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We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.

Site-selective cyclic AMP (cAMP) analogues have been shown to inhibit growth and induce differentiation in several human leukemia cell lines. However, detailed studies of the effects exerted by cAMP analogues on cell cycle kinetics have been lacking. We have examined the effects of 8-Cl-cAMP and N6-benzyl-cAMP on the cell cycle kinetics of the HL-60 human promyelocytic leukemia cell line. A cell cycle study was performed by univariate DNA analysis after 24-72 h of treatment with noncytotoxic concentrations of 8-Cl-cAMP and N6-benzyl-cAMP capable of inducing 50-60% growth inhibition in these cells. HL-60 cells treated with 5 microM 8-Cl-cAMP showed no significant change in the cell distribution in the cycle as compared to the untreated control cells, whereas the treatment with 10 microM N6-benzyl-cAMP transiently increased the percentage of cells in the G0/G1 phase after 48 h, followed by a partial recovery at 72 h. Combined treatment with low doses of 8-Cl-cAMP and N6-benzyl-cAMP, each of which alone produced 20% growth inhibition, exerted a growth inhibitory effect of 65% and delayed increase of the G0/G1 phase by 72 h. To better understand the cell cycle effects induced by 8-Cl-cAMP, flow cytometric analysis of bromodeoxyuridine incorporation was also performed. 8-Cl-cAMP treatment exhibited a slowing down of the cell cycle; thus, the delayed appearance of the G0/G1 cell accumulation after combined treatment could be due to this effect of 8-Cl-cAMP on the HL-60 cell cycle. At a toxic dose, 8-Cl-cAMP brought about a G2M block, whereas N6-benzyl-cAMP brought about an increase of the G0/G1 compartment. G2M block produced by toxic doses of 8-Cl-cAMP was not related to its adenosine metabolite since 8-Cl-adenosine did not produce any specific block in the cell cycle. Our results show, for the first time, that these site-selective cAMP analogues could affect cell cycle kinetics at different points. These data may provide the basis for combination treatments involving cAMP analogues and other agents in the treatment of human leukemia.

Soluble products of either Epstein-Barr virus (EBV)-infected B cells or activated monocytes promote the proliferation of EBV-infected B cells and permit their growth at low cell densities. This suggests that growth factors are important for B-cell immortalization by EBV. In this study, a monocyte-derived factor that promotes the growth of EBV-infected b cells was purified and identified as interferon-beta 2 (IFN-beta 2), which is also known as 26-kilodalton protein, B-cell differentiation factor (BSF-2), and interleukin-6 (IL-6). The purified protein has a specific activity of approximately 4 X 10(7) units per milligram of protein in assays of B-cell growth. Thus, IFN-beta 2/BSF-2 is a B-cell growth factor that promotes the proliferation of human B cells infected with EBV.

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.

Lymphoblastoid cell lines were derived from patients with active systemic lupus erythematosus by allowing spontaneous transformation of peripheral B lymphocytes (B cells) harboring endogenous Epstein-Barr virus or by superinfecting peripheral lymphocytes with exogeneous Epstein-Barr virus. Results of extensive studies aimed at identifying type C oncornaviruses in these lymphoblastoid cells were entirely negative by electron microscopy, DNA-DNA hybridization, reverse transcriptase assays, and cocultivation experiments. These results do not support the postulated association of oncornavirus infection in human systemic erythematosus.

An electron microscopic study of the morphology of Herpesvirus macaca, a serologically distinct infectious agent isolated from the leukocytes of rhesus monkeys, was performed. WI-38 fibroblast monolayers were infected with the virus and examined 18 days later. The morphology of Herpesvirus macaca was, in general, typical of the herpesvirus group. Enveloped virus particles observed via negative-stain technique had a diameter of 145-155 nm. An inner capsid composed of hexagonal capsomeres had a diameter of 100-110 nm and surrounded a central core. While enveloped forms appeared to be present within the nuclei of infected cells, they were not found in the cytoplasm except within vacuolar structures. Associated changes were found in the morphology of infected cells, including intracytoplasmic myelin figures.