Polyclonal rATG induces complement-independent apoptosis of naive, activated, and plasma B cells. This effect appears to involve the caspase- and cathepsin-mediated apoptosis pathways.

Unmethylated CpG DNA activation of naive CD27 ؊ B cells has been reported to require B-cell-receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell-independent activation of naive CD19 ؉ CD27 ؊ human peripheral blood B cells that induces efficient CD138 ؉ plasma-cell differentiation. CD27 ؉ and CD27 ؊ B cells were cultured in a 3-step system: (1)

Current monoclonal antibody therapies for multiple myeloma have had limited success, perhaps due to narrow target specificity. We have previously described the ability of polyclonal rabbit antithymo-cyte globulin (rATG) to induce caspase-and cathepsin-mediated apoptosis in human B and plasma cells. We now extend this observation to myeloma cells. Complement independent cell death was measured after addition of rATG (1-1000 g/mL) to cultures of myeloma cell lines or primary CD138 isolates from patient bone marrow aspirates. rATG induced significant levels of apoptosis in my-eloma cells as assayed by caspase induction , annexin V binding, subdiploid DNA fragmentation, plasma-membrane per-meability, and loss of mitochondrial-membrane potential. Addition of complement greatly augmented myeloma-cell death. Binding of rATG to individual my-eloma cell-surface proteins, primarily CD38, CD52, CD126, and CD138, was demonstrated by competitive inhibition experiments with targeted monoclonal antibod-ies. Three pathways of cell death were identified involving caspase activation, cathepsin D, and the genistein sensitive tyrosine kinase pathway. F(ab) 2 fragments of rATG had reduced proapoptotic activity, which was restored by coincuba-tion with Fc fragments, and anti-CD32 or anti-CD64 antibodies. We conclude that rATG is an effective agent for in vitro induction of apoptosis in multiple myeloma, and that exploratory clinical trials may be warranted. (Blood. 2006;107:2895-2903)

During the recall response by CD27+ IgG class-switched human memory B cells, total IgG secreted is a function of the following: 1) the number of IgG-secreting cells (IgG-SC), and 2) the secretion rate of each cell. In this study, we report the quantitative ELISPOT method for simultaneous estimation of single-cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgM− memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. BCR cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27high B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27high B cell blasts. In contrast, the addition of IL-21 to CD40L plus IL-4-activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG plus cytokine-activated B memory B cell populations, is suggestive of an on/off switch regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG-SC and the distribution of their IgG secretion rates.

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We have previously described two flow cytometry-based in vitro genotoxicity tests: micronucleus (MN) scoring (MicroFlow®) and a multiplexed DNA damage response biomarker assay (MultiFlow®). Here, we describe a strategy for combining the assays in order to efficiently supplement MN analyses with a panel of biomarkers that comment on cytotoxicity (i.e. relative nuclei count, relative increased nuclei count, cleaved PARP-positive chromatin and ethidium monoazide-positive chromatin) and genotoxic mode of action (MoA; i.e. γH2AX, phospho-histone H3, p53 activation and polyploidy). For these experiments, human TK6 cells were exposed to each of 32 well-studied reference chemicals in 96-well plates for 24 continuous hours. The test chemicals were evaluated over a range of concentrations in the presence and absence of a rat liver S9-based metabolic activation system. MultiFlow assay data were acquired at 4 and 24 h, and micronuclei were scored at 24 h. Testing 32 chemicals in two metabolic activation arms translated into 64 a priori calls: 42 genotoxicants and 22 non-genotoxicants. The MN assay showed high sensitivity and moderate specificity (90% and 68%, respectively). When a genotoxic call required significant MN and MultiFlow responses, specificity increased to 95% without adversely affecting sensitivity. The dose–response data were analysed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesise the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesising multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.