Multivesicular bodies in intestinal epithelial cells: responsible for MHC class II-restricted antigen processing and origin of exosomes.

In normal conditions intestinal epithelial cells (IECs) constitutively stimulate regulatory CD4(+) T cells. However, in Crohn's disease (CD), this major histocompatibility complex (MHC) class II-restricted antigen presentation results in stimulation of proinflammatory CD4(+) T cells. We hypothesized that these alternative functions might be mediated by differential sorting and processing of antigens into distinct MHC II-enriched compartments (MIICs). Accordingly, we analysed the endocytic pathways of lumenally applied ovalbumin (OVA) in IECs of the jejunum and ileum of wild-type (WT) and TNFDeltaARE/WT mice that develop a CD-resembling ileitis. Using quantitative reverse transcription polymerase chain reaction, we found that messenger RNA levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-17 and interleukin-10 were significantly up-regulated in the inflamed ileum of TNFDeltaARE/WT mice, confirming CD-like inflammation. Fluorescence and immunoelectron microscopy revealed the presence of MHC II and invariant chain throughout the late endocytic compartments, with most molecules concentrated in the multivesicular bodies (MVB). OVA was targeted into MVB and, in contrast to other MIICs, accumulated in these structures within 120 min of exposure. The IEC-specific A33 antigen localized to internal vesicles of MVB and A33/class II-bearing exosomes were identified in intercellular spaces. Remarkably, the expression pattern of MHC II/invariant chain molecules and the trafficking of OVA were independent of mucosal inflammation and the specific region in the small intestine. MVB seem to be principally responsible for class II-associated antigen processing in IECs and to constitute the origin of MHC II-loaded exosomes. The distinctive functions of IECs in antigen presentation to CD4(+) T cells might arise as a result of differential processing within the MVB identified here.

[Frequent causes of diarrhea: celiac disease and lactose intolerance]. / Häufige Ursachen von Durchfall: Sprue und Lactoseintoleranz.

Celiac disease and lactose intolerance are both relatively frequent diseases with symptoms occurring after ingestion of certain food components. In celiac disease wheat gluten and related proteins of other cereals induce an inflammatory disease of the small intestine in predisposed individuals, leading to gastrointestinal and extraintestinal symptoms. Moreover, there is an association with many other diseases and besides classic symptoms (diarrhea, weight loss, malabsorption) atypical courses with less or lacking gastrointestinal symptoms exist. The prevalence is about 1 : 100 (Europe, USA) and higher than supposed earlier. Diagnostic criteria include serologic tests (tissue transglutaminase antibody, endomysial antibody) and characteristic small bowel histology (lymphocytic infiltration, villous atrophy). Therapy is a strict and lifelong gluten-free diet. Rarely, refractory disease or lack of compliance are associated with increased risk of malignancy and worse prognosis. Lactose intolerance is attributed to low intestinal lactase levels, due to reduced genetic expression or mucosal injury and consequent intolerance to dairy products. The frequency is varying in different ethnic groups, occurring in 10-15% of Northern European people. Intensity of clinical symptoms (diarrhea, abdominal pain, bloating) depends on the amount of ingested lactose and individual activity of intestinal lactase. The capacity of lactose malabsorption can be measured using the noninvasive lactose breath hydrogen test. The treatment is based on a reduced dietary lactose intake or in case of secondary form treatment of the underlying disease.

Delayed complications after placement of self-expanding stents in malignant esophageal obstruction: treatment strategies and survival rate.

