Meryl A. Forman

Detection of JC virus DNA in peripheral blood cell subpopulations of HIV-1-infected individuals

While it has been suggested that JC virus (JCV) migrates in B-lymphocytes from the kidney to the central nervous system where it initiates demyelination, this phase of JCV pathogenesis has not been systematically explored. To determine the peripheral blood cell subpopulation(s) infected with JCV, monocytes, granulocytes, and T and B lymphocytes from HIV-1-infected individuals and uninfected controls were purified by flow cytometry. JCV DNA could be detected by PCR amplification in all of these cell subpopulations. This finding suggests that JCV lacks specificity in its interaction with leukocytes.

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

ZAP‐70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B‐cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP‐70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.

Simian Immunodeficiency Virus (SIV)-Specific CTL Are Present in Large Numbers in Livers of SIV-Infected Rhesus Monkeys

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.

Effect of complement consumption by cobra venom factor on the course of primary infection with simian immunodeficiency virus in rhesus monkeys.

Cobra venom factor (CVF)-induced consumption of complement proteins was used to investigate the role of complement in vivo in the immunopathogenesis of simian immunodeficiency virus of macaques (SIVmac) infection in rhesus monkeys. Repeated administration of CVF was shown to deplete complement to <5% of baseline hemolytic activity of serum complement for 10 days in a normal monkey. Three groups of SIVmac-infected animals were then evaluated: monkeys treated with CVF resulting in complement depletion from days -1 to 10 postinfection, monkeys treated with CVF resulting in complement depletion from days 10 to 21 postinfection, and control monkeys that received no CVF. CD8+ SIVmac-specific cytotoxic T lymphocyte (CTL) generation and CD4+ T lymphocyte depletion during primary infection were not affected by CVF treatment. Viral load, assessed by measurements of plasma p27gag antigen and viral RNA, was transiently higher during the first 4 weeks following infection in the CVF-treated monkeys and the subsequent clinical course in these treated animals was accelerated. These results suggest that complement proteins may participate in immune defense mechanisms that decrease virus replication following the initial burst of intense viremia during primary SIVmac infection. However, we cannot rule out that the observed increased virus replication was induced by immune activation resulting from the administration of a foreign antigen to these monkeys.

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.

Optimized Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP‐70 protein expression in whole‐blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP‐70 protein with significantly improved signal‐to‐noise ratio, an optimized combination of antibodies‐fluorophores to maximize ZAP‐70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter‐ and intra‐laboratory reproducibility, and a method to index ZAP‐70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP‐70 protein expression levels in the CLL population.