Loss of CD103+ intestinal dendritic cells during colonic inflammation.

AIM: To investigate possible differences in dendritic cells (DC) within intestinal tissue of mice before and after induction of colitis. METHODS: Mucosal DC derived from intestinal tissue, as well as from mesenteric lymph nodes and spleen, were analyzed by fluorescence activated cell sorting (FACS) analysis. Supernatants of these cells were analyzed for secretion of different pro- and anti-inflammatory cytokines. Immunohistochemistry and immunofluorescence were performed on cryosections of mucosal tissue derived from animals with colitis as well as from healthy mice. RESULTS: It was shown that DC derived from healthy intestinal lamina propria (LP) represented an immature phenotype as characterized by low-level expression of costimulatory cytokines. In contrast to DC from spleen and mesenteric lymph nodes (MLN) that secreted proinflammatory cytokines, LP-DC produced high levels of the anti-inflammatory cytokine IL-10. After induction of murine colitis in a CD4(+)CD62L(+) transfer model or in chronic dextran sulfate sodium-colitis, a marked increase of activated CD80(+) DC could be observed within the inflamed colonic tissue. Interestingly, in contrast to splenic DC, a significant population of DC within MLN and colonic LP expressed the mucosal integrin CD103 which was lost during colitis. CONCLUSION: The constitutive secretion of anti-inflammatory cytokines by immature DC within the intestinal LP might regulate the homeostatic balance between mucosal immunity and tolerance. CD103(+) DC could mediate this important function.

Calcitriol analog ZK191784 ameliorates acute and chronic dextran sodium sulfate-induced colitis by modulation of intestinal dendritic cell numbers and phenotype.

AIM: To investigate the effects of ZK1916784, a low calcemic analog of calcitriol on intestinal inflammation. METHODS: Acute and chronic colitis was induced by dextran sodium sulfate (DSS) according to standard procedures. Mice were treated intraperitoneally with ZK1916784 or placebo and colonic inflammation was evaluated. Cytokine production by mesenterial lymph node (MLN) cells was measured by ELISA. Immunohistochemistry was performed to detect intestinal dendritic cells (DCs) within the colonic tissue, and the effect of the calcitriol analog on DCs was investigated. RESULTS: Treatment with ZK191784 resulted in significant amelioration of disease with a reduced histological score in acute and chronic intestinal inflammation. In animals with acute DSS colitis, down-regulation of colonic inflammation was associated with a dramatic reduction in the secretion of the proinflammatory cytokine interferon (IFN)-gamma and a significant increase in intereleukin (IL)-10 by MLN cells. Similarly, in chronic colitis, IL-10 expression in colonic tissue increased 1.4-fold when mice were treated with ZK191784, whereas expression of the Th1-specific transcription factor T-beta decreased by 81.6%. Lower numbers of infiltrating activated CD11c+ DCs were found in the colon in ZK191784-treated mice with acute DSS colitis, and secretion of proinflammatory cytokines by primary mucosal DCs was inhibited in the presence of the calcitriol analog. CONCLUSION: The calcitriol analog ZK191784 demonstrated significant anti-inflammatory properties in experimental colitis that were at least partially mediated by the immunosuppressive effects of the derivate on mucosal DCs.

The effect of chromoendoscopy on the diagnostic improvement of gastric ulcers by endoscopists with different levels of experience.