PURPOSE: Placement of self-expanding metal stents is regarded as a safe and effective treatment in patients with incurable malignant esophagogastric obstruction. However, proceeding and possible benefit of re-interventions in patients with recurrent dysphagia due to delayed complications (>4 weeks after stent insertion) is unclear. PATIENTS AND METHODS: In 133 patients with malignant stricture of the esophagus or the esophagogastric junction 164 expandable metal stents were placed. About 89 patients were followed up until death. All tumor- or stent-related complications and consequent re-interventions were recorded. RESULTS: The overall incidence of delayed complications was 53.4% (71 of 133 pts.), with 34 patients (25.6%) experiencing more than one complication. Recurrent dysphagia due to tumor ingrowth (22%) or overgrowth (15%), bolus obstruction (21%), stent migration (9%), and development of esophagorespiratory fistula (9%) was successfully treated by dilatation (24%), placement of a second/third stent (27%), laser therapy (16%), and/or placement of a feeding tube (PEG, 19%). The median survival of patients with endoscopic therapy was significantly longer (222 +/- 26 days) compared to patients without re-intervention (86 +/- 14 days, P < 0.0001). CONCLUSIONS: Delayed complications after metal stent placement for malignant esophageal stricture are common, but can be treated successfully by endoscopic re-intervention in most cases. Regular interventional therapy may also improve survival.

Premedication with orally administered lorazepam in adults undergoing ERCP: a randomized double-blind study.

BACKGROUND: Restlessness often complicates ERCP and may be a reason for premature termination of the procedure. OBJECTIVE: Our purpose was to evaluate whether a premedication with orally administered lorazepam could reduce the need for sedatives and improve sedation quality. DESIGN: Randomized double-blind trial. SETTING: Therapeutic ERCP with an intravenous sedation containing midazolam, propofol, and S(+)-ketamine. PATIENTS: 95 inpatients (aged 20-91 years). INTERVENTIONS: 1 mg of lorazepam (n=47) or placebo (n=48) given orally before ERCP. MAIN OUTCOME MEASUREMENT: Total amount of administered propofol. RESULTS: Heart rate, blood pressure, number of oxygen desaturations, and states of restlessness did not differ between the groups. The same amount of midazolam was administered in both groups. There was no significant difference in the total amount of propofol to achieve adequate sedation (lorazepam vs placebo: 71+/-5 vs 63+/-4 microg/kg/min, mean+/-SE). Paradoxically, patients pretreated with lorazepam even needed more propofol in the early phase of sedation (275+/-39 vs 159+/-37 microg/kg in minutes 5-10, P<.05) and the total amount of ketamine administered was higher in this group as well (15.8+/-1.4 vs 11.3+/-1.2 microg/kg/min, P<.05). In both groups there were high rates of satisfaction with the course of the procedure evaluated both by the endoscopists and the patients. CONCLUSION: The trial failed to show an advantage of an oral premedication with lorazepam. The amount of sedatives administered in the lorazepam group even tended to be higher. A premedication with lorazepam may be counterproductive when followed by sedation containing another benzodiazepine.

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis.

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.

Oral p38 mitogen-activated protein kinase inhibition with BIRB 796 for active Crohn's disease: a randomized, double-blind, placebo-controlled trial.

BACKGROUND & AIMS: The p38 mitogen-activated protein kinase (MAPK) regulates the expression of proinflammatory cytokines, which play a critical role in the pathophysiology of Crohn's disease (CD). This study investigated the efficacy and safety of BIRB 796, a highly potent inhibitor of p38 MAPK, in chronic active CD. METHODS: In a multicenter, multinational trial, 284 patients with moderate to severe CD were randomized to receive placebo, or 10, 20, 30, or 60 mg of BIRB 796 twice daily for 8 weeks. Clinical endpoints were based on standard safety assessments, CD Activity Index, C-reactive protein levels, and quality of life (Inflammatory Bowel Disease Questionnaire). In a substudy, the Crohn's Disease Endoscopic Index of Severity and histologic results of biopsy specimens were assessed. RESULTS: No clinical efficacy (primary end point, clinical remission; secondary end point, clinical response; Inflammatory Bowel Disease Questionnaire; Crohn's Disease Endoscopic Index of Severity) was seen for BIRB 796 in comparison with placebo. A significant, dose-dependent decrease of C-reactive protein level was observed transiently after BIRB 796 after 1 week with a return to baseline level over time. The incidence of adverse events was comparable between all treatment groups, with the exception of a mild increase of transaminase levels that was seen more frequently in the BIRB 796 groups. Geographic center effects were observed with Russian centers producing distinctly higher remission and response rates and lower adverse event rates than in other countries in both placebo and active treatment groups. CONCLUSIONS: There was no evidence for clinical efficacy of BIRB 796 in CD. A remarkable difference in the course of CD exists between Russia and non-Russian centers.

