Juergen Stein

FTY720 Ameliorates Th1-Mediated Colitis in Mice by Directly Affecting the Functional Activity of CD4+CD25+ Regulatory T Cells

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4+CD25+ regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFβ, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4+CD25+ Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4+ T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4+CD25+ Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.

The New Low Calcemic Vitamin D Analog 22-Ene-25-Oxa-Vitamin D Prominently Ameliorates T Helper Cell Type 1-Mediated Colitis in Mice

In addition to its well defined role as a key regulator of calcium and bone metabolism, 1,25-dihydroxyvitamin D3 (calcitriol) has been established as a potent modulator of immune cell function. Still, because of the hypercalcemic toxicity occurring after systemic application of the parent compound, its clinical application as an immunosuppressant has been hampered. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs with well preserved immunosuppressive activity in vitro. Here, in vivo colitis was induced by applying a rectal enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to male BALB/c mice, and calcitriol (0.2 μg/kg) or ZK156979 (0.1–2.0 μg/kg) was given i.p. from days 0 to 3 or 3 to 5. Body mass and clinical activity score of colitis were recorded daily. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity and cytokine levels [tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4] were determined by enzyme-linked immunosorbent assay, and T-box transcription factor (T-bet) expression was determined by immunoblot analysis. We found that treatment with ZK156979 clearly reduced the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated body weight loss, diarrhea, and macroscopic intestinal inflammation with a potency comparable with that of calcitriol. The therapeutic effect of ZK156979 was accompanied by a down-regulation of myeloperoxidase activity, TNF-α, IFN-γ, and T-bet expression decreased, whereas local tissue IL-10 and IL-4 protein levels increased. To conclude, our data provide the first clear evidence that ZK156979 exhibits a beneficial prophylactic as well as therapeutic profile in T helper cell type 1-like experimental colitis, offering new therapeutic options for the treatment of human inflammatory bowel diseases.

Does secretin-stimulated MRCP predict exocrine pancreatic insufficiency?: A comparison with noninvasive exocrine pancreatic function tests.

Background Data on magnetic resonance cholangiopancreatography with secretin stimulation (S-MRCP) for the assessment of exocrine pancreatic insufficiency (EPI) are limited. We compared pancreatic function tests with the findings of S-MRCP in patients with chronic pancreatitis (CP) and disease controls. Methods S-MRCP was performed in 23 patients (18 CP, 5 disease controls). MRCP images were analyzed for secretin-induced duodenal liquid filling (0=no filling; 1=duodenal bulb; 2=up to lower flexure; 3=beyond lower flexure). EPI was evaluated by fecal elastase, fecal fat concentration, and a 13C mixed chain triglyceride breath test. Clinically relevant EPI was stated if 2 of 3 tests were pathologic. Results EPI was diagnosed in 10 of 18 patients with CP. Patients without EPI showed either grade 2 (n=4) or grade 3 (n=9) duodenal filling, whereas only 1/10 patients with EPI showed grade 3 duodenal filling. Sensitivity and specificity of S-MRCP for the diagnosis of EPI were 69% and 90%, respectively. Conclusions Assessment of duodenal filling should be performed in patients who undergo S-MRCP for the evaluation of pancreatic morphology. However, minor degrees of duodenal filling are equivocal and require further diagnostic evaluation.

Current practice in the diagnosis and management of IBD-associated anaemia and iron deficiency in Germany: The German AnaemIBD Study☆

BACKGROUND/AIM Anaemia is a common complication in inflammatory bowel disease (IBD), frequently resulting from iron deficiency. IBD guidelines advocate intravenous iron administration although some patients respond to oral supplementation. This non-interventional study investigates the current status of anaemia management in German IBD patients. METHODS Baseline data on pre-study treatment for anaemia were retrospectively analysed in IBD patients with anaemia participating in a prospective trial of the efficacy and safety of ferric carboxymaltose. Data were collected from 55 German gastroenterological centres up to August 2010. Subjects had received care at their centre for at least 12 months prior to baseline. RESULTS 193 cases of IBD-associated anaemia (115 Crohns disease, 77 ulcerative colitis) were analysed (mean age: 39 years (18-83), 79 (41%) males). Anaemia and iron status were usually assessed by haemoglobin (100%), serum ferritin (97%), and transferrin saturation (82%). In the previous 6 months, only 84 patients (43.5%) had been treated for anaemia: 47 (56%) with oral iron, 13 (15%) parenteral iron, 16 (19%) oral plus parenteral iron and 8 (10%) transfusions. No patients received erythropoietin stimulating agents. CONCLUSION Although intravenous iron supplementation is recommended in IBD patients, current German practice still relies on oral therapy, even in severe anaemia. The high incidence of severe anaemia in this cohort reflects inadequate iron replacement and status monitoring. While the proportion of IBD patients with inadequately treated anaemia/iron deficiency is unknown, greater awareness of existing guidelines for iron deficiency management in IBD patients appears necessary.

Patient age is a strong independent predictor of 13C-aminopyrine breath test results: a comparative study with histology, duplex-Doppler and a laboratory index in patients with chronic hepatitis C virus infection.

1 Noninvasive tests for the staging of chronic hepatitis C virus (HCV) infection would be an attractive alternative to liver biopsy. The 13C‐aminopyrine breath test (ABT) has been proposed for the noninvasive assessment of hepatic function and partly correlates with fibrosis. We aimed to investigate causes for the lack of discriminatory power for different degrees of hepatic fibrosis. 2 Eighty‐three patients (median age 49 years (28–78 years)) with chronic HCV infection underwent the ABT after an oral load of 75 mg N,N‐dimethyl‐13C‐aminopyrine. Portal vein flow was assessed by duplex‐Doppler and a laboratory index (aspartate aminotransferase to platelet ratio index or APRI) was calculated. Parameters were compared with liver histology. 3 The cumulative 13C‐recovery differed significantly between patients without relevant fibrosis (fibrosis score 0–2) and cirrhosis (5–6), beginning after 30 min of sampling (P < 0.05). The ABT did not discriminate patients with fibrosis scores 3–4 from the remaining two patient groups. Sensitivity and specificity for the prediction of cirrhosis was 73.4–82.8% and 63.2–68.4%, depending on the sampling time. Compared with the fibrosis score (P = 0.04), patient age was a highly significant independent predictor for the 13C‐recovery (P < 0.0001). Aspartate aminotransferase to platelet ratio index and duplex‐Doppler predicted cirrhosis with 76.6%vs. 87.5% sensitivity and 63.2%vs. 68.4% specificity. 4 Our data suggest an age‐dependent decrease of cytochrome P450 activity which probably accounts for the large overlap of ABT results that preclude clear differentiation. This is also consistent with former pharmacodynamic trials. Age‐adapted reference ranges could improve ABT results.

Regulation of α1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells

α1‐Proteinase inhibitor (α1‐PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti‐inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of α1‐PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage‐derived cytokines on α1‐PI secretion from intestinal epithelial cells, we cultured Caco‐2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco‐2 cells were then co‐cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, α1‐PI secretion from Caco‐2 cells was enhanced by 45%, probably by a direct action of macrophage‐derived cytokines on Caco‐2 cells. To extend this observation further, we treated differentiated Caco‐2 cells with macrophage‐derived proinflammatory cytokines (IL‐1β, IL‐8, TNF‐α), as well as with lymphocyte‐derived cytokines IL‐2, IL‐6 and IFN‐γ. As early as after 24h treatment, IL‐2 and IL‐8 induced a significant and dose‐dependent increase of α‐1‐PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL‐2 and IL‐8 α1‐PI secretion was only slightly decreased after treatment with IFN‐γ, while IL‐1β, IL‐6 and TNF‐α had no effect. α1‐PI secretion correlated well with the expression of this protein in differentiated Caco‐2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, α1‐PI secretion in enterocyte‐like Caco‐2 cells is up‐regulated by IL‐2 and IL‐8. Our results suggest that both lymphocyte‐ and macrophage‐derived cytokines regulate secretion of the anti‐inflammatory protein α1‐PI in intestinal epithelial cells.

Fluorometric High-Performance Liquid Chromatography of Free Fatty Acids Using Panacyl Bromide

Abstract The application of panacyl bromide, a fluorescence labeling reagent for carboxylic acids, was examined. Fatty acids are separated by reversed-phase high-performance liquid chromatography. The derivatives of series of both saturated and unsaturated fatty acids (C10:0-C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection is linear over a range of 10–500 pmol per injection. The method was demonstrated to be able to quantify free fatty acids in blood samples from normal subjects and diabetic patients.

P380 Safety and efficacy of bolus administered ferric carboxymaltose (500 mg) in the treatment of iron deficiency anaemia in IBD patients

P380 Safety and efficacy of bolus administered ferric carboxymaltose (500mg) in the treatment of iron deficiency anaemia in IBD patients D. Jakobsen1 *, M. Wiesenthal2, F. Hartmann3, A. Dignass4, S. Weber-Mangal5, J. Stein2. 11Krankenhaus Sachsenhausen, Crohn Colitis Center, Frankfurt, Germany, 2Crohn Colitis Zentrum Rhein-Main, Frankfurt, Germany, 3Marien Krankenhaus, Frankfurt, Germany, 4Apaglesion Markus Krankenhaus, Frankfurt, Germany, 5Vifor Pharma Germany, Munich, Germany

A prospective cohort study to assess the relevance of vedolizumab drug level monitoring in IBD patients

Abstract Background: Vedolizumab (VDZ) drug monitoring strategies in inflammatory bowel disease (IBD) patients have not been systematically investigated so far. We evaluated the correlation between VDZ trough levels (VTL) and the treatment response in IBD. Methods: Fifty-one patients with active IBD on or starting a therapy with VDZ were enrolled in this prospective and observational single centre study. Disease activity indices, blood tests, and anthropometric parameters were assessed over a time period of 6 months. One hundred and fifty-five VDZ serum trough levels were measured directly before the next scheduled application using liquid chromatography mass spectrometry (LC-MS/MS). Results: VDZ treatment was found to be clinically effective (Harvey Bradshaw Index (HBI) dropping from 10 to 5.5 points (p < .0005) in Crohn’s disease (CD) patients; partial Mayo score (pMS) from 4.4 to 2.1 points (p < .0005) in ulcerative colitis patients (UC). CRP levels tended to decrease and haemoglobin levels to increase under VDZ therapy. CD patients with a serum CRP level lower than 5 mg/l exhibited significantly higher VTL than those with elevated CRP levels (34.9 versus 21.7 µg/ml, p = .00153). UC patients with haemoglobin levels higher 12 g/dl at the time of VTL measurement had significantly higher VTL compared to patients with lower haemoglobin levels (35.4 versus 15.6 µg/ml, p < .0005). Conclusions: Our data suggest a significant correlation between VTL and response to therapy in IBD patients (higher VTL associated with better response).

Editorial: which iron preparation for patients with IBD? Authors’ reply

edly raised CRP levels, tend to respond poorly to oral iron, and like those intolerant of oral iron or having Hb <100 g/L, should usually proceed directly to intravenous iron. Whether ferric maltol, a new, more expensive and better tolerated oral iron will be an option in this situation is under further evaluation (ClinTrials.gov NCT02680756). For intravenous treatment, we do not feel that the current analysis establishes beyond doubt the superior efficacy of FCM. We agree with Aksan that we should be cautious about applying rank probabilities to important decision-making, and that further head-to-head efficacy and safety trials are needed. Meanwhile, the intravenous preparation selected in most centres will be guided mainly by its local cost, convenience of administration and individual patients’ tolerance.