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. The analysis of human cell lines revealed low but significant levels of delta opioid receptor transcripts in T, B or monocyte cell lines while the kappa transcript was found in B cell lines only. Investigation of murine cells showed the presence of transcript for the delta receptor in splenocytes and in some T and B cell lines. Unexpectedly, no expression of the mu receptor was detected. Sequence analysis of PCR products demonstrated nucleotide identity between immune and neuronal transcripts, indicating that they derive from the same genes. In conclusion, our results lead to the identification of kappa and delta opioid receptor transcripts in immune cells.

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. Some of these derivatives (e.g., 15a, SDZ 280.446) belong to the most potent resistance modulating compounds known so far.

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). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.

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). Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o. were otherwise ineffective doses of anticancer drugs given i.p. This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o. 4 h before a doxorubicin i.p. injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times. Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.

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. The MDR cells employed displayed the typical MDR phenotype. However, we found no correlation between the immunosuppressive activity of Cs derivatives and their capacity to reverse MDR and all four possible combinations of these two activities could indeed be shown among the tested Cs derivatives. This study demonstrates for the first time that some immunosuppressive Cs can be devoid of chemosensitising activity.

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.

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 assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.

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 0.1 microgram/ml for most TCDs, of 1 microgram/ml for TPA and of 20 micrograms/ml for one TCD (olivoretin A). These molecules also decreased the amount of cell-bound IgE detectable by CELISA on cells that had been coated with IgE prior to TCD treatment. When incubated with RBL-2H3 cells for 30 min-2 h, the TCDs and TPA stimulated serotonin release. Depending on their concentration, they had various effects on IgE-plus antigen-induced serotonin release. It is suggested that the down-regulation of IgE receptor expression by these tumor promoters is mediated through protein kinase C activation and phosphorylation of the Fc epsilon R.

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. It permits the comparison of the expression of specific glycans in a large number of different cell samples. As an example, it was able to detect alterations of cell surface glycan expression caused by inhibitors of N-linked oligosaccharide trimming.

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. Since the epsilon chain specific recognition is achieved by the biological receptor of IgE, the detection of cell-bound IgE does not need the use of epsilon chain specific antibodies. Instead, one can use any enzyme-coupled antibody capable to recognize the IgE through its light-chain epitopes. Interestingly, when the IgE bound to the RBL cells has a known specificity, it can be detected through its paratopes using the cognate antigen coupled to an enzyme.

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. The IgE produced by mouse hybridoma cells in the presence of deoxynojirimycin (dNM), a glucosidase I inhibitor, keeps intact both its antigen and Fc epsilon R-binding capacities. Rat basophilic leukemia (RBL) cells cultured in presence of dNM or of another glucosidase I inhibitor, castanospermine (Cs), show marked decreases in their capacity to bind mouse monoclonal IgE. In contrast, their culture in the presence of either deoxymannojirimycin (dMM) or swainsonine (Sw) either does not affect or even slightly increases the membranous expression of Fc epsilon R capable to bind mouse IgE.

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). More convenient for screening was an ELISA method performed on cells, in which the IgE bound to the RBL cell surface could be detected either by enzyme-anti-Ig or by enzyme-antigen conjugates with a similar, albeit low, sensitivity of 10 ng IgE/ml (500 pg/assay). Using the same antibody and cell sources, a very convenient and more sensitive method for screening purposes was found to be the measurement of antigen-induced basophil granule beta-N-acetylglucosaminidase release by IgE sensitized RBL cells: 0.5 ng IgE/ml and 50 pg/assay. For the same anti-TNP IgE source, this compares with a detection limit by ELISA of 0.2 ng IgE/ml (10 pg/assay) and by passive cutaneous anaphylaxis in rats of 2 ng IgE/ml (100 pg/assay).

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 T-cell-dependent B-cell pokeweed mitogen did not increase the mean level of mAlPase activity per cell, although there was an increase per culture. The T cell mitogen ConA did not cause an increase in mAlPase activity, although it was able to stimulate both cell proliferation and blast transformation. Several other mitogens and differentiating agents were tested, but did not detectably affect mAlPase expression. LPS high responder mouse strains C57BL/6 and CBA/J showed a higher LPS-induced mAlPase expression response to LPS than did LPS low responder strains BALB/c or CBA/N. These data suggest a preferential expression of mAlPase by stimulated cycling B cells. However, mAlPase expression appeared restricted to a subpopulation of cycling B cells and could not be elicited by every B-cell stimulus.

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 +/+, +/+). B cell hyperactivity/autoimmunity was also evaluated by serum antibody analyses: higher serum immunoglobulin levels, anti-nuclear antibodies, anti-native DNA, anti-single stranded DNA, rheumatoid-like factors (anti-rabbit IgG), and natural antibodies against dinitrophenol and trinitrophenol haptens and their non cross reactive carriers: bovine serum albumin, hen egg albumin and keyhole limpet haemocyanin. Interestingly the levels of serum immunoglobulins and of some specific antibodies were somewhat higher in B6 nu/nu, lpr/lpr than in 'normal' B6 nu/nu, +/+, though they remained much lower than in B6 +/+, lpr/lpr animals. This suggests that the lpr gene may express its influence on the level of B cell activity in the absence of T lineage cells that would have normally matured in a thymus and that this effect of the lpr gene does not require the massive proliferation of T lineage cells observed in B6 +/+, lpr/lpr mice.