and interleukin-13 (IL-13) have been correlated with the onset of EMT-associated organ fibrosis and high levels of TGFß have been shown in transitional cells (TCs) lining CD fistula tracts. This study investigated whether IL-13 could be involved in the pathogenesis of CD-associated fistulae. DESIGN: Protein or mRNA levels in HT29 intestinal epithelial cells (IECs) or colonic lamina propria fibroblasts (CLPFs) were studied by western blotting or real-time PCR. CLPFs were isolated from non-inflammatory disease controls or patients with CD with or without fistulae and IL-13 levels were analysed in surgically removed fistula specimens by immunohistochemistry. RESULTS: TGFß induced IL-13 secretion in CLPFs from patients with fistulising CD. In fistula specimens high levels of IL-13 were detected in TCs covering fistula tracts. In HT29 IEC monolayers, IL-13 induced SLUG and ß6-integrin mRNA, which are associated with cell invasion. HT29 spheroids completely disintegrated when treated with TGFß for 7 days, whereas IL-13-treated spheroids did not show morphological changes. Here, TGFß induced mRNA expression of SNAIL1 and IL-13, whereas IL-13 elevated SLUG and ß6-integrin mRNA. An anti-IL-13 antibody was able to prevent IL-13-induced SLUG expression in HT29 IECs. CONCLUSIONS: TGFß induces IL-13 expression and an EMT-like phenotype of IECs, while IL-13 promotes the expression of genes associated with cell invasion. These findings suggest that TGFß and IL-13 play a synergistic role in the pathogenesis of fistulae and inhibition of IL-13 might represent a novel therapeutic approach for fistula treatment.

Potential role for SNAIL family transcription factors in the etiology of Crohn's disease-associated fistulae.

BACKGROUND: Fistulae represent an important clinical complication of Crohn's disease (CD). The fistula tracts are covered by flat, myofibroblast-like cells with an epithelial origin (transitional cells, TC). We recently demonstrated a role of epithelial mesenchymal transition (EMT) in the pathogenesis of CD-associated fistulae. EMT is associated with an increased migratory and invasive potential of epithelial cells in different tissues. Here we investigated whether cytokines or growth factors as well as EMT-associated SNAIL family transcription factors are expressed in CD fistulae. METHODS: By immunohistochemistry we analyzed seven perianal fistulae from seven CD and two perianal fistulae from two non-inflammatory bowel disease (IBD) control patients. Hematoxylin and eosin staining or immunohistochemistry for the expression of tumor necrosis factor (TNF), TNF-receptor I (TNF-RI), SNAIL1, SLUG, fibroblast growth factors (FGF) 1, 2, 4, 7, epidermal growth factor (EGF), and TWIST were performed using standard techniques. RESULTS: Immunohistochemical staining of surgical specimens from CD patients revealed a strong expression of TNF and TNF-RI in and around fistula tracts. While SNAIL1 was also heavily expressed in the nuclei of TC, indicative of transcriptionally active protein, SLUG, FGF-1, and FGF-2 were detected rather in the fibrotic periphery of CD fistulae than in TC. In contrast, we did not detect considerable protein staining for FGF-4 and FGF-7 nor of EGF or the transcription factor, TWIST. CONCLUSIONS: Our data demonstrate that SNAIL1 and TNF are strongly expressed in TC of CD-associated fistulae. These observations support our previous data and indicate the onset of EMT-associated events in the pathogenesis of CD fistulae.

Asymmetric sphincter innervation is associated with fecal incontinence after anal sphincter trauma during childbirth.

AIMS: Functional asymmetry of pelvic floor innervation has been shown to exist in healthy subjects, and has been proposed to be a predictor of increased risk for fecal incontinence in case of trauma. However, this remains to be shown for different clinical conditions such as traumatic childbirth. METHODS: A conventional surface EMG system was used to assess the innervation of the external anal sphincter. A symmetry index was used to define the relative EMG amplitude asymmetry of the external anal sphincter between 0 (symmetric) and 1 (asymmetric). Three cohorts were studied: 40 nulliparous women in the third trimester (Study 1), 15 primiparous women within 6 months following vaginal delivery without clinically apparent anal sphincter trauma (Study 2), and 50 women after childbirth-related third or fourth degree perineal tear 6-12 months postpartum (Study 3). Furthermore, all women underwent conventional anorectal manometry. RESULTS: Sixteen or forty nulliparous women reported signs of fecal incontinence; however, relative asymmetry was not correlated to symptom severity (P = 0.345), and not to manometric measures (Study 1). In Study 2, Women who had suffered clinically apparent anal sphincter trauma (P = 0.07) tended to have a stronger association between incontinence and asymmetry. In Study 3, 19/50 women reported moderate to severe incontinence. Asymmetry and symptom severity were significantly correlated (P < 0.001). Patients with incontinence had a significantly higher asymmetry score than their continent counterparts. CONCLUSION: Functional asymmetry of anal sphincter innervation is significantly associated with incontinence symptoms, but only after childbirth-related sphincter injuries and therefore, should be regarded as an additional risk factor.

Percutaneous MR-guided radiofrequency ablation of recurrent sacrococcygeal chordomas.

