Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing

Milk fat globule-epidermal growth factor 8 (MFG-E8) plays an important role in maintaining intestinal barrier homeostasis and accelerating intestinal restitution. However, studies of MFG-E8 expression in humans with ulcerative colitis are lacking. We examined MFG-E8 expression in colonic mucosal biopsies from ulcerative colitis patients and healthy controls (n = 26 each) by real-time quantitative polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry. MFG-E8 mRNA and protein expression was lower in ulcerative colitis patients than in controls. MFG-E8 expression was inversely correlated with mucosal inflammatory activity and clinical disease activity in patients. MFG-E8 was present in human intestinal epithelial cells both in vivo and in vitro. Apoptosis induction was also detected in the intestinal epithelium of ulcerative colitis patients by terminal-deoxynucleoitidyl transferase mediated nick-end labeling assay. We used lentiviral vectors encoding human MFG-E8 targeting short hairpin RNA to obtain MFG-E8 knockdown intestinal epithelia cell clones. MFG-E8 knockdown could promote apoptosis in intestinal epithelial cell lines, accompanied by a decrease in level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and induction of the proapoptotic protein BCL2-associated protein X (BAX). The addition of recombinant human MFG-E8 led to decreased BAX and cleaved caspase-3 levels and induction of BCL-2 level in intestinal epithelia cells. MFG-E8 knockdown also attenuated wound healing on scratch assay of intestinal epithelial cells. The mRNA level of intestinal trefoid factor 3, a pivotal factor in intestinal epithelial cell migration and restitution, was downregulated with MFG-E8 knockdown. In conclusion, we demonstrated that decreased colonic MFG-E8 expression in patients with ulcerative colitis may be associated with mucosal inflammatory activity and clinical disease activity through basal cell apoptosis and preventing tissue healing in the pathogenesis of ulcerative colitis.     

Apoptosis: one of the mechanisms that maintains unresponsiveness of the intestinal mucosal immune system

Intestinal mucosa is constantly exposed to environmental AGS: Activation of lamina propria (LP) T cells by luminal Ags may lead to the production of inflammatory cytokines and subsequent mucosal inflammation and tissue damage. However, in normal circumstances, LP T cells do not respond to antigenic stimulation. The mechanisms of this unresponsiveness in healthy subjects are not fully understood. In this study, we found by in vivo analysis that, except for T cells in lymph nodules of the mucosa, 15% of LP T cells underwent apoptosis in normal individuals. In contrast, there was a marked reduction in apoptosis of LP T cells in patients with inflammatory bowel disease (Crohn's disease and ulcerative colitis) and those with specific colitis. Our findings suggest that apoptosis might be a mechanism that turns off mucosal T cell responses to environmental Ags in healthy subjects, and resistance to apoptosis could be an important cause of mucosal immune dysregulation and tissue inflammation in colitis.     

Preventive effects of Goji berry on dextran-sulfate-sodium-induced colitis in mice

Goji berry (Lycium barbarum) exerts immune modulation and suppresses inflammation in vitro and in vivo. We hypothesized that Goji berry had beneficial effects on dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice through suppressing inflammation. Six-week-old male C57BL/6 mice were supplemented with a standard AIN-93G diet with or without 1% (w/w) Goji berry for 4 weeks. Then, colitis was induced by supplementing 3% DSS in drinking water for 7 days, followed by 7 days of remission period to mimic ulcerative colitis symptoms. Goji berry supplementation ameliorated DSS-induced body weight loss, diminished diarrhea and gross bleeding, and resulted in a significantly decreased disease activity index, as well as DSS-associated colon shortening. Moreover, 30% mortality rate caused by DSS-induced colitis was avoided because of Goji berry supplementation. Histologically, Goji berry ameliorated colonic edema, mucosal damage and neutrophil infiltration into colonic intestinal tissue in response to DSS challenge, which was associated with decreased expression of chemokine (C-X-C motif) ligand 1 and monocyte chemoattractant protein-1, as well as inflammatory mediators interleukin-6 and cyclooxygenase-2. In conclusion, Goji supplementation confers protective effects against DSS­induced colitis, which is associated with decreased neutrophil infiltration and suppressed inflammation. Thus, dietary Goji is likely beneficial to inflammatory bowel disease patients as a complementary therapeutic strategy.     

