Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K i ‫؍‬ 0.19 M). SP600125 is a reversible ATPcompetitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-␥, TNF-␣, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharideinduced expression of tumor necrosis factor-␣ and inhibited anti-CD3-induced apoptosis of CD4 ؉ CD8 ؉ thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein-and phycoerythrin-conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics.We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.

Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

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The production of hybridomas between immunologically activated T cells and malignant T-cell lines offers a potentially unlimited source of soluble T-cell-derived products. Recently, human T-T hybrids have been described; however, their use has been hampered by slow growth and chromosomal instability due at least in part to the presence of thymidine in the traditional hypoxanthine/aminopterin/thymidine (HAT) selection medium. In this report, we describe the development of a rapidly growing hypoxanthine phosphoribosyltransferase-deficient human T-cell line designated J3R7, the use of azaserine/hypoxanthine (AH) medium as an alternative selection medium to HAT medium, and the production of functional T-T hybrids by using the J3R7 line and the AH selection technique. Hybrids selected in AH medium were 4-fold greater in number and 3-fold faster in growth rate than hybrids grown in HAT medium. No stable clones were obtained from HAT cultures whereas AH-derived hybrids could be readily cloned by the method of limiting dilution. Evidence for hybridization included (i) the presence of approximately twice the number of chromosomes in hybrids than in J3R7 cells; (ii) the presence on hybrid cells of the Leu-3a surface antigen, present on normal helper T cells but not on J3R7 cells; (iii) the expression of HLA antigens of both the normal T-cell partner and the J3R7 line; and (iv) the constitutive secretion of interleukin 2 from multiple hybrid clones but not from the J3R7 cell line. Thus far, these clones have maintained their rapid growth, chromosome number, surface phenotype, and constitutive secretion of interleukin 2 for 4 months.T lymphocytes produce a variety of immunologically active mediators (lymphokines) on stimulation with antigens or mitogens (1, 2). These mediators can be divided into two broad categories: antigen nonspecific, such as growth factors for T cells (interleukin 2, IL-2) and B cells (3), and antigen-specific helper and suppressor factors (4). Due to the limited availability of these factors, knowledge of their structure and function lags far behind that of immunoglobulin, the major B-cell product. Typically, the T-cell products are derived from bulk cultures ofnormal lymphocytes stimulated with mitogens such as phytohemagglutinin (PHA) or concanavalin A (Con A). Although this approach has yielded biologically active factors, the presence of multiple lymphokines as well as the mitogens themselves have hindered large-scale preparation of homogeneous material. In mice, the propagation of T-cell clones in vitro has overcome some ofthese problems (5). In man, however, such clones have been difficult to maintain and have not yet proved to be reliable sources of lymphokines.The use of T-T hybridomas produced by the fusion of an activated T cell with a malignant T-cell line offers the theoretical advantages ofa single cell source offactor, rapid and continuous growth in the absence of exogenous growth promoters, and an unlimited supply ofproduct free ofmitogen. This technique has been used successfu...