Current immunological tools

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Although lymphoproliferation (LPA) has remained largely an immunological tool for the research laboratory, there are now a variety of new methodologies that do not require use of radioactive materials or the prolonged incubation times of LPA. These include flow cytometry assays to quantify subsets of circulating cells based on cell surface characteristics, ELISPOT to assess expression of cytokines, and newer cytokine genotype microarray methodologies. Genotype microarray technologies offer tremendous potential for identifying the activation of a wide array of genes throughout the course of disease, but so far these methods have not translated easily into the clinical lab setting and have been hampered by high cost and intense laboratory processing within hours of sample collection. While all of these methods offer insight into mechanisms of action of the immune system, there is a concomitant pressing need for clinical assays that can be standardized for the routine monitoring of patients over time. In addition, such methods must be amenable to use in the reference laboratory by providing results that are comparable from laboratory to laboratory and can withstand shipping conditions. In this context, a general biomarker for the immunosuppressive state would be a useful adjunct in the monitoring of HHV-6 susceptible and recently infected individuals. The FDA has recently cleared an assay for the detection of cell-mediated immunity in immunosuppressed populations, ImmuKnowTM, which measures global T-cell function by stimulating whole blood cells with the plant mitogen, PHA, and then quantifying lymphocyte activation by the intracellular content of ATP in the selected CD4+ cell subset.

Disease associations between HHV-6 infection and multiple sclerosis, fibromyalgia, and chronic fatigue syndrome

Ablashi et al. (2005) have performed a meta-analysis of over 85 published papers in which the relationship between HHV-6 and patients with multiple sclerosis or fibromyalgia, or chronic fatigue syndrome (CFS) was examined. Although a clear causal relationship has not been established between HHV-6 and these chronic illnesses, his study discerned that there is evidence of correlation in more than 75% of those papers that distinguish between active and latent virus. Since both in vitro studies and in vivo clinical experience provide evidence that HHV-6 is immuno-suppressive, patients with a history of CFS (data provided through the courtesy of Daniel Peterson, MD) were tested for the functionality of their global T-cell response using an FDA-cleared test for cell-mediated immunity assessment (Cylex Immune Function Assay, ImmuKnowTM) and these cellular immune responses were compared with individuals infected with HIV and stable transplant patients on immunosuppressive regimens.

Method: rapid immune function assay

Whole blood was collected into an 8 ml sodium heparin vacutainer tube during inpatient or routine clinical visits, stored at room temperature, and tested within 30 h of draw at Viracor (Kansas City, MO). The ImmuKnow™ assay (Catalog No. 4400, Cylex, Inc. Columbia, MD), which is FDA-cleared, was performed according to manufacturer's package insert. Briefly, 250 ml of whole blood was diluted with sample diluent, added to wells of a 96-well microtiter plate and incubated for 15-18h with PHA in a 37oC, 5% CO2 incubator. CD4+ cells were then positively selected within the microwells using magnetic particles coated with anti-human CD4 monoclonal antibodies (Dynabeads®, Dynal, Oslo, Norway) and a strong magnet (Cylex® Magnet Tray 1050, Cylex Inc®. Columbia, MD). Microwells were washed to remove residual cells, and the isolated CD4+ cells were lysed to release intracellular ATP. ATP was measured using luciferin/luciferase and a luminometer (Turner Biosystems, Sunnyvale, CA). A patient's level of immune response was assessed based on the amount of ATP expressed in ng/ml.

Results: comparing healthy adults, stable transplant patients, HIV-infected patients, and CFS patients

In these preliminary studies, the immune function characteristics of healthy adults, stable transplant recipients, HIV-infected individuals and CFS patients were compared on the basis of their distribution into low, moderate, or strong immune response zones as assessed by the intracellular ATP (ng/ml) of their T cells shown in

Remarkably, all three immunosuppressed populations had median immune function values that were not statistically different. Transplant patients ran the lowest at 259 ng/ml ATP, followed by HIV patients at 262 ng/ml ATP, and CFS patients at 281 ng/ml ATP. In addition, the relative distributions of patients in the low, moderate, and strong zones were statistically equivalent. Distribution of transplant recipients into three zones of immune response was initially defined by a three center clinical trial of stable patients (Kowalski et al., 2003). The majority (52%) of stable transplant patients ran in the moderate zone with a significant percentage in the low zone (40%) and only 8% in the strong zone (Table 1). By comparison, HIV patients distributed nearly identically to solid organ transplant with the majority in the moderate zone (55%), followed by the low zone (37%), and a minor proportion in the strong zone (8%). The CFS patients showed remarkable consistency with both the transplant patients and the HIV patients with the

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