Loss of miR-24-3p promotes epithelial cell apoptosis and impairs the recovery from intestinal inflammation

While apoptosis plays a significant role in intestinal homeostasis, it can also be pathogenic if overactive during recovery from inflammation. We recently reported that microRNA-24-3p (miR-24-3p) is elevated in the colonic epithelium of ulcerative colitis patients during active inflammation, and that it reduced apoptosis in vitro. However, its function during intestinal restitution following inflammation had not been examined. In this study, we tested the influence of miR-24-3p on mucosal repair by studying recovery from colitis in both novel miR-24-3p knockout and miR-24-3p-inhibited mice. We observed that knockout mice and mice treated with a miR-24-3p inhibitor had significantly worsened recovery based on weight loss, colon length, and double-blinded histological scoring. In vivo and in vitro analysis of miR-24-3p inhibition in colonic epithelial cells revealed that inhibition promotes apoptosis and increases levels of the pro-apoptotic protein BIM. Further experiments determined that silencing of BIM reversed the pro-apoptotic effects of miR-24-3p inhibition. Taken together, these data suggest that miR-24-3p restrains intestinal epithelial cell apoptosis by targeting BIM, and its loss of function is detrimental to epithelial restitution following intestinal inflammation.Subject terms: Apoptosis, Drug delivery     

Dyospiros kaki phenolics inhibit colitis and colon cancer cell proliferation,but not gelatinase activities

Polyphenols from persimmon (Diospyros kaki) have demonstrated radical-scavenging and antiinflammatory activities; however, little is known about the effects of persimmon phenolics on inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Therefore, we aimed in this work to characterize the antiinflammatory and antiproliferative effects of a persimmon phenolic extract (80% acetone in water), using an in vivo model of experimental colitis and a model of cancer cell invasion. Our results show, for the first time, a beneficial effect of a persimmon phenolic extract in the attenuation of experimental colitis and a potential antiproliferative effect on cultured colon cancer cells. Administration of persimmon phenolic extract to mice with TNBS-induced colitis led to a reduction in several functional and histological markers of colon inflammation, namely: attenuation of colon length decrease, reduction of the extent of visible injury (ulcer formation), decrease in diarrhea severity, reduced mortality rate, reduction of mucosal hemorrhage and reduction of general histological features of colon inflammation. In vitro studies also showed that persimmon phenolic extract successfully impaired cell proliferation and invasion in HT-29 cells. Further investigation showed a decreased expression of COX-2 and iNOS in the colonic tissue of colitis mice, two important mediators of intestinal inflammation, but there was no inhibition of the gelatinase MMP-9 and MMP-2 activities. Given the role of inflammatory processes in the progression of CRC and the important link between inflammation and cancer, our results highlight the potential of persimmon polyphenols as a pharmacological tool in the treatment of patients with IBD.     

NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats

Although substance P (SP) has been implicated as a mediator of neurogenic inflammation in the small intestine, little information is available regarding the role of SP in the pathogenesis of chronic ulcerative colitis. In this study, our aim was to investigate whether the intraperitoneal administration of a nonpeptide neurokinin-1 (NK-1) antagonist, CP-96345, which antagonizes the binding of SP to its NK-1 receptor, results in the attenuation of colonic inflammation induced in rats by 5% dextran sodium sulfate (DSS) in drinking water for 10 days compared with an inactive enantiomer, CP-96344. Disease activity was assessed daily for 10 days, after which colonic tissue damage was scored and myeloperoxidase activity and colon and urinary 8-isoprostanes were measured. Animals receiving DSS exhibited marked physical signs of colitis by day 5 compared with controls. Chronic administration of the NK-1 antagonist significantly reduced the disease activity index, mucosal myeloperoxidase activity, colonic tissue damage score, and mucosal and urinary levels of 8-isoprostanes compared with inactive enantiomer- or vehicle-injected (saline) animals receiving DSS alone. These data indicate that the administration of an NK-1 antagonist can attenuate colonic inflammation and oxidative stress and suggest a novel therapeutic approach in the treatment of chronic ulcerative colitis.     

