Maarten Cozijnsen

73 Chapter 4 Infliximab impacts leukocyte RNA expression and serum inflammatory proteins Materials & methods Patient population This pilot study is based on the ITSKids study, an investigator initiated RCT (Clinicaltrials.gov Identifier: NCT01880307), that aimed to compare the efficacy of two treatment strategies in newly diagnosed children with CD. Patients were eligible when diagnosed with Crohn’s disease, with moderate-to-severe disease activity – determined by a Pediatric CD Activity Index (PCDAI) above 30 – and if they had not received prior CD related therapy. Patients who needed surgery or had severe comorbidity were excluded. Patients were randomized into two treatment groups: the experimental group received 5 infliximab infusions (5mg/ kg at week 0, 2, 6, 14 and 22) and simultaneously started with AZA treatment (2-3 mg/kg once daily) and the control group received oral prednisolone (daily 1mg/kg, max 40mg for 4 weeks, then tapering of prednisolone in 6 weeks until stop) and simultaneously started AZA treatment (2-3 mg/kg once daily). Both at baseline and week 10, patients underwent clinical diagnostic work-up including blood tests, fecal calprotectin measurement and endoscopic examination. Blood samples were taken for RNA and protein analyses. Eleven patients were randomized: 6 patients started with infliximab and 5 started with prednisolone (Table 1). Disease activity measures, such as clinical disease activity and inflammatory markers in blood and feces were comparable. However, the small sample size did leave some differences in disease location and age at onset between the two groups. All patients were followed up until week 10. In one patient that started infliximab and azathioprine, azathioprine was stopped at week 6 because of neutropenia and in one patient that started prednisolone, infliximab was started at week 6 for perianal disease. All patients were analyzed in the group to which they were randomized according to the intention to treat principle. Endoscopy was not performed at week 10 in one patient of each group because of patient refusal. Peripheral blood RNA expression analysis Blood was collected in PAXgene tubes before start of treatment and after 10 weeks of treatment. The PAXgene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland) was used according to the manufacturer’s instructions to isolate total RNA from the PAX gene Blood RNA tubes. The RNA concentration was measured on a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, Waltham, MA, United States of America (USA)). The integrity of the RNA samples was verified with the TapeStation (Agilent Technologies, Santa Clara, CA, USA) using the RNA Screentapes (Agilent Technologies). 2.5 ug of total RNA was used for Globin depletion using the human GLOBINclear Kit (Thermo Fisher Scientific). 100 ng globin depleted mRNA was labelled using the GeneChip® 3’ IVT PLUS Reagent Kit (Affymetrix, Thermo Fisher Scientific). The labelled samples were hybridized to GeneChip HT HG-U133+