BACKGROUND: In recent years chromoendoscopy has become popular as a diagnostic enhancement tool in endoscopy. Using the macroscopic description of gastric ulcers, experienced endoscopists may be able to differentiate malignant and benign lesions. The aim of our study was to determine whether indigo carmine staining improves the ulcer differentiation by experienced and inexperienced endoscopists. METHODS: 50 patients were enrolled, 7 with malignant gastric ulcers and 43 with benign gastric ulcers. Gastroscopy was initially videotaped native, then on a second tape after staining with 0.2% indigo carmine. Later on biopsies were taken for histology. Subsequently the tapes were randomly evaluated by three experienced (>2000 gastroscopies; group A) and by three inexperienced (<100 gastroscopies; group B) investigators blinded from any personal data of the patients. The investigators had to classify the ulcers, using published criteria, native as well as stained. The results were compared within each group and with the histology. RESULTS: The endoscopic native diagnosis showed a sensitivity of 66.3%, a specificity of 86.3%, a positive predictive value of 48.1% and a negative predictive value of 94% for group A, respectively 66%, 62.5%, 22.7% and 92% for group B. After staining, the values of these parameters were reduced insignificantly to a sensitivity of 60.2%, a specificity of 78.5%, a positive predictive value of 36.1% and a negative predictive value of 92.8% for group A. Group B, on account of one investigator who demonstrated excellent skills, showed a significant better sensitivity (79.9%) and a slight improvement of the positive and negative predictive values to 25.7% respectively 94.8%, whereas the specificity very slightly decreased to 61.3%. The diagnostic accuracy before and after staining was 83.6%, respectively 76.5%, in group A and 63.2%, respectively 63.9% in group B. The correlation with the histology, determined by Cohen's kappa coefficient (median value), decreased from 0.46 for the native to 0.30 for the chromoendoscopic diagnosis in group A and remained unchanged (0.17) in group B. CONCLUSION: We concluded that chromoendoscopy does not improve the classification of gastric ulcers with respect to malignant or benign origin. The role of endoscopic experience could only be proved in the native macroscopic diagnosis of the investigators. After staining, with the exception of one investigator, experienced as well as inexperienced endoscopists lost their diagnostic accuracy.

Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo.

Probiotic microorganisms are defined as viable nutritional agents conferring benefit to the health of the human host. Especially, Escherichia coli strain Nissle 1917 (EcN) was shown to be equally effective as mesalazine in the maintenance of remission in ulcerative colitis (UC). Presumably, the therapeutic effect of EcN is linked to the presence of the strain in the region of interest; however, it remains difficult to follow the orally administered strain on its passage through the complex microbial environment of the intestine in vivo, inhabited dominantly by various E. coli strains, using traditional culturing methods. In this study we transformed EcN and a wild-type E. coli from a laboratory rat (EcR) with a plasmid carrying a gfp gene (pUC-gfp) to obtain EcN- and EcR-GFP to allow in vivo detection without alteration of strain-specific characteristics. Analysis of different strain-specific characteristics included the measurement of stimulation of IL-8 secretion and adhesion in vitro using the epithelial cell line HT-29. The kinetics of intestinal distribution in mice and colonization properties in rats following oral administration was studied in vivo. Detectability of the strain in histologic specimens was analysed using fluorescence microscopy and immunohistochemistry. The identity of fluorescent E. coli strains isolated from stool samples, Peyer's patches (PP) and mesenteric lymph nodes (MLN) was determined by REP-PCR. We were able to demonstrate that EcN and EcN-GFP do not differ in stimulation of IL-8 secretion or adhesion to HT-29 cells. In vivo, EcN-GFP colonies were readily detectable by fluorescence microscopy in luminal samples and also by immunohistochemistry in histological sections allowing analysis of the kinetics of the intestinal passage following oral administration. Translocation of fluorescent and non-fluorescent bacteria into PP and MLN was noted at 6 h post oral administration. EcN-GFP was detectable initially for 14 days in faecal samples of rats, while EcR-GFP was detectable throughout the whole experiment (45 days). Challenge with ampicillin at day 45 demonstrated continuing presence of EcN-GFP in small numbers by reappearing fluorescent colonies. The plasmid was not stable in vivo since non-fluorescent EcN colonies were detected also in faecal samples by REP-PCR. In summary, transformation of EcN to obtain EcN-GFP in our study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allows the detection in mixed microbial environments in vivo but the stability of EcN-GFP in vivo is limited.

Comparison of interobserver agreement for different scoring systems for reflux esophagitis: Impact of level of experience.

BACKGROUND: The Savary-Miller, the Los Angeles, and the MUSE (metaplasia, ulcer, stricture, erosion) scoring systems have been developed to assess esophageal lesions related to GERD. Interobserver agreement for these systems was compared, with particular reference to the experience of the endoscopist. METHODS: By using videoendoscopes, videotapes were made of the gastroesophageal junction of 60 patients who presented with symptoms suggestive of GERD. The Savary-Miller, the Los Angeles, and the MUSE systems were used to score all video clips by 9 endoscopists who were subgrouped by level of experience (3 levels, 3 endoscopists per level). Agreement was assessed by using weighted kappa statistics (kappa). RESULTS: The Savary-Miller scoring system revealed moderate agreement for the experienced group (kappa=0.41) but performed poorly when applied by inexperienced raters (kappa=0.16). The Los Angeles system was most reproducible in all subgroups, irrespective of the level of experience (kappa=0.49 to 0.65). The MUSE scoring system was highly similar to the Los Angeles scoring system with respect to erosions and, in addition, allowed assessment of complications of GERD. CONCLUSIONS: The Los Angeles and the MUSE scoring systems are most reliable for the assessment of erosions caused by GERD. Because of low reliability, use of the Savary-Miller scoring system is not recommended. For all scoring systems, interobserver agreement varies with the level of experience in the performance of upper endoscopy.