OBJECTIVE: Radiofrequency ablation is emerging as a therapeutic technique for the treatment of an increasing variety of tumors. Exact visual guidance to the tumor and controlled delivery of energy is pivotal for ablation success. CONCLUSION: Introducing MRI as a guidance technique ideally uses tumor-specific tissue characteristics, allows direct multiplanar reconstruction for precise needle positioning, and permits real-time monitoring and assessment of treatment-induced tissue signal changes to increase the safety of the procedure.

Serotonin receptor blockade increases food intake and body weight after total gastrectomy in rats.

BACKGROUND: Total gastrectomy often results in early satiety and loss of body weight. Serotonin inhibits food intake, and postprandial serotonin release is increased after total gastrectomy. Serotonin might contribute to early satiety and loss of body weight after total gastrectomy. METHODS AND MATERIALS: Food intake and body weight were investigated with an automated recording system in gastrectomized rats 1-12 months postoperatively. Rats were treated with metergoline, a 5-hydroxytryptamine (5-HT)(1/2) receptor antagonist, two different 5-HT(3) receptor antagonists, a combination of metergoline and devazepide, a cholecystokinin (CCK) a receptor antagonist, or vehicle. In addition, metergoline or vehicle was applied continuously by an intraperitoneal osmotic minipump for 7, 28, or 84 days after total gastrectomy. RESULTS: Metergoline treatment resulted in a dose-dependent increase in food intake in gastrectomized rats. 5-HT(3) receptor antagonist treatment had no effect, and devazepide in addition to metergoline did not further stimulate food intake. Metergoline increased food intake at 1, 3, and 6 months postoperatively by up to 45% (24-h cumulative food intake [FI], 6 months: vehicle 3.83 +/- 0.10, metergoline 5.52 +/- 0.15 g/100 g body weight (BW), P < 0.0001). Chronic metergoline treatment for 7, 28, or 84 days significantly increased food intake after total gastrectomy compared to vehicle treatment (FI 7 days: vehicle 30.83 +/- 0.71, metergoline 36.27 +/- 0.85 g/100 g BW; P < 0.0002; average weekly FI during 28 days; vehicle 31.23 +/- 0.22, metergoline 36.83 +/- 0.33 g/100 g BW, P < 0.0001; average weekly FI during 84 days: vehicle 33.02 +/- 0.59, metergoline 35.07 +/- 0.48 g/100g BW, P < 0.008), and there was a significant body weight increase compared to vehicle treatment (7 days: DeltaBW vehicle -0.7 +/- 1.2 g vs DeltaBW metergoline 9.0 +/- 2.1 g, P < 0.001; 28 days: DeltaBW vehicle 0.3 +/- 2.2 vs DeltaBW metergoline 13.0 +/- 2.3, P < 0.001; 84 days: DeltaBW vehicle 25.7 +/- 10.2 vs DeltaBW metergoline 49.5 +/- 7.2, P < 0.04). Treatment for 84 days resulted in a significant body weight gain, while vehicle treatment had no effect (vehicle: 438 +/- 11 g vs 464 +/- 12 g, P < 0.2, n.s.; metergoline: 448 +/- 9 g vs 498 +/- 10 g, P < 0.007). CONCLUSIONS: Inhibition of food intake by serotonin might contribute to early satiety and loss of body weight after total gastrectomy.

Total gastrectomy severely alters the central regulation of food intake in rats.

OBJECTIVE: To investigate the central regulation of food intake by quantifying neuron activation of the nucleus of the solitary tract (NTS) after injection of cholecystokinin (CCK) or food intake in gastrectomized rats. SUMMARY BACKGROUND DATA: Total gastrectomy is followed by early satiety, low calorie intake, and weight loss in the majority of patients. The etiology of these effects is unknown. Sixty percent to 70% of patients remain underweight after total gastrectomy, the weight loss averaging 25% of preoperative body weight. About two thirds of gastrectomized patients report early satiety, and about 60% do not reach the recommended daily calorie intake. The NTS is a brain stem center involved in the regulation of food intake; thus, the extent and pattern of neuronal activation provide information on the process involved in the initiation of satiation and the regulation of food intake. METHODS: The authors investigated neuronal activation in the NTS using c-fos immunohistochemistry following CCK injection or food intake in healthy control rats, sham-operated control rats, age-matched control rats, weight-matched control rats, and vagotomized or gastrectomized rats. RESULTS: Neuronal activation in the NTS after CCK injection was significantly decreased 21 days after total gastrectomy, but increased by up to 51% 3 months and by up to 102% 12 months after surgery compared to age-matched unoperated control rats. Neuronal activation in the NTS in response to feeding was markedly increased up to fivefold in gastrectomized rats. This increase was early in onset and sustained, and occurred despite significantly reduced food intake. Administration of MK329, a CCK-A receptor antagonist, significantly reduced the number of postprandially activated neurons in both gastrectomized and control rats. CONCLUSIONS: The early postprandial activation of NTS neurons after total gastrectomy in rats may correspond to early satiety reported by patients, while the sustained activation of NTS neurons after a meal could contribute to a reduced daily calorie intake. These data suggest that a disturbed central regulation of food intake might contribute to early satiety, reduced food intake, and weight loss after total gastrectomy.