The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4 ؉ and CD8 ؉ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at ؊70°C for <3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at ؊70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

The newly identified type III interferon (IFN-) has antiviral activity against a broad spectrum of viruses. We thus examined whether IFN-has the ability to inhibit human immunodeficiency virus type 1 (

Cytokines and neuropeptides are modulators of neuroimmunoregulation in the central nervous system (CNS). The interaction of these modulators may have important implications in CNS diseases. We investigated whether interleukin-1β (IL-1β) modulates the expression of neurokinin-1 receptor (NK-1R), the primary receptor for substance P (SP), a potent neuropeptide in the CNS. IL-1β upregulated NK-1R expression in human astroglioma cells (U87 MG) and primary rat astrocytes at both mRNA and protein levels. IL-1β treatment of U87 MG cells and primary rat astrocytes led to an increase in cytosolic Ca 2+ in response to SP stimulation, indicating that IL-1β-induced NK-1R is functional. CP-96,345, a specific non-peptide NK-1R antagonist, inhibited SP-induced rise of [Ca 2+ ] i in the astroglioma cells. Investigation of the mechanism responsible for IL-1β action revealed that IL-1β has the ability of activating nuclear factor-κb (NF-κB). Caffeic acid phenethyl ester (CAPE), a specific inhibitor of NF-κB activation, not only abrogated IL-1β-induced NF-κB promoter activation, but also blocked IL-1β-mediated induction of NK-1R gene expression. These findings provide additional evidence that there is a biological interaction between IL-1β and the neuropeptide SP in the CNS, which may have important implications in the inflammatory diseases in the CNS.