Several studies have reported the successful use of Rituximab in patients with active disease [5,41]. patients with vasculitis. Results We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC ANCA production is induced by CpG-B but not by other B cell stimulants. In addition to CpG-B + IL2, PBMCs from 5 different pairs were also stimulated with either IL2 alone, LPS + IL2, pokeweed mitogen (PWM) + IL2, Soblidotin or inactivated em staphylococcus aureus /em + IL2. A representative set of results is shown from a PR3+ ANCA patient (A) and a MPO+ ANCA patient (B). Table 1 Clinical and laboratory data of the patients at the time of study thead Patient NumberAgeGenderANCA TitreClinical StatusTreatment em Organ Involvement /em /thead 137MPR3 60 URemissionNilR, J269MPR3 34 UNewly DiagnosedPrednisolone 5 mg/dayR, J356MPR3 100 URelapseNilR, J, S, P473FPR3 100 UNewly DiagnosedNilR, L, E550MPR3 33 UNewly DiagnosedNilL, E, J663MMPO 100 UNewly DiagnosedNilR, L, C, J777MMPO 54 UNewly DiagnosedNilR, L867MMPO 38 URelapseNilR, L957MMPO 47URelapseNilR, S, L1082MMPO 100 UNewly DiagnosedNilR, J Open in a separate window R = Renal, E = ENT, L = Lung, C = Cardiac, J = Joints, S = Skin, P = Peripheral Neuropathy Detection of peripheral blood circulating B cells capable of producing ANCA in response to CpG-B The production of ANCA autoantibodies by PBMCs in ANCA+ vasculitis patients suggested the presence of circulating ANCA autoreactive B cells Soblidotin in these patients. In order to test this possibility, we attempted to detect peripheral blood circulating B cells that are capable of producing ANCA by ELISpot. PBMCs isolated from a MPO+ ANCA patient who had had a relapse of vasculitis disease (patient no.8 in Table ?Table1)1) were cultured with CpG-B and IL-2 for 5 days. Cells were then transferred into wells previously coated with either myeloperoxidase antigen or control antigen, and cultured overnight. Antibody producing cells that had produced IgG antibody against these antigens were detected by an anti-human IgG antibody. The ELISpot assay shows the presence of MPO-reactive B cells within the PBMC population of the MPO+ ANCA vasculitis patients but not of the control individuals (Fig. ?(Fig.3).3). Together, the above data indicate that ANCA+ vasculitis patients have in Soblidotin their peripheral circulation B cells which are capable of Soblidotin producing ANCA in response to CpG stimulation. Open in a separate window Figure 3 Detection of circulating B cells capable of producing ANCA in response to CpG-B. PBMCs from 2 MPO+ ANCA vasculitis patients were cultured with CpG-B and IL-2. These PBMCs had not undergone enrichment for B cells prior to culture. After 5 days culture, cells were transferred into ELISpot wells which had been coated with either myeloperoxidase (MPO) in duplicates or foetal calf serum (FCS) as a control antigen. After overnight culture, IgG antibody producing cells against these antigens were detected by anti-human IgG conjugate. The total number of IgG producing B cells was measured by coating the wells with polyclonal anti-human IgG. The results from a patient as shown in this figure are representative of results from 2 patients. Fig A shows ELIspot plate with total IgG producing cells in the first column followed by the detection of anti-MPO B cells in duplicates in the middle columns and finally cells against the control antigen. The numbers of spots counted are depicted in Figure B. In spite of both patient and control having similar number of IgG producing cells, the number of anti-MPO B cells is higher in the MPO+ patient. This result coincide with those from a parallel experiment where PBMCs from this pair of individuals were cultured Rabbit Polyclonal to Sirp alpha1 in the presence of CpG-B to measure their em in vitro /em production of anti-MPO by ELISA as shown in (C). CpG-B also induced production of the relevant IgG autoantibodies in patients with other autoimmune diseases em in vitro /em To test if the CpG-B effect of inducing autoantibody production may be seen in other autoimmune diseases besides ANCA+ vasculitis, the same experimental procedure was performed in patients presenting with other types of autoimmune diseases, namely autoimmune thyroiditis and anti-phosphoslipid antibody syndrome. These patients were similar to most of our ANCA+ vasculitis patients in that they were not.