Berberine ameliorates DSS-induced intestinal mucosal barrier dysfunction through microbiota-dependence and Wnt/β-catenin pathway

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colon, and it has become one of the world-recognized medical problems as it is recurrent and refractory. Berberine (BBR) is an effective drug for UC treatment. However, the underlying mechanism and targets remain obscure. In this study, we systematically investigated the therapeutic effect and its mechanism of BBR in ameliorating DSS-induced mouse colitis. Expectedly, the colon inflammation was significantly relieved by BBR, and microbiota depletion by antibiotic cocktail significantly reversed the therapeutic effect. Further studies showed that BBR can regulate the abundance and component of bacteria, reestablish the broken chemical and epithelial barriers. Meanwhile, BBR administration dramatically decreased ILC1 and Th17 cells, and increased Tregs as well as ILC3 in colonic tissue of DSS-induced mice, and it was able to regulate the expression of various immune factors at the mRNA level. Moreover, a proteomic study revealed that Wnt/β-catenin pathway was remarkably enhanced in colonic tissue of BBR-treated mice, and the therapeutic effect of BBR was disappeared after the intervention of Wnt pathway inhibitor FH535. These results substantially revealed that BBR restores DSS-induced colon inflammation in a microbiota-dependent manner, and BBR performs its protective roles in colon by maintaining the structure and function of the intestinal mucosal barrier, regulating the intestinal mucosal immune homeostasis and it works through the Wnt/β-catenin pathway. Importantly, these findings also provided the proof that BBR serves as a potential gut microbiota modulator and mucosal barrier protector for UC prevention and therapy.     

Lipoxin biosynthesis in inflammatory bowel disease

BACKGROUND AND AIMS: Lipoxins are anti-inflammatory lipid mediators that are produced in gut mucosa, which serve to limit and resolve persistent inflammation. The purpose of this study was to evaluate colonic lipoxin biosynthesis in patients with ulcerative colitis (UC) to establish a possible biochemical basis for persistent inflammation in UC. METHODS: Colonic mucosa from patients with UC or organ donors (controls) was placed into tissue culture for 90 min. The conditioned media was assayed (ELISA) for lipoxin A4 (LXA) and the biologically active isomer 15-epi-LXA4 (aspirin triggered lipoxin, ATL). Mucosal tissue 15-lipoxygenase protein was determined by Western blot. RESULTS: Patient colonic mucosa produced significantly lower (12-fold) amounts of LXA, relative to organ donors. This occurred irregardless of patient steroid treatment. However, patient tissue responded to in vitro aspirin by synthesizing biologically active ATL. For the first time, human colonic mucosa was found to synthesize 15-lipoxygenase-2, an epithelial-derived isoenzyme used for lipoxin synthesis. These levels were significantly lower in UC patients compared to the control tissue. Finally, mice chronically treated with a putative selective 15-lipoxygenase inhibitor (PD 146176) experienced significantly worse intestinal function during experimental colitis, relative to untreated mice. CONCLUSION: Colonic mucosa from UC patients demonstrated defective lipoxin biosynthesis, which may contribute to the inability of these patients to resolve persistent colonic inflammation.     

Osteopontin as two-sided mediator of intestinal inflammation

Osteopontin (OPN) is characterized as a major amplifier of Th1-immune responses. However, its role in intestinal inflammation is currently unknown. We found considerably raised OPN levels in blood of wild-type (WT) mice with dextran sodium sulfate (DSS)-induced colitis. To identify the role of this mediator in intestinal inflammation, we analysed experimental colitis in OPN-deficient (OPN(-/-)) mice. In the acute phase of colitis these mice showed more extensive colonic ulcerations and mucosal destruction than WT mice, which was abrogated by application of soluble OPN. Within the OPN(-/-) mice, infiltrating macrophages were not activated and showed impaired phagocytosis. Reduced mRNA expression of interleukin (IL)-1 beta and matrix metalloproteinases was found in acute colitis of OPN(-/-) mice. This was associated with decreased blood levels of IL-22, a Th17 cytokine that may mediate epithelial regeneration. However, OPN-(/-) mice showed increased serum levels of tumour necrosis factor (TNF)-alpha, which could be due to systemically present lipopolysaccharide translocated to the gut. In contrast to acute colitis, during chronic DSS-colitis, which is driven by a Th1 response of the lamina propria infiltrates, OPN(-/-) mice were protected from mucosal inflammation and demonstrated lower serum levels of IL-12 than WT mice. Furthermore, neutralization of OPN in WT mice abrogated colitis. Lastly, we demonstrate that in patients with active Crohn's disease OPN serum concentration correlated significantly with disease activity. Taken together, we postulate a dual function of OPN in intestinal inflammation: During acute inflammation OPN seems to activate innate immunity, reduces tissue damage and initiates mucosal repair whereas during chronic inflammation it promotes the Th1 response and strengthens inflammation.     

