Identification of kappa- and delta-opioid receptor transcripts in immune cells.

To investigate the role of opioids as direct modulators of the immune response, we have searched for expression of the recently cloned delta, mu and kappa opioid receptors in immune cells. We have devised a reverse transcriptase-polymerase chain reaction strategy which specifically detects a region spanning putative transmembrane regions 2 to 7 for each transcript in both human and mouse immune cells. In human peripheral blood lymphocyte and monocyte preparations, delta was undetectable while the kappa transcript was present.

Derivatives of a novel cyclopeptolide. 2. Synthesis, activity against multidrug resistance in CHO and KB cells in vitro, and structure-activity relationships.

A series of derivatives of the novel cyclopeptolide 1 was prepared, and their ability to chemosensitize multi drug resistant CHO and KB cells in vitro was evaluated. In contrast to the parent compound, several of the derivatives were found to be highly active. In particular, conversion of the R-lactic acid residue of 1 into its S-isomer via lactone ring cleavage and recyclization with inversion resulted in a marked enhancement of activity.

SDZ 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the P-glycoprotein-mediated tumour cell multidrug resistance.

SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal).

In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.

The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier. In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines. In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice).

Overcoming multidrug resistance in Chinese hamster ovary cells in vitro by cyclosporin A (Sandimmune) and non-immunosuppressive derivatives.

Cyclosporin A (Sandimmune) increased the in vitro susceptibility of 'parental' and 'multidrug-resistant' (MDR) chinese hamster ovary (CHO) cell lines to three anti-tumour drugs: colchicine, daunomycin, and vincristine. Several immunosuppressive or non-immunosuppressive derivatives of cyclosporin (Cs) were compared for their ability to sensitise both parental and MDR cells to chemotherapeutic agents. Although 5-10-fold increases of sensitivity to anti-tumour drugs could be obtained for cells of the parental line with several Cs-derivatives, the largest 'gains' of sensitivity (chemosensitisation) were obtained for the cells of the MDR line and with only some of the Cs derivatives.

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses.

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion.

The C57BL/6 nude, beige mouse: a model of combined T cell and NK effector cell immunodeficiency.

This article describes the construction and establishment of a double congenic nude, beige C57BL/6 (B6 nu, bg) mouse strain. The mice do not show higher fragility than C57BL/6 nude mice and the double congenic strain can be maintained under conventional mouse housing conditions. Although the B6 nu, bg display a very low natural killer activity which cannot be enhanced by an interferon inducer (poly(I-C], they lack responsiveness to a T cell mitogen (concanavalin A); and they also show extremely low responsiveness to a B cell mitogen (0128: B12 Escherichia coli lipopolysaccharide) probably as a result of combined effects of the beige and nude genes in the C57BL/6 genetic context.

Proliferation of interleukin 2 dependent cytotoxic T cell line cells. Different protein kinase C activators achieve the same maximal promotion, but with distinct dose-response profiles.

Protein kinase C activators of the teleocidin family are claimed to be able to replace interleukin 2 for inducing short-term proliferation of the interleukin 2 dependent murine cytotoxic T cell line. While teleocidin B4 was found to be much more active than 12-O-tetradecanoyl-phorbol-13-acetate, a related molecule showing a substitution of a single OH by OCH3, olivoretin A, lacked detectable activity in this assay. However, the maximal proliferation achievable by protein kinase C activators did not exceed one third of the one given by recombinant interleukin 2.

Protein kinase C activators of the teleocidin family decrease the IgE-binding capacity of rat basophilic leukemia cells.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and the teleocidins (TCDs) had similar inhibitory effects on IgE binding onto the membrane of rat basophilic leukemia (RBL)-2H3 cells. The level of expression of the functional IgE Fc receptor (Fc epsilon R), as measured by CELISA, was decreased up to a maximum of 60% within 5 min-1 h of treatment. This inhibition was obtained at concentrations of 0.

An enzyme-linked lectin-binding assay on cells (CELLBA) for the comparison of lectin receptor expression on cell surfaces.

Lectins can be used to specifically detect some cell surface glycans. Their expression on different cells or on cells of a given lineage throughout differentiation or following treatment with drugs can be compared using lectins labelled with radioactive, fluorescent or enzymatic probes. We describe a new method which, by analogy with CELISA (ELISA on cells), is called CELLBA (or ELLBA on cells) for cellular, enzyme-linked lectin-binding assay.

Detection of mouse IgE by CELISA.

The detection and quantitation of mouse IgE is usually impaired by the difficulty to obtain reliable antibody reagents which are fully specific for the epsilon chain - and reactive enough - to be used in an enzyme-linked immunosorbent assay (ELISA). An ELISA on cells (CELISA) was developed for the detection of mouse IgE, using rat basophilic leukemia (RBL) cells. It is based on the high affinity of the receptors for the Fc of IgE (Fc epsilon R) displayed on the surface of the RBL cells.

Effect of glycoprotein-processing inhibitors on the mouse IgE binding capacity of rat basophilic leukemia cells.

The basophile surface high affinity receptors for IgE (Fc epsilon R) are heavily glycosylated glycoproteins like the IgE Fc itself. Their functional expression in their physiological environment can be studied with the help of a recently developed CELISA (ELISA on cell) methodology. The relevance of the IgE and Fc epsilon R glycans for their interaction in situ has been probed using inhibitors of discrete trimming steps of N-linked carbohydrate processing.

A comparison of five different methods for the detection of TNP specific mouse IgE: ELISA, ELISA on cells, rosetting, granule enzyme release assay and passive cutaneous anaphylaxis.

Using the same anti-TNP hybridoma supernatant pool as IgE antibody source (2 micrograms/ml), several methods were compared for their sensitivity in IgE detection and convenience for screening purposes. Using rat basophilic leukemia (RBL) cells as specific receptor cells, one out of two rosetting methods with TNP-sheep red blood cells allowed the detection of 0.5 ng IgE/ml (50 pg/assay) while the other was much less sensitive (60 ng/ml).

Membrane alkaline phosphatase activity: an enzymatic marker of B-cell activation.

The expression of membrane alkaline phosphatase (mAlPase) activity was studied on viable cells from mouse lymphoid organs. The low mAlPase activity level of ex vivo mouse spleen cells was markedly increased by in vitro culture in the presence of the direct B-cell mitogen lipopolysaccharide (LPS). This increase occurred nearly simultaneously with increased uptake of 3H-thymidine and an increased percentage of blasts in the culture.

The C57B1/6 nu/nu, lpr/lpr mouse. III. Autoimmunity status.

C57B1/6 mice homozygous at the lpr (lymphoproliferation) locus display evident lymphadenopathy (in our B6 colony, primordially cervical lymph node enlargement) and autoimmunity (various autoantibodies). Four groups of mice corresponding to the diverse combinations of the lpr gene and the nu (nude, athymic) gene on the B6 genetic background have been compared for signs of lymphadenopathy. It occurred in all tested B6 +/+, lpr/lpr mice and none of the other groups (B6 nu/nu, +/+; B6 nu/nu, lpr/lpr and B6 +/+, +/+).