Juergen Schoelmerich

Development of the Crohn's disease digestive damage score, the Lemann score.

Crohns disease (CD) is a chronic progressive destructive disease. Currently available instruments measure disease activity at a specific point in time. An instrument to measure cumulative structural damage to the bowel, which may predict long-term disability, is needed. The aim of this article is to outline the methods to develop an instrument that can measure cumulative bowel damage. The project is being conducted by the International Program to develop New Indexes in Crohns disease (IPNIC) group. This instrument, called the Crohns Disease Digestive Damage Score (the Lémann score), should take into account damage location, severity, extent, progression, and reversibility, as measured by diagnostic imaging modalities and the history of surgical resection. It should not be “diagnostic modality driven”: for each lesion and location, a modality appropriate for the anatomic site (for example: computed tomography or magnetic resonance imaging enterography, and colonoscopy) will be used. A total of 24 centers from 15 countries will be involved in a cross-sectional study, which will include up to 240 patients with stratification according to disease location and duration. At least 120 additional patients will be included in the study to validate the score. The Lémann score is expected to be able to portray a patients disease course on a double-axis graph, with time as the x-axis, bowel damage severity as the y-axis, and the slope of the line connecting data points as a measure of disease progression. This instrument could be used to assess the effect of various medical therapies on the progression of bowel damage. (Inflamm Bowel Dis 2011)

Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-κB pathway in adipocytes links nutritional signalling with innate immunity

To study the effects of fatty acids and the involvement of the Toll‐like receptor‐4/nuclear factor‐κB (TLR‐4/NF‐κB) pathway with respect to the secretion of adipokines from adipocytes 3T3‐L1 adipocytes were stimulated with increasing doses of fatty acids. The secretion of adiponectin, resistin and monocyte chemoattractant protein‐1 (MCP‐1) was measured by enzyme‐linked immunosorbent assay. The NF‐κB p65 nuclear translocation and TLR‐4 expression were investigated by Western blot. The effects mediated by NF‐κB were tested using a specific NF‐κB‐inhibitor and TLR‐4‐induced effects were analysed with a neutralizing TLR‐4 antibody. Binding of 14C‐labelled fatty acids to TLR‐4/MD‐2 was investigated using a FLAG‐tagged extracellular part of TLR‐4 fused to full‐length MD‐2 via a linker (lipopolysaccharide‐Trap). The messenger RNA (mRNA) expression of adipokines in abdominal adipose tissue of rats fed a standard chow or a high‐fat diet was investigated by reverse transcription–polymerase chain reaction. The TLR‐4 is induced during adipocyte differentiation and its expression is enhanced following fatty acid stimulation. The stimulatory effects of stearic and palmitic acids on MCP‐1 secretion and of palmitoleic acid on resistin secretion are mediated via NF‐κB. The stimulatory effects of stearic, palmitic and palmitoleic acids on resistin secretion and the stimulatory effect of stearic acid on MCP‐1 secretion are mediated via TLR‐4. Fatty acid‐mediated effects are caused by an endogenous ligand because fatty acids were shown not to bind directly to TLR‐4/MD‐2. Adipose tissue mRNA expression and serum levels of adipokines did not differ in rats fed a high‐fat diet. These data provide a new molecular mechanism by which fatty acids can link nutrition with innate immunity.

The Accuracy of Abdominal Ultrasound in the Assessment of Bowel Disorders

BACKGROUND Little is known about the sensitivity, specificity, and predictive values of transabdominal ultrasonographic (US) findings in a teaching hospital setting. METHODS We carried out a prospective study including 227 patients with symptoms suggestive of inflammatory bowel disorder. The Picker 9200 CS equipment (5-mHz curved-array probe) was used to obtain bowel images. Gastrointestinal endoscopy, enteroclysis, bowel enema, computed tomography scan, or bowel surgery was used as reference. RESULTS Of 227 patients, 168 had pathologic findings of the bowel as final diagnosis. The overall sensitivity of US was 76%, whereas the positive predictive value was 98%. Overall specificity was 95%. The negative predictive value for bowel disorders was only 58%, since US missed pathologic findings in 48 patients. Subgroup analysis showed a sensitivity of 84% for Crohns disease, 66% for ulcerative colitis, 46% for bowel tumors, and 60% for diverticulitis. Topographic comparisons showed that US detected inflammatory bowel-wall alterations preferentially in the terminal ileum and colon, whereas abnormalities in the duodenum, jejunum, and rectum were frequently missed (sensitivity, 10%-20%). CONCLUSIONS Positive US findings are useful for the diagnosis of bowel processes. US is highly predictive albeit not disease-specific. Negative US examinations, however, do not exclude pathologic bowel processes. A topographic location of pathologic US findings is mostly confined to the colon.

