Francis Loor

RANKING OF P-GLYCOPROTEIN SUBSTRATES AND INHIBITORS BY A CALCEIN-AM FLUOROMETRY SCREENING ASSAY

In order to compare the capacities of a variety of compounds to interfere with P-glycoprotein (Pgp) function, a novel assay was set up to work on a large screening scale. The model assay measures the capacity of parental sensitive (Par) and multidrug-resistant (MDR) cells to efflux a small fixed amount of acetoxymethyl calcein (calcein-AM) after their pretreatment with concentration ranges of known Pgp modulators. This microplate cytometry-based assay was performed with two different pairs of cell lines, the human lymphocytic leukemia CEM cells and the murine monocytic leukemia P388 cells. For a given Pgp-expressing MDR cell line, a Pgp modulator EC50 was defined as the concentration required to restore half of the calcein retention shown by similarly treated Par cells. With both MDR-P388 and MDR-CEM cells, EC50 comparisons ranked five reference Pgp modulators as follows: SDZ 280-446 > SDZ PSC 833 > cyclosporin A > verapamil > vinblastine. Further use of the MDR-CEM cells could rank 15 Pgp modulators for their capacity to interfere with calcein-AM efflux as follows: SDZ 280-446 1.9 x > SDZ PSC 833 8.3 x > cyclosporin A 3.8 x > amiodarone 1.1 x > quinacrine 1.6 x > verapamil 1.4 x > quinidine 1.1 x > vinblastine 11 x > vincristine 2 x > chloroquine > beta-lumicolchicine > or = gamma-lumicolchicine > or = colchicine > etoposide > or = doxorubicin. This calcein-AM assay should open the way for ranking large numbers of novel structures for their potential Pgp modulator properties, particularly for an efficient screening of Pgp function antagonists, but it does not allow defining whether their inhibition may be competitive or not.

The abamectin derivative ivermectin is a potent P-glycoprotein inhibitor

Among the compounds endowed with the capacity to reverse the P-glycoprotein (Pgp)-mediated multidrug resistance of cancer cells, a powerful agent was found to be the cyclosporin D derivative SDZ PSC 833. After in vivo treatment with SDZ PSC 833, mice showed a decreased tolerability to cyclosporin A (CsA), but also to ivermectin, a widely used polycyclic lactone pesticide of Streptomyces avermitills origin. The sequels were suggestive of CsA- or ivermectin-induced central nervous system dysfunction; they were interpreted as caused by the neutralization of the Pgp at the blood-brain barrier level, implying that CsA and ivermectin were Pgp substrates. CsA was already known to display both Pgp substrate and Pgp inhibitor properties. It now appears that ivermectin may also inhibit Pgp function. When compared in short-term assays for Pgp function inhibition, which measure the restoration of the retention of two Pgp probes in multidrug-resistant (MDR) cells to their parental (Par) cell levels, ivermectin appeared only a few fold weaker than SDZ PSC 833 in the case of murine monocytic leukemia MDR-P388 cells and nearly as active as SDZ PSC 833 in the case of human lymphocytic leukemia MDR-CEM cells. Therefore, like CsA or FK-506, ivermectin may be both a substrate and an inhibitor of Pgp.

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.

SDZ PSC 833 and SDZ 280-446 are the most active of various resistance-modifying agents in restoring rhodamine-123 retention within multidrug resistant P388 cells

Multidrug resistance (MDR) of tumor cells may result from overexpression of P-glycoprotein (Pgp) but may be down-modulated by resistance-modifying agents (RMAs). The cyclosporin SDZ PSC 833 and the cyclopeptolide SDZ 280–446 were found to be the strongest RMAs known to date for restoring the sensitivity of MDR cells to anti-cancer drugs, as well as for restoring their retention of daunomycin, a fluorescent anthracycline. Using rhodamine-123 (Rhod-123), another fluorescent probe of Pgp function which also differentiates sensitive and MDR cells, several RMAs were compared for their capacity to inhibit Pgp function. At variance with the data obtained with the daunomycin probe, a series of RMAs did not detectably restore Rhod-123 retention by the MDR cells. With the remaining RMAs, achieving the same levels of Rhod-123 retention required 3 times lower RMA concentrations when the RMA was added to the MDR cells for both the initial uptake and the efflux of Rhod-123 rather than for its uptake only. Nevertheless, the data emphasized the large superiority of SDZ PSC 833 and SDZ 280–446 over all other RMAs.

An indirect asymmetrical sandwich ELISA using anti-allotype antibodies for the specific and quantitative measurement of mouse IgG2a of Igh-1b allotype

We recently described an indirect double sandwich ELISA (Klein-Schneegans et al., J. Immunol. Methods (1989) 119, 117) which permits the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses with one major exception: IgG2a of the b allotype (Igh-1b in mouse strains such as C57BL/6) could not be reliably quantitated even by a very specific and sensitive asymmetrical sandwich ELISA (using two different anti-IgG2a isotype antibodies for capture and for detection). We now describe a similar method based on the use of two different anti-IgG2a allotype antibodies for the capture and detection of IgG2a in the serum of Igh-1b mouse strains.

Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL6 mice and their congenics at the lpr (lymphoproliferation) locus

The serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA were determined in mice of C57BL/6 background, from weaning to one year of age, by quantitative isotype-specific, indirect double sandwich enzyme-linked immunosorbent assays (ELISAs). Only limited data could be obtained for the IgG2a isotype in the present study. The mean serum Ig levels found for 6-month-old B6 mice were 0.22 mg/ml for IgM, 0.28 mg/ml for IgG1, 1.22 mg/ml for IgG2b, 0.18 mg/ml for IgG3, 0.075 mg/ml for IgA and about 0.7 mg/ml for IgG2a. In comparison with mice of the wild strain, C57BL/6 mice homozygous at the lpr (lymphoproliferation) locus showed very high increases in serum Ig levels when older than 20 weeks. With 6-month-old B6 lpr mice, increases in concentration were found for all tested heavy chain isotypes: 6 to 6.5-fold for IgA (0.45 mg/ml) and IgG1 (1.82 mg/ml), 9-fold for IgG3 (1.6 mg/ml), 11 to 11.5-fold for IgM (2.44 mg/ml) and IgG2b (13.8 mg/ml) and about 8-fold for IgG2a (5.5 mg/ml). Therefore homozygosity at the lpr locus provides the conditions for generalized, poly-isotypic rather than isotype-specific restricted Ig enhancement. This observation may be more compatible with hyperinducibility of all B-cell subclasses than with excessive production of T-cell-derived factors whose activity would be expected to be restricted to some T-dependent subclasses, and at least to affect IgM-committed B cells to a lesser extent than other B-cell classes.

Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives

FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux. The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype. Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones. FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity. One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.

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.

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).

A quick procedure for identifying doubly homozygous immunodeficient scid beige mice

This article describes a rapid and reliable procedure for identifying mice which are doubly homozygous at the scid and beige (bg) loci starting from CB17 scid (no T and B cells) and B6 bg mice (no NK activity). The [scid, bg] mice are directly identified in the F2 progeny by monitoring (1) the hypogammaglobulinemia for the scid gene and (2) the prolonged bleeding associated with the bg gene. Like CB17 scid mice, the [scid, bg] mice show a high susceptibility to infections and die early in life unless they are protected against potential infections. This is achieved by a graft of splenocytes plus bone marrow cells from (B6 bg x CB17 scid) F1 mice. These [scid, bg] mice combine the bg and scid immunodeficiencies and should be better recipients for xenografts than classical scid mice.