Hydroethidine (HE) and 2',7'-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2',7'-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2-. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2-. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.

Objective. Disease activity in systemic lupus erythematosus (SLE) is usually assessed with complex disease activity scores comprising a variety of different parameters. In order to determine whether SLE disease activity correlates with abnormal B lymphocyte activity, B cell subsets were analyzed, and their relationship to clinical and humoral measures of disease activity was assessed.Methods. The distribution of B cell subsets was determined by fluorescence-activated cell sorting analysis and assessed in relation to the autoantibody profile, disease activity measured by the SLE Disease Activity Index (SLEDAI) and the European Consensus Lupus Activity Measure scores, disease duration, and therapy.Results. The number and frequency of CD27 high plasma cells were significantly correlated with the SLE disease activity indices and with the titer of antidouble-stranded DNA (anti-dsDNA) autoantibodies. Circulating B cell subsets were not influenced by age or sex, but appeared to relate to the duration of disease and the therapeutic regimen, with the number and frequency of CD27 high plasma cells increasing and those of CD27؊ naive B cells decreasing over time. Patients were divided into those with a SLEDAI score of 0-8 (low disease activity) and those with SLEDAI score >8 (high disease activity). Patients with high disease activity had an increased frequency of both CD19؉ B cells and CD27 high plasma cells. By using a nonparametric data sieving algorithm, we observed that these B cell abnormalities provided predictive values for nonactive and active disease of 78.0% and 78.9%, respectively. The predictive value of the B cell abnormalities (78.9%) was greater than that of the humoral/clinical data pattern (71.4%), including anti-dsDNA antibody levels, circulating immune complexes, increased erythrocyte sedimentation rate, mucocutaneous involvement, and acute renal involvement.Conclusion. Flow cytometric monitoring of B cell subsets in the peripheral blood provides new insights into abnormalities of B cell function in SLE and may also be a diagnostically valuable option for monitoring the activity of this autoimmune disease.

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Wilson's disease results from mutations in the P‐type Cu2+‐ATPase causing Cu2+ toxicity. We previously demonstrated that exposure of mixed neuronal/glial cultures to 20μM Cu2+ induced ATP loss and death that were attenuated by mitochondrial substrates, activators, and cofactors. Here, we show differential cellular sensitivity to Cu2+ that was equalized to 5 μM in the presence of the copper exchanger/ionophore, disulfiram. Because Cu2+ facilitates formation of oxygen radicals (ROS) which inhibit pyruvate dehydrogenase (PDH) and alpha‐ketoglutarate dehydrogenase (KGDH), we hypothesized that their inhibition contributed to Cu2+‐induced death. Toxic CU2+ exposure was accompanied by early inhibition of neuronal and hepatocellular PDH and KGDH activities, followed by reduced mitochondrial transmembrane potential, ΔΨM. Thiamine (1–6mM), and dihydrolipoic acid (LA, 50μM), required cofactors for PDH and KGDH, attenuated this enzymatic inhibition and subsequent death in all cell types. Furthermore, liver PDH and KGDH activities were reduced in the Atp7b mouse model of Wilson's disease prior to liver damage, and were partially restored by oral thiamine supplementation. These data support our hypothesis that Cu2+‐induced ROS may inhibit PDH and KGDH resulting in neuronal and hepatocellular death. Therefore, thiamine or lipoic acid may constitute potential therapeutic agents for Wilson's disease.