In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis.

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage‐oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti‐oxidative potential. Using the dextran sulphate sodium (DSS)‐induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 µg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti‐CD3 antibody in the presence of interleukin (IL)‐2 (final concentration 10 U/ml). After incubation for 24 h, IL‐1β, IL‐6, IL‐12 tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ levels in supernatants were analysed by the beadlyte® cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3·2 with acteoside versus 5·2 with phosphate‐buffered saline (PBS) (P < 0·02). In chronic colitis both 120 µg (3·3 versus 5·2) or 600 µg acteoside (3·0 versus 5·2) significantly ameliorated colitis (both P < 0·02). Stimulated MLN from mice with chronic DSS‐induced colitis treated with acteoside showed a significant down‐regulation of IFN‐γ secretion (195 pg/ml with 600 µg acteoside versus 612 pg/ml with PBS, P < 0·02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl4‐induced ascitic cirrhosis and 2‐day portal vein–ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α‐cryptdins and β‐defensins was determined by real‐time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence‐activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α‐cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver–gut axis including the underlying mechanisms could help us to find new treatment avenues. (HEPATOLOGY 2012)

Soluble galectin-3 is a strong, colonic epithelial-cell-derived, lamina propria fibroblast-stimulating factor

Background: Colonic lamina propria fibroblasts (CLPFs) play an important role in the pathogenesis of fibrosis and strictures in Crohn’s disease. Aim: To identify colonic epithelial cell (CEC)-derived factors that activate CLPFs. Methods: Primary human CECs and CLPFs were isolated from control mucosa and interleukin 8 (IL8) of CLPF cultures was quantified by ELISA. Activation of nuclear factor κB (NF-κB) was shown, and translocation of NF-κB was inhibited by a dominant-negative IκB-expressing adenovirus. The major CLPF-activating and IL8 inducing protein was purified using fast-performance liquid chromatography (HiPrep 16/60 Sephacryl S-200 High Resolution Column) and sodium dodecyl sulphate gel electrophoresis. Results: A considerable increase in IL8 secretion by CLPFs cultured in CEC-conditioned media compared with that in unconditioned media (155.00 (10.00) pg/µg v 1.434 (0.695) pg/µg) was found. The effect of CEC-conditioned media on CLPF IL8 secretion was NF-κB dependent. A protein or DNA array confirmed the involvement of NF-κB and activator protein-1. Purification of a candidate band isolated with the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequent sequencing showed soluble galectin-3 to be a strong CLPF-activating factor. Depletion of galectin-3 from conditioned media by immunoprecipitation abolished the CLPF stimulatory effect. Conclusions: Using a classical biochemical approach, soluble galectin-3 was identified as a strong activator of CLPFs produced by CEC. Galectin-3 induced NF-κB activation and IL8 secretion in these cells and may be a target for future therapeutic approaches to reduce or avoid stricture formation.

Effects of the New C1q/TNF‐related Protein (CTRP‐3) “Cartonectin” on the Adipocytic Secretion of Adipokines

Background: Cartonectin (collagenous repeat‐containing sequence of 26‐kDa protein; CORS‐26) was described as a new adipokine of the C1q/TNF molecular superfamily C1q/TNF‐related protein‐3 (CTRP‐3), secreted by the adipocytes of mice and humans. The receptor and function of cartonectin are unknown and the recombinant protein is not commercially available.

Bilberries and their anthocyanins ameliorate experimental colitis

Bilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% of anthocyanins (ACs), which have anti-oxidative, anti-carcinogenic, and anti-inflammatory properties. We investigated whether experimental colitis can be ameliorated by dried bilberries or ACs. Acute and chronic dextrane sodium sulphate (DSS) colitis were induced in Balb/c mice by 2.5% DSS in the drinking water. Mice were fed with dried bilberries or ACs, respectively. Cytokines were determined in supernatants from mesenteric lymph nodes (MLNs) by ELISA and apoptosis was investigated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Oral administration of bilberries during acute DSS-induced colitis ameliorated disease severity and reduced secretion of IFN-γ and tumor necrosis factor from mesenteric lymph node cells. Dried bilberries also improved chronic DSS-colitis. Ingestion of ACs reduced intestinal inflammation in acute and chronic DSS-colitis with decreased histological scores and cytokine secretion. Both bilberries and ACs prevented inflammation-induced apoptosis in colonic epithelial cells. Taken together, ingestion of dried bilberries had positive effects on various parameters especially in acute DSS-colitis. Oral administration of ACs resulted in an amelioration of acute colitis as well as chronic colitis. These promising results justify a clinical study on their therapeutic effect in inflammatory bowel disease patients.

