Alterations in exhausted and classical memory B cells in lupus nephritis - Relationship with disease relapse.

INTRODUCTION: B cell exhaustion is a functional abnormality of B lymphocytes observed in chronic infections and shows association with autoreactivity. The role of exhausted and classical memory B cells in maintaining disease stability of lupus nephritis (LN) remains unclear. METHODS: We measured classical memory (CD19+CD21+CD27+), exhausted B cells (CD19+CD21-CD27-), and related cytokines in LN patients with multiple relapses (MR) (n = 15) and no relapse (NR) (n = 15) during disease remission. The expression of inhibitory/adhesion molecules, cell proliferation and calcium mobilization in classical memory and exhausted B cells were also assessed. RESULTS: The MR group had higher proportion of circulating exhausted and classical memory B cells compared to the NR group and healthy controls (HC) (p all <0.05 for MR vs. NR or HC). Blood levels of IL-6, BAFF, IL-21, CD62L, CXCR3 and Siglec-6 were all higher in the MR group (p < 0.05, for all). Exhausted B cells from the MR group showed higher FcRL4, CD22, CD85j and CD183 but lower CD62L expression than NR and HC groups. Exhausted B cells from MR patients exhibited reduced proliferation compared to NR patients and HC, while classical memory B cell proliferation in MR group was higher than the other two groups. Exhausted B cells from both MR and NR patients showed impaired calcium mobilization. CONCLUSION: Alterations in exhausted and classical memory B cells are related to disease relapse in LN. These findings may help devise new strategies for monitoring disease activity and preventing relapse in LN.

Genotyping with a dried blood spot method: a useful technique for application in pharmacogenetics.

Several commercial DNA isolation kits are available for extracting the genomic DNA from the ethylene diamine tetra-acetic acid (EDTA) whole blood samples. To obtain DNA from whole blood these DNA isolation procedures require quite some hands on time and are rather expensive. An alternative technique could be dried blood spot (DBS) sampling, with which DNA isolation is faster, cheaper and logistics are easier. We have developed a non-commercial DBS method and examined its performance in practice. DNA isolation from EDTA blood samples and made blood spots on filter paper from 106 renal transplant recipients were compared. Additionally, DNA isolation with a column method and two different DBS method was performed for 10 healthy volunteers and compared. Also DNA isolation with only capillary blood using both DBS methods from another 100 healthy volunteers has been investigated. Real-time PCR FRET assays for the CYP3A4 A-392G, CYP3A5 A6986G, ABCB1 C1236T, G2677T/A and C3435T polymorphisms were used and the melting curves of both DNA isolation methods were compared. In all cases DNA extracted with the column method corresponded completely with the results of the DNA isolated with the DBS procedure. Hence, DNA isolation from filter paper appears to be a useful alternative for the commercially available DNA isolation kits.