Cyclosporin G and metabolite binding to cyclophilin and a 50-kDa binding protein related to in vitro immunosuppression.

Seven purified metabolites of cyclosporin G (CsG) were studied for binding to cyclophilin and a 50-kDa binding protein (50-kDa BP). The ratios of the metabolite dissociation constants with respect to CsG were compared with in vitro immunosuppression by using the primary mixed lymphocyte suppression assay. The immunosuppressive potency ratio of the parent compounds, both cyclosporin A (CsA) and CsG, compared favorably with the drug dissociation constants for cyclophilin and the 50-kDa BP.

Development of a radioreceptor assay to measure glucocorticoids.

We have developed a radioreceptor assay to measure glucocorticoids. The assay employs the partially purified 95-kDa receptor isolated from human liver and purified by size fractionation on high-performance liquid chromatography (HPLC). In the assay [3H]prednisolone competes with steroids (endogenous and exogenous) for binding to the receptor.

cis-urocanic acid down-regulates the induction of adenosine 3',5'-cyclic monophosphate by either trans-urocanic acid or histamine in human dermal fibroblasts in vitro.

It has been demonstrated that UVB radiation (290-320 nm) suppresses mammalian cell-mediated immunity by effecting the trans to cis isomerization of urocanic acid (UCA) in the stratum corneum, the uppermost layer of the skin. Trans-urocanic acid has been shown to be the photoreceptor for UVB-induced immune suppression and the cis-isomer has been demonstrated to be immunosuppressive. Little is known, however, about how the isomerization of UCA may affect the proximal or distal cells of the skin or the immune system.

Correlation of cyclosporine and metabolite binding to cyclophilin and a 50 kDa binding protein with in vitro immunosuppression: a preliminary report.

Seven purified cyclosporine (CsA) metabolites were analyzed for binding to cyclophilin and to a 50 kDa protein purified from a JURKAT cell line. In addition, the potency of the seven metabolites, relative to CsA, was obtained using a primary mixed lymphocyte culture (MLC) suppression assay. CsA, M1, 17, and 21 were found to be immunosuppressive in the concentration range used (0-500 ng/mL).

Purification and characterization of cyclosporine and FK-506 binding proteins from a human T-helper cell line.

Cytosolic proteins that specifically bind cyclosporine A and FK-506 were isolated and purified from the JURKAT human T-helper cell line. These binding proteins were purified by affinity, molecular weight exclusion and weak cation exchange column chromatography. Radiolabeled cyclosporine A specifically bound to a approximately 17 kDa molecule which is cyclophilin and also bound to a approximately 50 kDa protein(s).

A preliminary study to evaluate an in vitro assay for determining patient whole blood immunosuppressive cyclosporine A and metabolite activity: comparison with cytosolic binding assays using cyclophilin or a 50-kilodalton binding protein, and the Abbott TDx cyclosporine A parent, and parent and metabolites assays.

Thirty-five allograft recipients undergoing cyclosporine A (CsA) therapy were randomly selected to evaluate a "novel" in vitro assay that determines CsA and metabolite immunosuppressive activity in whole blood. The assay uses a third party mixed lymphocyte culture (MLC) system to which patient whole blood extracts containing CsA and metabolites are added. The ability of the extracted CsA and metabolites to inhibit proliferation in this system is proportional to the immune suppressive activity in whole blood.

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors.

Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues.

The potency of 3 reduced peptide bond analogues of bombesin (BN) was investigated using small cell lung cancer (SCLC) cell lines. (Psi13,14, Leu14)BN, (Psi9,10, Leu14)BN and (Psi12,13, Leu14)BN inhibited specific binding of 125I-GRP with IC50 values of 15, 90, and 600 nM. (Psi13,14, Leu14)BN and (Psi9,10, Leu14)BN did not elevate cytosolic Ca2+ levels but antagonized the increase in cytosolic Ca2+ caused by BN.

Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth.

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines.

Synthetic C-terminal peptide of IL-1 functions as a binding domain as well as an antagonist for the IL-1 receptor.

Fragments of IL-1 beta were chemically synthesized and tested for biological activity as well as binding of radiolabelled peptides to the IL-1 receptor. A peptide from the extreme C-terminal region of IL-1 beta was found to antagonize intact, native IL-1 beta in the thymocyte bioassay. In addition, this C-terminal region peptide, when radiolabelled, can function as a ligand for the IL-1 receptor on murine cell lines and effectively compete with intact radiolabelled recombinant IL-1 beta.

Mast cells induced in vitro by interleukin 3 from native murine thymus cells.

To shed further light on the induction and characterization of thymus-derived mast cells, we cultured a variety of cell populations from murine thymus tissues (Balb/c) in the presence or absence of interleukin 3 (IL-3). The whole cell population and the non-adherent T cell-depleted population developed mast cells. The morphological studies revealed granulated cells; the granules were stained with toluidine blue, alcian blue (pH 3.

Correlation of cell-surface phenotype with the establishment of interleukin 3-dependent cell lines from wild-mouse murine leukemia virus-induced neoplasms.

The wild mouse ecotropic virus, Cas-Br-M murine leukemia virus, induces myeloid and erythroid leukemias as well as T-cell and B-cell lymphomas in NFS mice. The ability to establish long-term cell lines from these tumors in the presence or absence of the T-cell-derived lymphokine interleukin 3 (IL-3) was examined. IL-3-dependent cell lines were readily obtained from the majority of the myeloid or erythroid leukemias and immunoblastic lymphomas.

Biologic effects of prolonged melphalan treatment of murine long-term bone marrow cultures and interleukin 3-dependent hematopoietic progenitor cell lines.

The mechanism of alkylating agent-induced leukemia is unknown. For the determination of whether chronic alkylating agent treatment of hematopoietic stem cells in vitro was detectably leukemogenic, murine long-term bone marrow cultures (LTBMC) and clonal interleukin 3 (IL-3)-dependent multipotential hematopoietic progenitor cell lines [B6SUtA clone (cl) 27 and Ro cl 3-1] derived from LTBMC were chronically pulse treated in vitro with the alkylating agent melphalan [L-phenylalanine mustard (L-PAM)]. Weekly treatment of C3H/HeJ or CD-1 Swiss mouse LTBMC with 3 X 10(-6)M L-PAM significantly decreased cumulative production of nonadherent granulocytes and granulocyte-macrophage progenitor cells responsive to L-cell or WEH1-3 cell colony-stimulating factor compared to the production seen in untreated control cultures; it also significantly reduced the hematopoietic longevity (13 wk compared to greater than 20 wk for untreated control cultures).

Evidence for specific receptors for interleukin 3 on lymphokine-dependent cell lines established from long-term bone marrow cultures.

Utilizing 125I-labelled IL 3, an assay for specific IL 3 receptors on continuous cell lines was developed. The 32D-cl 23 and FDC-P1 cell lines examined were originally derived from long-term cultures of murine bone marrow supplemented with WEHI-3-conditioned media. Both of these cell lines have been shown to be absolutely dependent on IL 3, a component of WEHI-3-conditioned media, for growth in vitro.

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland.

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons).

Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity.

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.

Procedures for the purification of interleukin 3 to homogeneity.

A procedure is described for the routine purification of IL 3 to homogeneity from WEHI-3-conditioned media. The techniques employed include ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite, and G-75 Sephadex column chromatography. The last step in purification involves chromatography on C18 hydrophobic supports in RP-HPLC systems, which results in the coelution of a protein peak and IL 3 activity.