Antigen targeting to MHC class II-enriched late endosomes in colonic epithelial cells: trafficking of luminal antigens studied in vivo in Crohn's colitis patients.

In Crohn's disease (CD), colonic epithelial cells (CECs) are suggested to stimulate pro-inflammatory CD4+ T cells. However, the endocytic pathways of luminal antigens involved in underlying MHC class II presentation by CECs remain unknown. Our aim was to elucidate antigen trafficking and associated MHC class II expression in CECs of CD patients in vivo. In CD patients (Crohn's colitis and remission) and healthy controls undergoing colonoscopy, ovalbumin (OVA) was sprayed onto inflamed or healthy mucosa. The subcellular localization of OVA and MHC class II was visualized in biopsies taken from OVA-incubated mucosa using fluorescence and cryoelectron microscopy. Targeting of OVA into late endosomes of CECs was found in healthy (controls and CD in remission) and inflamed mucosa (Crohn's colitis). MHC class II expression in CECs was not detected in healthy mucosa but strongly up-regulated during CD inflammation. Induced MHC class II in CECs was predominantly seen at basolateral membranes and in late endosomes, which were efficiently accessed by internalized OVA. Our data provide in vivo evidence that the endocytic pathway of luminal antigens in CECs of Crohn's colitis patients intersects MHC class II-enriched late endosomes and support the postulated role of CECs in MHC class II-associated antigen presentation during CD.

Mean corpuscular volume and ADH1C genotype in white patients with alcohol-associated diseases.

BACKGROUND: Alcohol abuse is associated with several gastrointestinal diseases, such as esophageal carcinoma, chronic alcoholic pancreatitis, and liver cirrhosis. Increased mean corpuscular volume (MCV) has been recognized as a biomarker for alcohol abuse and heavy drinkers. Recent studies from Japan revealed that macrocytosis is related to ALDH-2/2 genotype, leading to increased acetaldehyde accumulation. It has also demonstrated that increased MCV values could also be an independent biomarker for esophageal cancer in Asians. Therefore, the aim of the current study was to investigate possible associations of MCV value with polymorphisms of ADH1C in white patients with alcohol-associated esophageal carcinoma, chronic alcoholic pancreatitis, and alcoholic cirrhosis as well as in heavy drinkers without organ damage. METHODS: In this study, a total of 510 alcoholic patients were enrolled with esophageal cancer (n = 98), chronic pancreatitis (n = 98), alcoholic liver cirrhosis (n = 151), and alcohol abuse without gastrointestinal disease (n = 163). ADH1C genotyping was performed by PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis from whole blood. The relation between MCV and ADH1C gene polymorphisms (ADH1C*1 and 1C*2) controlled for the amount of drinking, smoking, and age were investigated using both univariate and multivariate analysis. RESULTS: In univariate analysis, higher alcohol consumption was associated with increased MCV. Other variables were not associated with macrocytosis. In multiple linear regression analysis, after adjustment for age and smoking, higher alcohol consumption and female sex were independently associated with higher MCV values. No other variables, including which alcohol-associated disease the patient had, had an independent effect. Adding ADH genotype rendered no independent significant effect on MCV value. CONCLUSIONS: In a white population, MCV values were not associated with genotype polymorphisms of ADH1C. In contrast to findings in Asians, macrocytosis does not seem to be an independent biomarker for esophageal cancer. The role of ADH1C polymorphism in increasing MCV and the potential use of MCV as a marker for esophageal carcinoma are still pending.

Interferon-gamma mediates antigen trafficking to MHC class II-positive late endosomes of enterocytes.