蒲公英多糖对溃疡性结肠炎大鼠IL-6/STAT3信号通路的影响

目的：观察蒲公英多糖对溃疡性结肠炎大鼠模型IL-6/STAT3信号通路的调控作用。方法：清洁级SD大鼠40只,雌雄各半,随机分为4组（n=10）：空白组、模型组、阳性对照组、蒲公英多糖组。采用2,4,6-三硝基苯磺酸（TNBS）诱导结肠炎大鼠模型,阳性对照组采用美沙拉嗪10 mg/kg·d灌胃,蒲公英多糖组采用蒲公英多糖10 mg/kg·d灌胃,治疗4周后处死,观察大鼠结肠粘膜病理改变,检测大鼠血清白介素-6（IL-6）含量、结肠髓过氧化物酶（MPO）、白介素-6受体（sIL-6Rα）、糖蛋白130（gp130）、转录活化因子3（STAT3）、IL-6 mRNA表达。结果：与正常组比较,模型组大鼠血清IL-6含量明显升高（P<0.01）,MPO阳性密度明显增高（P<0.01）,sIL-6Rα、gp130含量明显增高（P<0.01）,肠组织STAT3、IL-6 mRNA相对表达量明显增高（P<0.01）;与模型组比较蒲公英多糖组、美沙拉嗪组大鼠血清IL-6含量明显降低（P<0.01）,MPO阳性密度明显降低（P<0.01）,sIL-6Rα、gp130含量明显降低（P<0.01）,肠组织STAT3、IL-6 mRNA相对表达量与模型组比较明显降低（P<0.05）。结论：蒲公英多糖能够降低溃疡性结肠炎大鼠IL-6水平,下调IL-6/STAT3通路中sIL-6Rα、gp130蛋白表达量,进而下调大鼠肠组织STAT3、IL-6 mRNA的转录水平,缓解结肠组织的炎症状态,保护和修复粘膜组织,起到治疗溃疡性结肠炎的作用。     

X-ray colonometry in the diagnosis of colonic dyskinesias and inflammatory diseases

Clinical and X-ray studies were made in 316 patients, which revealed intestinal dyskinesia, chronic colitis, and nonspecific ulcerative colitis in 105, 133, and 78 patients, respectively. Irrigoscopy (administration of a contrast enema, examination of the mucosal contour, and double contrasting) was performed in all the patients, by analyzing X-ray planimetric indices. X-ray colonoplanimetry makes it possible to objectify the interpretation of the X-ray pattern in chronic inflammatory diseases of the large bowel and colonic dyskinesia.     

MicroRNA-193a-3p Reduces Intestinal Inflammation in Response to Microbiota via Down-regulation of Colonic PepT1

Intestinal inflammation is characterized by epithelial disruption, leading to the loss of barrier function, recruitment of immune cells, and host immune responses to gut microbiota. PepT1, a di/tripeptide transporter that uptakes bacterial products, is up-regulated in inflamed colon tissue, which implies its role in bacterium-associated intestinal inflammation. Although microRNA (miRNA)-mediated gene regulation has been found to be involved in various processes of inflammatory bowel disease (IBD), the biological function of miRNAs in the pathogenesis of IBD remains to be explored. In this study we detected miRNA expression patterns in colon tissues during colitis and investigated the mechanism underlying the regulation of colonic PepT1 by miRNAs. We observed an inverse correlation between PepT1 and miR-193a-3p in inflamed colon tissues with active ulcerative colitis, and we further demonstrated that miR-193a-3p reduced PepT1 expression and activity as a target gene and subsequently suppressed the NF-κB pathway. Intracolonic delivery of miR-193a-3p significantly ameliorated dextran sodium sulfate-induced colitis, whereas the overexpression of colonic PepT1 via PepT1 3′-untranslated region mutant lentivirus vector abolished the anti-inflammatory effect of miR-193a-3p. Furthermore, antibiotic treatment eliminated the difference in the dextran sodium sulfate-induced inflammation between the presence and absence of miR-193a-3p. These findings suggest that miR-193a-3p regulation of PepT1 mediates the uptake of bacterial products and is a potent mechanism during the colonic inflammation process. Overall, we believe miR-193a-3p may be a potent regulator of colonic PepT1 for maintaining intestinal homeostasis.     

