Decreased levels of CD54 (ICAM-1)-positive lymphocytes in the peripheral blood in untreated patients with active juvenile dermatomyositis.

Significant abnormalities are observed in the peripheral blood of juvenile dermatomyositis (JDM) patients with active disease. In this study, we confirm that there is a significant increase in the relative percentage of B lymphocytes in the peripheral blood of a group of untreated children with newly diagnosed active JDM compared to healthy children (P < 0.0001). In order to investigate if properties intrinsic to B cells contributed to their relative increase in JDM, the percentage of B cells expressing activation markers (CD23, CD25, CD54, and CD69) was measured and compared to pediatric controls. Compared to healthy children less than 10 years of age (not significantly different from the JDM group), the JDM patients had an increase in the proportion of lymphocytes expressing CD19 (B cells; P = 0.0017) and decreases in the percentage of lymphocytes that were CD3(-) CD16(+) and/or CD56(+) (NK cells; P = 0. 01) and CD3(+) CD8(+) (T suppressor/cytotoxic cells; P = 0.02). There were no significant differences in any of the B-cell activation markers assessed. Of note, the percentage of CD54(+) non-B lymphocytes (i.e., T cells and NK cells expressing CD54) was significantly lower in the JDM patients (25% +/- 5%) than in the "age-related" healthy control group (43% +/- 4%; P = 0.013). These results suggest the following for untreated children with active JDM: (i) the increase in the percentage of peripheral blood B cells is not due to intrinsic B-cell activation, and (ii) CD54/ICAM-1(+) non-B cells, CD8(+) T cells, and NK cells are being removed from circulation and may be participating in the pathophysiology of the disease.

Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers.

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface of in vitro activated CD4+ T cells. Approximately 90% of in vitro activated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% of in vitro activated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of the in vitro activated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.

Flow cytometric analyses of the lymphocyte subsets in peripheral blood of children with untreated active juvenile dermatomyositis.

Juvenile dermatomyositis (JDMS) is a vasculopathy affecting primarily skin and muscle. Although the etiology is unknown, immunopathogenetic mechanisms appear to play a role in both the susceptibility to the disease and its progression. We measured the percentage and absolute numbers of B cells and T-cell subsets in the peripheral blood of untreated JDMS patients with active early disease and compared the results with those obtained from a study of peripheral blood obtained from a heatlhy age-related control group. The absolute number of total lymphocytes in the peripheral blood of the JDMS patients was significantly lower (P < 0.002) than that observed in the healthy control population, with an associated decrease in the absolute number of all T-cell subsets. No concomitant decrease in the absolute number of B lymphocytes was observed in the JDMS patients. In contrast, the percentage of B lymphocytes and the T-helper/T-suppressor cell ratio were significantly higher in the JDMS group than in the control group (P < 0.001 and P < 0.002, respectively). Retrospective analysis of JDMS patients' serum samples obtained within 1 month of the flow cytometric evaluation indicated that 79% of the sera contained an antinuclear antibody and 46% had immunoglobulin G values above age-adjusted reference ranges. The increased percentage of B cells, the increased T-helper/T-suppressor cell ratio, the positive antinuclear antibody results, and the increased concentration of serum immunoglobulin suggest that humoral immune dysregulation may contribute to the pathogenesis of JDMS.

Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of the NADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the leukocytes of affected patients. Recently, several flow cytometry-based procedures which measure various reactive oxygen intermediates generated by the NADPH oxidase system have been developed. Most of the procedures developed to date require time-consuming granulocyte isolation, washing, and counting procedures, or they lack sensitivity. We have modified an existing procedure such that cell labelling and stimulation are performed directly in whole blood. Optimization of this procedure and its use in the diagnosis of patients with CGD or X-linked carriers are presented.