Immunophenotyping and assessment of cell function by three-color flow cytometry of peripheral blood lymphocytes.

Infection by the human immunodeficiency virus type 1 (HIV-1) is known to cause a number of changes in the immunophenotypic profile of patients even in the early asymptomatic stages of disease. Such "surrogate markers" are known to correlate with the stage of HIV disease and often are predictive of outcomes. The best known of these is the absolute count of T helper lymphocytes, or CD4 cells, which undergoes a gradual decline as the virus infects greater and greater numbers of these cells (1). A number of other markers have been found, some of which also are predictive of outcome in many cases. These include prevalence and intensity of the CD38 marker on CD8 T cells, percentage of CD4 cells exhibiting loss of the CD26 and CD28 markers, and percentage of CD4 cells with the CD95 marker (2). The CD38 intensity of CD3/CD8 cells has, in fact, been more closely correlated with future disease progression in patients than the CD4 count (3). The following method is a comprehensive immunophenotyping panel that incorporates all of these markers and provides several parameters by which to monitor disease progression and advise clinicians on treatment options.

CD38 expression on cryopreserved CD8+ T cells predicts HIV disease progression.

Previous studies have revealed that the expression of CD38 on CD8+ T cells is a strong predictor of disease progression in human immunodeficiency virus (HIV)-infected individuals. Those studies were performed using fresh patient samples over an extended trial period. After demonstrating the validity of assay results on cryopreserved cells, we performed a retrospective study using frozen cell samples to determine the predictive value of CD38 expression in patients with CD4 counts above 400 cells/microl. The CD38 expression as measured by antibody binding capacity and the CD38 median channel were shown to be associated with time to new opportunistic infection or death (both P < 0.001). These results suggest that CD38 expression on CD8+ T cells, whether fresh or frozen, provides a useful predictor of HIV disease progression.

Impaired induction of the apoptosis-protective protein Bcl-xL in activated PBMC from asymptomatic HIV-infected individuals.

Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = -0.695, P = 0.05). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.

Investigating stalking crimes.

1. Three types of stalkers that are identified in this crime classification are the non-domestic stalker who may know the target through social contact or from a random meeting in a public place; the domestic stalker who may be known to the target and had a close personal relationship with the target; and the erotomania stalker whose target is typically a public figure. 2. Stalking is conceptualized on a continuum from nondelusional to delusional behavior. 3. Classification of a crime is the first step in the investigative process.