Association of the NOD2 genotype with bacterial translocation via altered cell-cell contacts in Crohn's disease patients.

Background: Recent insights into the pathogenesis of Crohns disease (CD) point to an important role of the mucosal barrier and intestinal microflora that may induce a chronic inflammation after crossing the intestinal barrier. The first detected susceptibility gene for CD, NOD2, is a pattern recognition receptor (PRR) for the recognition of the bacterial cell wall component muramyldipeptide (MDP). Binding of MDP to NOD2 is followed by activation of proinflammatory pathways mainly regulated by nuclear factor kappa B (NF‐&kgr;B). In this study we investigated whether impaired recognition of MDP via NOD2 variants is associated with increased bacterial translocation across the epithelial barrier and whether this is followed by increased or decreased NF‐&kgr;B activation. Methods: NOD2 variants were analyzed in 36 CD patients and 30 controls. Endotoxin was stained by immunohistochemistry in 30 intestinal biopsies from patients carrying NOD2 variants (NOD2‐mut) or being NOD2 wildtype (WT). Junctional proteins were visualized by immunofluorescence and quantified by Western blotting. NF‐&kgr;B activation was analyzed by immunohistochemistry in specimens from NOD2‐WT and NOD2‐mut CD and control patients. Results: We demonstrated the increased presence of endotoxin in the mucosal lamina propria of CD patients carrying NOD2 variants. This was associated with an altered composition of epithelial cell–cell contacts. Patients carrying NOD2 variants displayed increased NF‐&kgr;B activation in the mucosa. Conclusions: This study for the first time demonstrates that translocation of luminal bacteria and/or bacterial products into the intestinal mucosa is increased in patients carrying NOD2 variants, leading to higher activation of proinflammatory signaling cascades. (Inflamm Bowel Dis 2010)

Antimicrobial peptide response to blood translocation of bacterial DNA in Crohn's disease is affected by NOD2/CARD15 genotype

Background: Blood translocation of bacterial‐DNA has been described in patients with Crohns disease (CD). The hosts immune cell types cooperate to respond against bacterial insults. Some antimicrobial peptides are inducible after culture with bacterial products and a linkage has been established between them and NOD2/CARD15. The aim was to test whether defensins and cathelicidin (LL‐37) expression and NOD2/CARD15 mutations in blood neutrophils are related to molecular bacterial translocation events in CD patients. Methods: Fifty consecutively admitted CD patients and 15 healthy controls were included. Clinical and analytical characteristics of patients were considered. NOD2/CARD15 genotyping, presence of bacterial‐DNA, defensin and cathelicidin gene, and protein levels in neutrophils and serum cytokine levels were studied. Results: Twenty patients (40%) presented bacterial‐DNA in blood. Eleven were active and 9 were in remission. Bacterial‐DNA was not present in controls. NOD2/CARD15 mutations were identified in 25 patients (50%), 15 of which were in remission. Sixty percent of bacterial‐DNA(+) and 43% of bacterial‐DNA(−) patients showed a NOD2/CARD15 mutation. &bgr;‐Defensin 2 and LL‐37 mRNA and protein levels were upregulated in bacterial‐DNA(+) patients. &bgr;‐Defensin 2 and LL‐37 expression correlated with bacterial‐DNA concentration only in patients with a wildtype NOD2/CARD15 genotype. Cultured neutrophils of bacterial‐DNA(‐) patients confirmed the muramyl dipeptide‐independent association between DEFB2 and LL‐37 with bacterial‐DNA concentration in wildtype NOD2/CARD15 patients. Cytokine levels were increased in bacterial‐DNA(+) patients and correlated with bacterial‐DNA concentration. NOD2/CARD15 genotype did not influence this correlation. Conclusions: &bgr;‐Defensin 2, LL‐37, and proinflammatory cytokines are increased in CD patients with bacterial‐DNA in a concentration‐dependent manner. NOD2/CARD15 plays a key role in the regulation of this response. (Inflamm Bowel Dis 2010;)