Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates ‘migration inhibition of neural crest cells.’ Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration–response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-β and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay.     

Rearrangement and expression of immunoglobulin genes and expression of Tac antigen in hairy cell leukemia.

The origin and exact stage of differentiation of the neoplastic cells that comprise hairy cell leukemia have remained uncertain. As Ig heavy and light chain genes must both undergo a DNA rearrangement during B-cell development but rarely do so within other hematopoietic lineages, we examined these genes in this leukemia. The neoplastic cells of all eight cases demonstrated rearranged heavy and light chain genes and, in two cases examined, contained the corresponding mRNA for heavy and light chain Ig. Consistent with this B-cell genotype, all cases displayed cell surface HLA-DR and B-cell-associated antigens. Unexpectedly, all cases demonstrated cell surface Tac antigen, which previously had been restricted predominantly to select T-cell malignancies and activated T cells. Prior studies suggested that the anti-Tac monoclonal antibody recognized a peptide associated with the binding of interleukin 2 (T-cell growth factor) in such T cells. Immunoprecipitation with anti-Tac and NaDodSO4/polyacrylamide gel electrophoresis revealed an antigen on leukemic hairy cells with a Mr of 53,000-57,000, identical in size to the receptor on activated T cells. This apparent biphenotypic status might reflect a transformation-associated expression of the Tac antigen in this leukemia. Alternatively, hairy cell leukemia may be a malignancy of a unique stage of normal B-cell differentiation in which the Tac antigen is expressed.     

The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.

Late-phase human T-cell lymphotropic virus I-associated adult T-cell leukemia cells express IL-2 receptors (IL-2R) but no longer produce IL-2. We have reported that the IL-2-independent adult T-cell leukemia line HuT-102 secretes a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and lymphokine-activated killer cell activity. Stimulation of proliferation of the cytokine-dependent human T-cell line Kit-225 mediated by HuT-102-conditioned medium or by 3200-fold-purified IL-T was not blocked by the addition of antibodies against IL-2 or IL-2R alpha subunit. However, IL-T-mediated stimulation of this human T-cell line was inhibited by addition of Mik-beta 1, an antibody that binds specifically to IL-2R beta subunit. In addition, the activation of large granular lymphocytes to lymphokine-activated killer cells mediated by IL-T-containing conditioned medium was not blocked by antibodies directed toward IL-2 or IL-2 alpha but was inhibited by an antibody to IL-2R beta, suggesting the requirement of this receptor subunit for IL-T action. This conclusion was confirmed using an IL-3-dependent murine myeloid precursor cell line, 32D, that expresses IL-2R alpha and IL-2R gamma, but not IL-2R beta. Neither IL-2 nor IL-T stimulated 32D cell proliferation. However, after transfection with the gene encoding human IL-2R beta, 32D beta cells proliferated on addition of either cytokine. The IL-T-mediated stimulation of 32D beta proliferation was inhibited by an anti-IL-2R beta antibody but not by an anti-IL-2 antibody. Thus, the IL-T-mediated stimulation of T-cell and lymphokine-activated killer cell activation requires the expression of the IL-2R beta subunit.     

Blockade of the interleukin-2 receptor by anti-Tac antibody inhibits the generation of antigen-nonspecific suppressor T cells in vitro.

The role of interleukin 2 (IL-2) in the activation of suppressor T cells was investigated by using the monoclonal antibody anti-Tac, which blocks the binding of IL-2 to the 55-kDa peptide of the high-affinity IL-2 receptor. Anti-Tac was added to an antigen-nonspecific suppressor system in which Con A-induced suppressor T cells were generated during a preculture period, and their effects on immunoglobulin production were assessed in second, indicator cultures containing pokeweed mitogen and peripheral blood mononuclear cells. Anti-Tac added during the preculture period inhibited Con A-induced suppressor T-cell generation. Cells activated by a short (2-day) preculture period to become effectors of suppression were primarily of the Tac-positive, T8 (CD8)-positive phenotype. Tac-positive, T8-negative T cells might also contribute to the suppressor activity. Our studies indicate that anti-Tac, by producing a functional blockade of human high-affinity IL-2 receptors, inhibits the generation of antigen-nonspecific suppressor T cells.     

In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation

A novel nonmyeloablative conditioning regimen was investigated in 44 patients with hematologic malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. Recipient conditioning consisted of CAMPATH-1H, 20 mg/day on days -8 to -4; fludarabine, 30 mg/m(2) on days -7 to -3; and melphalan, 140 mg/m(2) on day -2. Thirty-six recipients received unmanipulated granculocyte colony-stimulating factor-mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. GVHD prophylaxis was with cyclosporine A alone for 38 patients and cyclosporine A plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite polymerase chain reaction indicate that 18 of 31 patients studied were full-donor chimeras while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range 3 to 29 months), 33 patients remain alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and 4 of them subsequently died. Four patients died of regimen-related complications. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of nonrelapse mortality was 11%. Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD.