C. Ubezio

Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease

Objective External fistulas represent a disabling manifestation of Crohns disease with a difficult curability and a high relapse rate despite a large therapeutic armamentarium. Stem cell therapy is a novel and promising approach for treatment of chronic inflammatory conditions. We therefore investigated the feasibility, safety and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in the treatment of fistulising Crohns disease. Patients and methods We enrolled 12 consecutive outpatients (eight males, median age 32 years) refractory to or unsuitable for current available therapies. MSCs were isolated from bone marrow and expanded ex vivo to be used for both therapeutic and experimental purposes. Ten patients (two refused) received intrafistular MSC injections (median 4) scheduled every 4 weeks, and were monitored by surgical, MRI and endoscopic evaluation for 12 months afterwards. The feasibility of obtaining at least 50×106 MSCs from each patient, the appearance of adverse events, and the efficacy in terms of fistula healing and reduction of both Crohns disease and perianal disease activity indexes were evaluated. In addition, the percentage of both mucosal and circulating regulatory T cells expressing FoxP3, and the ability of MSCs to influence mucosal T cell apoptosis were investigated. Results MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohns disease and perianal disease activity indexes (p<0.01 for both), and rectal mucosal healing were induced by treatment without any adverse effects. The percentage of mucosal and circulating regulatory T cells significantly increased during the treatment and remained stable until the end of follow up (p<0.0001 and p<0.01, respectively). Furthermore, MSCs have been proven to affect mucosal T cell apoptotic rate. Conclusions Locally injected MSCs represent a feasible, safe and beneficial therapy in refractory fistulising Crohns disease.

Peripheral regulatory T cells and serum transforming growth factor‐β: Relationship with clinical response to infliximab in Crohn's disease

Background: CD4+Foxp3+ regulatory T cells (Treg) inhibit T‐cell proliferation in vitro and are effective in suppressing colitis in mouse models. Tumor necrosis factor (TNF)‐&agr;, which is centrally involved in Crohns disease (CD) pathogenesis, also impairs Treg function. Here we investigated the influence of anti‐TNF therapy on Treg frequency and function in CD. Methods: Twenty CD patients were treated with infliximab administered at weeks 0, 2, and 6. Blood was collected immediately before the first infusion and after 10 weeks. Treg frequency was quantified by flow cytometry. Treg function was measured using a standard coculture assay. Serum levels of transforming growth factor (TGF)‐&bgr;1 and interleukin (IL)‐10 were measured by enzyme‐linked immunosorbent assay (ELISA). Results: Pretreatment Treg frequency and serum TGF‐&bgr;1 levels were significantly higher in nonresponder than responder patients. Clinical improvement in 12 CD patients was associated with a significant increase of Treg frequency after 10 weeks. Treg were functionally active before and after treatment with infliximab, both in responder and nonresponder CD patients. In responder patients the restoration of Treg pool was accompanied by a parallel significant increase of serum TGF‐&bgr;1 and IL‐10. No significant change in the elevated Treg or serum TGF‐&bgr;1 was seen in nonresponder patients. Conclusions: This study suggests that there may be a relationship between numbers of Treg in the blood, serum TGF‐&bgr;1, and response to infliximab; however, further prospective studies are needed. (Inflamm Bowel Dis 2010)

Abnormal thymic stromal lymphopoietin expression in the duodenal mucosa of patients with coeliac disease

Objective The short isoform of thymic stromal lymphopoietin (TSLP), a cytokine constitutively expressed by epithelial cells, is crucial in preserving immune tolerance in the gut. TSLP deficiency has been implicated in sustaining intestinal damage in Crohns disease. We explored mucosal TSLP expression and function in refractory and uncomplicated coeliac disease (CD), a T-cell-mediated enteropathy induced by gluten in genetically susceptible individuals. Design TSLP isoforms—long and short—and receptors—TSLPR and interleukin (IL)-7Rα—were assessed by immunofluorescence, immunoblotting and qRT-PCR in the duodenum of untreated, treated, potential and refractory patients with CD. The ability of the serine protease furin or CD biopsy supernatants to cleave TSLP was evaluated by immunoblotting. The production of interferon (IFN)-γ and IL-8 by untreated CD biopsies cultured ex vivo with TSLP isoforms was also assessed. Results Mucosal TSLP, but not TSLPR and IL-7Rα, was reduced in untreated CD and refractory CD in comparison to treated CD, potential CD and controls. Transcripts of both TSLP isoforms were decreased in active CD mucosa. Furin, which was overexpressed in active CD biopsies, was able to cleave TSLP in vitro. Accordingly, refractory and untreated CD supernatants showed higher TSLP-degrading capacity in comparison to treated CD and control supernatants. In our ex vivo model, both TSLP isoforms significantly downregulated IFN-γ and IL-8 production by untreated CD biopsies. Conclusions Reduced mucosal TSLP expression may contribute to intestinal damage in refractory and untreated CD. Further studies are needed to verify whether restoring TSLP might be therapeutically useful especially in refractory patients with CD.

P.05.15 IS ULCERATIVE COLITIS AN ATYPICAL T HELPER 2-MEDIATED DISEASE CHARACTERISED BY EXCESSIVE PRODUCTION OF INTERLEUKIN-13?

The aim of this study was to compare the outcomes of therapy with IFX and ADA in CD patients in daily clinical practice. Material and methods: All CD patients treated with anti TNF-alpha antibodies form July 1999 to September 2011 at “Casa Sollievo della Sofferenza” Hospital were enrolled. Demographical and clinical data were collected in an electronic database for the analysis. Results: A total of 188 patients (113 treated with IFX and 75 with ADA) were enrolled. The mean age at diagnosis was 29±12 (IFX) vs 34±14 (ADA) (p=0.003). The mean duration of disease was 8±6 years for both groups. Ileocolonic disease was significantly more frequent in IFX patients (45 vs 19 pts, p=0.03). Luminal disease (74 IFX vs 61 ADA, p=0.02), fistulizing disease (28 IFX vs 12 ADA, p=0.004), and extraintestinal manifestations (11 IFX vs 2 with ADA, p=0.07) were the indications for therapy. All patients treated with ADA vs 66 (58%) treated with IFX received a maintenance therapy; the mean duration of treatment was 12±10 for ADA vs 6±6 months for IFX (p=0.0003). The response rate was similar (76% IFX and 67% ADA; p=0.17). Five of 6 patients (83%) non-responder to ADA and 7 of 10 patients refractory to IFX responded to the alternative anti-TNF (p=ns). 107 patients were treated with immunosuppressants (76 with IFX and 31 with ADA; p=0.0005). The response rate did not improve with the combo therapy in both groups. 74 patients were active smokers (49 treated with IFX and 25 with ADA; p=0.1). The response rate was significantly lower in smokers treated with IFX (71.4% vs 92%, p=0.005). 15 (13%) patients treated with IFX and 10 (14%) treated with ADA had adverse events (AEs) (p=ns). No differences in AE rate between patients in mono or combo therapy were found. Conclusions: IFX and ADA showed similar efficacy and safety. Current smokers showed a significant lower response rate only in patients treated with IFX. Most patients refractory to an antiTNF may respond to the other. Prospective, randomized head to head studies comparing IFX to ADA are strongly desiderable.