Lactobacillus GG in inducing and maintaining remission of Crohn's disease.

BACKGROUND: Experimental studies have shown that luminal antigens are involved in chronic intestinal inflammatory disorders such as Crohn's disease and ulcerative colitis. Alteration of the intestinal microflora by antibiotic or probiotic therapy may induce and maintain remission. The aim of this randomized, placebo-controlled trial was to determine the effect of oral Lactobacillus GG (L. GG) to induce or maintain medically induced remission. METHODS: Eleven patients with moderate to active Crohn's disease were enrolled in this trial to receive either L. GG (2 x 10(9) CFU/day) or placebo for six months. All patients were started on a tapering steroid regime and received antibiotics for the week before the probiotic/placebo medication was initiated. The primary end point was sustained remission, defined as freedom from relapse at the 6 months follow-up visit. Relapse was defined as an increase in CDAI of >100 points. RESULTS: 5/11 patients finished the study, with 2 patients in each group in sustained remission. The median time to relapse was 16 +/- 4 weeks in the L. GG group and 12 +/- 4.3 weeks in the placebo group (p = 0.5). CONCLUSION: In this study we could not demonstrate a benefit of L. GG in inducing or maintaining medically induced remission in CD.

Detection of dysplastic lesions by fluorescence in a model of chronic colitis in rats after local application of 5-aminolevulinic acid and its esterified derivatives.

5-Aminolevulinic acid (ALA) and ALA ester-induced protoporphyrin IX (PPIX) fluorescence are used for photodynamic diagnosis and therapy with promising results. The aim of the present study was to investigate the detection of dysplastic lesions by fluorescence after topical application of ALA and different esterified derivatives in a model of chronic colitis in rats. In female CD rats chronic colitis was induced by oral application of 5% dextrane sulfate sodium. ALA was used at different concentrations (0.072 and 0.036 mol/L). ALA-methylester (m-ALA), ALA-hexylester (h-ALA) and ALA-benzylester (b-ALA) were used at a concentration of 0.003, 0.002 and 0.002 mol/L, respectively. Fluorescence was examined under blue light, and histological findings of fluorescent and nonfluorescent biopsy specimens were recorded. Using ALA at a concentration of 0.072 mol/L, all dysplastic lesions (8/8) showed fluorescence (sensitivity 100%). Specificity was low at 57%. Reducing the concentration to 0.036 mol/L resulted in a sensitivity of only 56% (5/9) with an increase in specificity to 76%. On using h-ALA, sensitivity was 60% (3/5) with a specificity of 51%. Using m-ALA and b-ALA, sensitivity values were 25% and 33%, and values for specificity were 62% and 63%, respectively. Despite a low number of dysplastic lesions, the results of this study indicate that ALA ester-induced PPIX fluorescence has the potential for the detection of premaligant lesions but was not superior to ALA. ALA esters were used in 18- to 36-fold lower concentrations compared with ALA.

Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis.

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day -2 to day +7. Chronic colitis was induced by transfer of CD4(+) CD62L(+) T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-gamma], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-gamma, 32,477 +/- 6,377 versus 9,734 +/- 1,717 [P = 0.004]; IL-6, 231 +/- 35 versus 121 +/- 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 +/- 0.2 versus 1.9 +/- 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.

Functional expression of the interleukin-11 receptor alpha-chain and evidence of antiapoptotic effects in human colonic epithelial cells.

A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Ralpha monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Ralpha selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Ralpha mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Ralpha restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity.

