120 CHAPTER 6 recruited at the Amsterdam University Medical Centers and Reade, in Amsterdam, the Netherlands into the ‘Prevention of Rheumatoid Arthritis (RA) by B cell directed therapy’-trial (PRAIRI-trial, EudraCT Number: 2009-010955-29). In total 81 RA-risk individuals were included. RA-risk individuals in the rituximab group received rituximab 1000 mg i.v. on day 1 (RTX-group). Individuals in the placebo group received an infusion of matching placebo (placebo-group). In addition, all individuals in both groups on day 1 received 100 mg methylprednisolone i.v. as pre-medication to minimize infusion-related reactions of rituximab. During the screening visit, after blood was drawn and at least 4 weeks before baseline, all patients were vaccinated with a pneumococcal vaccine and dependent on the season also with an influenza vaccine. Moreover, some patients got a H1N1 vaccination due to the 2009 flu pandemic. Therefore, the main difference between the screening visit and the baseline visit is that at screening all patients were medication-naïve and not yet vaccinated. In 75 patients (93% of the total cohort), blood was drawn during the screening and baseline visits, after 6 and 12 months and in case arthritis developed, from which RNA was isolated for BCR repertoire analysis. One patient was excluded from BCR analyses due to the fact that she was diagnosed with chronic lymphocytic leukaemia after one month. Additionally, a study was conducted in a novel cohort that included individuals double positive for ACPA and IgM-RF with arthralgia, but without clinical signs of synovitis in the Amsterdam University Medical Centers from 2016 onwards, the “DOMINant clones in the Onset of RA”-study (DOMINO study). These individuals were not treated with rituximab or placebo and did not receive vaccinations prior to study inclusion (untreated-group). Individuals were followed-up with 6-monthly visits on which fluorescence activated cell sorting (FACS) was performed with subsequent sorted fraction and whole blood BCR sequencing. For this manuscript, all RA-risk individuals that attended at least 2 visits were selected (N=10). 9 individuals that were autoantibody negative, did not have arthralgia and did not have concomitant diseases or drug use were included as healthy controls. The BCR repertoire of these individuals was sequenced, but FACS was not performed. The studies were approved by the independent Medical Ethics Committee of the Amsterdam UMC, location AMC and performed according to the Declaration of Helsinki. All patients gave written informed consent. Flow cytometry Plasmablast/Plasma cells were identified as CD3-CD20-CD27+IgD-CD19+CD38++ and memory B cells as CD3-CD20+CD27+IgD-CD19+CD38-. To avoid artifacts, e.g. death of plasmablasts induced by freezing, cells were freshly isolated from peripheral
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