Upregulation of CFTR expression but not SLC26A3 and SLC9A3 in ulcerative colitis

In inflamed colonic mucosa, the equilibrium between absorptive and secretory functions for electrolyte and salt transport is disturbed. We compared the expression of three major mediators of the intestinal salt transport between healthy and inflamed colonic mucosa to understand the pathophysiology of diarrhea in inflammatory bowel disease. Expression levels of the cystic fibrosis transmembrane regulator (CFTR) (Cl- channel), SLC26A3 (Cl-/HCO exchanger) and SLC9A3 (Na+/H+ exchanger) mRNAs were measured by real-time quantitative RT-PCR in peroperative colonic samples from controls (n = 4) and patients with ulcerative colitis (n = 10). Several samples were obtained from each individual. Tissue samples were divided into three subgroups according to their histological degree of inflammation. Expression of CFTR and SLC26A3 proteins were determined by immunohistochemistry and Western blotting from the same samples, respectively. Increased expression of CFTR mRNA was observed in all three groups of affected tissue samples, most pronounced in mildly inflamed colonic mucosa (5-fold increase in expression; P < 0.001). The expression of the CFTR protein was detected from health and inflamed colon tissue. Although the expression of the SLC26A3 mRNA was significantly decreased in severe ulcerative colitis (P < 0.05), the SLC26A3 protein levels remained unchanged in all groups. The expression of SLC9A3 mRNA was significantly changed between the mild and severe groups. Intestinal inflammation modulates the expression of three major mediators of intestinal salt transport and may contribute to diarrhea in ulcerative colitis both by increasing transepithelial Cl- secretion and by inhibiting the epithelial NaCl absorption.     

Role of the enteric microbiota in intestinal homeostasis and inflammation

The mammalian intestine encounters many more microorganisms than any other tissue in the body thus making it the largest and most complex component of the immune system. Indeed, there are greater than 100 trillion (10¹⁴) microbes within the healthy human intestine, and the total number of genes derived from this diverse microbiome exceeds that of the entire human genome by at least 100-fold. Our coexistence with the gut microbiota represents a dynamic and mutually beneficial relationship that is thought to be a major determinant of health and disease. Because of the potential for intestinal microorganisms to induce local and/or systemic inflammation, the intestinal immune system has developed a number of immune mechanisms to protect the host from pathogenic infections while limiting the inflammatory tissue injury that accompanies these immune responses. Failure to properly regulate intestinal mucosal immunity is thought to be responsible for the inflammatory tissue injury observed in the inflammatory bowel diseases (IBD; Crohn disease, ulcerative colitis). An accumulating body of experimental and clinical evidence strongly suggests that IBD results from a dysregulated immune response to components of the normal gut flora in genetically susceptible individuals. The objective of this review is to present our current understanding of the role that enteric microbiota play in intestinal homeostasis and pathogenesis of chronic intestinal inflammation.     

思连康治疗溃疡性结肠炎的疗效及机制研究

目的研究微生态制剂思连康辅助治疗溃疡性结肠炎的疗效及其可能的机制。方法将溃疡性结肠炎患者79例随机分成治疗组及对照组,治疗组40例,对照组39例。对照组采用常规治疗,治疗组在常规治疗的基础上加用双歧四联活菌制剂——思连康。比较2组治疗前及治疗2个月后患者的临床症状评分、结肠黏膜炎症表现、细胞因子IL-10、IL-18的变化。结果治疗2个月后2组临床症状评分、结肠黏膜炎症评分均较治疗前有明显改善（P〈0.01）,且治疗组临床症状、结肠黏膜炎症改善程度优于对照组（P〈0.01）,IL-10、IL-18的含量治疗前后有明显的变化,IL-10增加,IL-18降低。结论思连康可辅助治疗溃疡性结肠炎,其机制可能是通过调节细胞因子的变化,进一步影响结肠黏膜的免疫功能。     