MHC class II-positive late endosomes of enterocytes are thought to be involved in antigen presentation to CD4(+) T cells. In contrast to enterocytes of BALB/c mice, severe combined immunodeficiency (SCID) enterocytes lack MHC class II expression and fail to transport internalized ovalbumin (OVA) into late endosomes. IFN-gamma is known to induce MHC class II in enterocytes and antigen targeting to late endosomes in macrophages. In this study, we investigated the influence of IFN-gamma and MHC class II on the processes of antigen traffic in enterocytes. Subcellular targeting of OVA and MHC class II expression within enterocytes were examined in SCID, IFN-gamma-treated SCID, BALB/c and C57BL/6 MHC class II knockout (KO) mice after a single feed with OVA. Sorting of OVA into late endosomes was found in enterocytes from BALB/c, C57BL/6 KO and IFN-gamma-stimulated SCID mice, but not from untreated SCID mice. MHC class II expression was restricted to enterocytes of IFN-gamma-treated SCID and BALB/c mice, present at basolateral membranes and within endosomal compartments. These enterocytes further revealed colocalization of class II antigens and OVA in endosomes. We suggest that antigen trafficking into late endosomes of enterocytes is mediated by IFN-gamma and occurs in the absence of MHC class II.

Mesenteric pulsatility index analysis predicts response to azathioprine in patients with Crohn's disease.

OBJECTIVE: Mesenteric blood flow measurement has been found to predict relapse after steroid-induced remission in patients with Crohn's disease (CD) and ulcerative colitis (UC). Therefore, we assessed prospectively the possible relationship between changes in mesenteric blood flow and prognosis in chronically active patients with need of immunosuppressive therapy with azathioprine (AZA) or 6-mercaptopurine (6-MP). METHODS: Doppler ultrasound (DUS) measurements of the pulsatility index (PI) of the superior mesenteric artery (SMA) and inferior mesenteric artery (IMA) were performed in 52 patients with chronically active inflammatory bowel disease (CD 31 patients; UC 21 patients) before beginning therapy with AZA/6-MP (US1) and during clinical remission (CD activity index <150, Truelove index score I) (US2). Patients were weaned from concomitant therapy with corticosteroids as soon as possible and were followed up for 12 months. RESULTS: After 1 year, 16 patients with CD (51.6%) and 13 patients with UC (61.9%) were in remission, whereas 23 patients had recurrent disease or had undergone surgery. A decreased SMA PI at US2 predicted clinical relapse in all patients with CD [100%; P < 0.001; mean (+/-SD) 77 +/- 67 d after US1], but only 4 of 8 patients (50%; difference not significant; mean 84 +/- 75 d after US1) with UC. Conversely, an increase of SMA PI was associated with sustained remission in the majority of CD patients (12/16 patients; 75%; P < 0.002), but in only 7 of 13 patients (54%) with UC. Flow measurements in the IMA and postprandial values for both arteries were less reliable. CONCLUSION: Repeated DUS measurements of the SMA PI predict response to AZA/6-MP in patients with chronic active CD.

Effectiveness and tolerability of repeated treatment with infliximab in patients with Crohn's disease: a retrospective data analysis in Germany.

AIM: The aim was to evaluate the efficacy and safety of repeated treatment with infliximab in patients with chronic active Crohn's disease under routine conditions in clinics and private practices. METHODS: Patients with active Crohn's disease were treated with a total of 567 infusions (420 re-infusions) of infliximab. The treatment schedule was at the discretion of the treating physician. Efficacy and tolerability were documented by a standardized questionnaire. RESULTS: There were indications for therapy in 46% of patients with chronic active disease, fistulas in 15% and combined symptoms in 38%. The mean disease duration was 9.4 years. At the beginning of therapy, 76% of patients were on corticosteroids, 67% received 5-aminosalicylates and 48% azathioprine. The average dose of infliximab administered was 300 mg; the mean interval between individual infusions was 8.7 weeks. Following treatment with infliximab, steroids could be withdrawn in 47% and reduced in 33% of patients, whereas the dosage of 5-aminosalicylates and azathioprine mostly remained unchanged. The efficacy and tolerability of infliximab were judged by the physicians as being very good or good in 73.4 and 88.4% of patients respectively. Further treatment with the anti-TNFalpha antibody was planned for 61% of patients. Improvement of Crohn's Disease Activity Index (CDAI), white blood cell (WBC) and C-reactive protein (CRP) levels was noted in almost all patients. CONCLUSION: Infliximab used in an individually adapted regimen induced a significant clinical response in the majority of patients with refractory and fistulating Crohn's disease. In nearly 80% of patients corticosteroids could be withdrawn or reduced and the majority of patients were on azathioprine at the end of follow-up procedures.

Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis.

BACKGROUND: The distinction between idiopathic inflammatory bowel disease (IBD) and infectious, usually self-limited enterocolitis is still a diagnostic dilemma. Procalcitonin (PCT) is the prohormone of calcitonin and is considered a specific marker of bacterial infection. The aim of this prospective study was to determine the value of PCT in differentiating flares of IBD from self-limited colitis. In addition, because standard laboratory inflammatory parameters are poorly correlated with disease activity in IBD, the relation between PCT levels and disease activity was investigated. METHODS: A total of 76 patients (26 Crohn's disease, CD; 25 ulcerative colitis, UC; and 25 patients with self-limited enterocolitis) were enrolled. Serum levels of PCT were measured by a sandwich immunoluminometric assay. C-reactive protein (CRP) levels, white blood cell counts, and stool cultures were obtained from all patients. Disease activity was assessed by the Crohn's disease activity index (CDAI) and the Truelove index for CD and UC, respectively. RESULTS: Patients with self-limited enterocolitis showed significantly higher PCT levels when compared with IBD patients (0.36 ng/mL, range 0.18-1.7 vs 0.10 ng/mL, range 0.08 0.5, p < 0.001). For a PCT value of > or =0.4, the sensitivity for self-limited colitis was 92% and specifity 96%. The positive predictive value (PPV) for self-limited colitis was 96%, whereas the negative predictive value (NPV) was 93%. In IBD patients, PCT levels were in the normal range although significantly higher in active disease when compared with inactive disease (0.13 ng/mL, range 0.08-0.5 vs 0.09 ng/mL, range 0.08-0.15, p < 0.001). This difference was less pronounced for CD (0.11 ng/mL, range 0.08-0.2 vs 0.09 ng/mL, range 0.08-0.15, p < 0.05) than for UC (0.14 ng/mL, range 0.08-0.5 vs 0.09 ng/mL, range 0.08-0.11, p < 0.01). In CD, PCT levels correlated significantly 0.5, p < 0.01). with the CDAI (r =0.05, p <0.01). CONCLUSIONS: The measurement of PCT offers two diagnostic options in IBD. Supranormal levels indicate self-limited enterocolitis. Furthermore, although within the normal range in IBD, PCT levels may serve as a new serological marker of disease activity.

Response of refractory colitis to intravenous or oral tacrolimus (FK506).

Intravenous cyclosporine has proven to be an alternative to emergency colectomy in steroid-refractory ulcerative colitis, whereas the experience with FK506 is limited. In this report we compare intravenous to oral FK506 treatment in 38 patients with refractory ulcerative (n = 33) or indeterminate (n = 5) colitis. FK506 was started intravenously in the first group (n = 18) at a dose of 0.01 to 0.02 mg/kg up to 14 days, followed by 0.1 to 0.2 mg/kg orally, or was started orally at this dose in a second group (n = 20). Additional azathioprine/6-mercaptopurine was given and steroids were tapered in responding patients, followed by a dose reduction of FK506. Clinical disease activity and laboratory parameters were assessed to evaluate efficacy and safety. Primary objectives were the induction of remission (Truelove index of mild) and colectomy-free survival. Treatment lasted for a mean of 7.6 months, and the mean observation period was 16.2 months. Eighteen of 38 patients improved within 14 days, and a complete remission was achieved in 13 patients after 1 month. A colectomy within 1 month was performed in 3 of 38 patients. The overall colectomy rate was 34%. One-half of the patients with a minimum follow-up of 2 years required a colectomy. Intravenous and per oral administration were equally safe and effective. The most frequent adverse events included tremor, hyperglycemia, hypertension, and infection, but none were severe. Renal impairment was rare and subsided upon drug withdrawal. In conclusion. FK506 is effective in the treatment of refractory colitis with per oral dosing being equivalent to intravenous administration.