Rationale for probiotic and antibiotic treatment strategies in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), commonly referred to as Crohn's disease and ulcerative colitis are chronic aggressive disorders which share many similarities concerning pathomechanism and clinical course, but have very distinct features. Both entities are mainly located in areas with high bacterial concentrations, such as the terminal ileum and cecum in Crohn's disease and the rectum in ulcerative colitis. In recent years, overwhelming evidence accumulated, supporting the hypothesis that IBD are characterized by a genetically determined, overly aggressive immune response towards ubiquitous luminal antigens, especially commensal bacteria and their products. Trials in both human IBD and experimental colitis have demonstrated that broad-spectrum antibiotics may influence the course of ulcerative colitis and Crohn's disease and antibiotics with narrow activity against the anaerobic fraction of the flora can prevent relapse in Crohn's disease after surgically induced remission. Since relevant antibiotic strategies can be associated with some side effects, the ongoing research recently focused on alternative methods to modify the intestinal flora in patients with IBD. Clinical observations including few controlled trials, basic research, and animal studies have suggested a potential role for probiotic bacteria within the treatment regimens for IBD. However, the mode of action of these organisms is still largely unclear and in vitro studies are inconclusive. This review summarizes recent in vitro and in vivo data regarding the role of the intestinal microflora in the pathogenesis of chronic intestinal inflammation and possible therapeutic mechanisms of probiotic bacteria relevant to IBD. Furthermore, we will review clinical trials examining the efficacy of antibiotic and probiotic treatment strategies in IBD.

The role of endogenous bacterial flora: bystander or the necessary prerequisite?

Abundant experimental and clinical data incriminate microbial factors in the pathogenesis of inflammatory bowel diseases. Commensal bacteria, and their products, provide the constant antigenic stimulus to disequilibrate the mucosal immune system towards an overly aggressive response in a genetically susceptible host with reduced tolerance towards the autologous flora. Not all bacteria have equal proinflammatory capabilities. Some may be even beneficial as demonstrated by the anti-inflammatory effects of so-called probiotics. Further investigations are needed to translate the clear experimental and clinical evidence into benefit for the patients.

Gastrointestinal disorders of the critically ill. Bacterial translocation in the gut.

The human gastrointestinal tract is colonized by a dense population of microorganisms, referred to as the bacterial flora. Although the gut provides a functional barrier between these organisms and the host, bacterial translocation is a common event in the healthy person. However, in critically ill patients, with various underlying diseases, this bacterial translocation may lead to infections and consequently to a further reduction in general health status. The mechanism of bacterial translocation is widely, and somehow controversially investigated in vitro and in animal models. In human studies, several diseases have been associated with bacterial translocation. However, methodological shortcomings, insufficient populations and conflicting results leave many open questions. This is also reflected in the various published therapeutic strategies. To overcome this problem more investigations in humans are needed, especially in techniques for detecting bacterial translocation.

Role of commensal bacteria in chronic experimental colitis: lessons from the HLA-B27 transgenic rat.

Rats on Lewis or Fischer background, transgenic for human HLA-B27 and beta(2)-microglobulin genes spontaneously develop colitis, gastritis, arthritis, dermatitis, orchitis, epididymitis, carditis, alopecia and nail changes. Disease susceptibility correlates with the gene copy number and is influenced by the genetic background. The pathomechanism in this model is still not completely understood. Cell transfer experiments indicate an essential role of HLA-B27 expression in bone marrow-derived cells. On Fischer background the onset of colitis occurs at 2 months of age, peaks at 3 months of age, and plateaus. Histologic findings include inflammatory cell infiltration, mostly limited to the mucosa, crypt hyperplasia, reduction of goblet cells, occasionally crypt abscesses and early ulcers. There is evidence that normal luminal bacteria play an essential role in initiating and perpetuating chronic colitis and gastritis in HLA-B27 transgenic rats: Transgenic rats raised under germ-free conditions do not develop gastrointestinal disease, whereas transgenic littermates exposed to specific pathogen-free bacteria develop colitis and gastritis within 2-4 weeks. Obligate anaerobic bacteria, especially Bacteroides spp., may play a predominant role since metronidazole prevents colitis and transgenic germ-free rats contaminated with a cocktail of six obligate and facultative anaerobic bacteria develop colitis and gastritis only in the presence of Bacteroides vulgatus. Luminal bacteria may also be involved in trafficking and homing of inflammatory cells into remote organs, since varying cecal bacterial composition does not only alter local inflammation but also influences gastritis. Lymphocyte transfer experiments indicate a specific response to luminal bacteria. In summary, this animal model is suitable for investigating the influence of normal luminal bacteria on the cellular immune mechanism in chronic intestinal inflammation.