白细胞介素23受体、白细胞介素17A在炎症性肠病患者中的表达 及临床意义

目的:通过检测白细胞介素23受体(IL-23R)及白细胞介素17A(IL-17A)在炎症性肠病(IBD)患者肠黏膜及血清中的表达水平,探讨其在IBD发病过程中的作用及意义。方法:收集32例克罗恩病(CD)患者、29例溃疡性结肠炎(UC)患者及27例对照者的内镜肠黏膜活检标本,采用荧光定量PCR技术检测肠黏膜内IL-23R、IL-17AmRNA的表达情况,免疫组化技术分析IL-23R、IL-17A在肠黏膜中的原位表达。结果:与健康对照组相比,CD及UC患者肠黏膜组织内IL-23R mRNA表达显著增高(P<0.05),CD及UC组间的表达量差异无统计学意义(P>0.05)。CD及UC患者肠黏膜组织内IL-17A mRNA表达显著增高(P<0.05),CD组肠黏膜组织内IL-17AmRNA表达显著高于UC组(P<0.05)。免疫组化分析显示IL-23R阳性细胞在CD与UC肠黏膜固有层内有较多表达,较正常黏膜内的肠上皮细胞相比,CD及UC患者肠黏膜IL-23R蛋白表达量最著增高(P<0.05),UC及CD组间的表达量差异无统计学意义(P>0.05)。IL-17A阳性细胞在CD与UC肠黏膜固有层内有较多表达,较正常黏膜内的肠上皮细胞相比,CD及UC患者肠黏膜IL-17A蛋白表达量最著增高(P<0.05)。结论:IL-23R及IL-17A在IBD患者肠黏膜中表达显著增高,提示IL-23R及IL-17A表达异常与IBD的发生发展密切相关,有可能成为IBD治疗的新靶点。     

Effects of ulcerative colitis activity on plasma and mucosal prostaglandin E2 concentration

The aim of this study was to determine effects of changes in ulcerative colitis activity on mucosal and plasma PGE2 concentrations measured with an EIA in 49 patients who underwent sigmoidoscopy. The disease was diagnosed in 37 patients. Twelve patients with normal colonic mucosa served as controls. Patients were divided into three groups depending on the changes of endoscopic picture during a three-month follow-up. Some laboratory markers of the disease activity, such as C-reactive protein, albumin, gamma-globulin and hemoglobin concentrations, sedimentation rate, and white blood and platelets counts, were also evaluated. Initial examination revealed a significant, positive correlation of mucosal and plasma PGE2 concentration with endoscopic score. Follow-up of patients without significant progression of mucosal changes revealed constant and close to normal concentration of mucosal PGE2. Plasma PGE2 was higher at the second examination, yet without significant difference. Improvement of endoscopic picture resulted in significant decrease of plasma and mucosal PGE2 concentrations. Worsening of mucosal changes reflected endoscopically was associated with significant increase of PGE2. There were no remarkable changes in the values of analyzed laboratory markers of the disease activity. These results indicate the usefulness of mucosal or plasma PGE2 measurement as a possible prognostic marker in patients with ulcerative colitis.     

Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease

The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation.     

Selective survival of calretinin- and vasoactive-intestinal-peptide-containing nerve elements in human chagasic submucosa and mucosa

Chronic Chagas' disease is frequently characterized by massive myenteric neuron loss resulting in megacolon with severely and irreversibly disturbed motility. Here, we focused on two submucosal neuron populations, immunoreactive for calretinin (CALR) or somatostatin (SOM), and their respective mucosal nerve fibres in chagasic megacolon. Surgically removed megacolonic segments of seven chagasic patients were compared with seven age- and region-matched non-chagasic control segments. Evaluation included immunohistochemical triple-staining of cryosections for CALR, SOM and peripherin or for CALR and vasoactive intestinal peptide (VIP) and of submucosal whole-mounts for CALR, SOM and the pan-neuronal marker anti-HuC/D. Submucosal neuron counts in chagasic tissue revealed neuron numbers reduced to 51.2?% of control values. In cryosections, nerve fibre area measurements revealed 8.6?% nerve fibre per mucosal area in control segments, but this value decreased to 1.5?% in megacolonic segments. In both evaluations, a disproportionate decrease of SOM-reactive nerve elements was observed. The proportions of SOM-positive neurons related to the total neuron number declined to 2?% (control 10?%) and the proportion of SOM-reactive mucosal nerve fibres related to the whole mucosal area to 0.014?% (control 1.8?%)in chagasic tissue. The second set of cryosections revealed extensive colocalization of CALR with VIP in both surviving submucosal perikarya and mucosal nerve fibres. We suggest that VIP, a neuroprotective and neuroeffectory peptide typically contained in submucosal neurons, allows both the VIP-containing neurons to endure and the patients to survive by maintaining their mucosal barrier, despite the almost complete loss of colonic motility for decades.