Effects of glucocorticoid and antiglucocorticoid hormones on leukocyte numbers and function.

Hormones were administered to mice in seven daily intraperitoneal injections of saline suspensions. Progesterone and cortexolone, which often fail to act as antiglucocorticoids in vivo, were found to have antiglucocorticoid effects on the immune system under these conditions. The effects seen were increases in numbers of lymphocytes, monocytes, neutrophils and total leukocytes in the blood, increases in the number of peritoneal exudate cells and splenic plaque-forming cells, and increased splenocyte responses to the mitogen phytohemagglutinin. Deoxycorticosterone, sometimes also considered to be an antiglucocorticoid, acted only as a glucocorticoid here. Both deoxycorticosterone and the glucocorticoid corticosterone had effects opposite to those produced by progesterone and cortexolone on these parameters.

Synthesis of 7 alpha- and beta-carboxymethyl derivatives of cortisol, corticosterone, deoxycorticosterone and cortisone. Immunogenic properties of cortisol, corticosterone and deoxycorticosterone derivatives.

7 alpha- and 7 beta-Carboxymethylderivatives of cortisol, corticosterone and deoxycorticosterone have been synthetized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. Highly specific antisera were obtained which may possibly be used for a direct radioimmunoassay of these steroids in human and rodent plasma. In the case of the derivatives of cortisol and corticosterone and stereoisomery of the coupling had an effect on the affinity and the specificity of the antisera. In all immunized rabbits the antisera obtained with the 7 alpha-derivative had a higher affinity and a narrower specificity than the antiserum obtained with the 7 beta-derivative.

Production of high affinity monoclonal antibodies to deoxycorticosterone.

Monoclonal antibodies to deoxycorticosterone were produced. Mice were immunised with deoxycorticosterone-3-mono-oxime-BSA conjugate and spleen cells were then hybridised with NS1/1Ag4-1 mouse myeloma cells using 1500 mol. wt polyethylene glycol. The hybrids were grown in RPMI 1640 medium containing HAT to facilitate selection of positive clones. The clones and subclones were screened by using deoxycorticosterone-3-mono-oxime-[125I]iodohistamine. Dextran-coated charcoal was used for separation of antibody bound and free fractions. Two independent clones producing antibody which specifically binds labelled deoxycorticosterone were obtained. Cells from the two best sub-clones were used to raise ascites fluid. Comparison of these antibodies with one of the best conventional antisera previously raised in rabbits showed that the affinity constants were almost comparable (0.49-1.4 X 10(10) l/mol). Cross-reactivity of monoclonal antibodies with cortisol, corticosterone, testosterone and pregnenolone was lower than for polyclonal antisera, but for progesterone the cross-reactivity was similar in both cases. The assay sensitivity obtained with ascites fluid was comparable to that of conventional antibody (2.5 pg/ml). The dilution of ascites fluid which produced 50% binding of the label was 1:4,000,000. These results confirm that it is possible to produce monoclonal antibodies to corticosteroids.

Production, characterization and clinical application of antisera to cortisol.

In the present paper the production of highly specific and sensitive antisera to cortisol is described. The antisera were generated in rabbits using both cortisol-3-monooxime and cortisol-3,20-dioxime derivatives. Bovine thyroglobulin was used as a carrier protein. Antibody characteristics were determined by radioimmunoassay procedure. Antibody titers observed after immunization with the monooxime conjugate were significantly higher (p less than 0.001) than those obtained with the dioxime conjugate. In addition, cross reactivity for various naturally occurring steroids was markedly lower and sensitivity was increased as compared to dioxime antisera (15 vs. 40-60 pg/tube). Accuracy and precision were calculated for the monooxime antiserum used in a RIA system for clinical application. A close correlation (r = 0,9802) was found between plasma cortisol values measured by radioimmunoassay and the competitive protein binding method. The concentrations obtained by the protein binding method were slightly but significantly higher than those measured by radioimmunoassay indicating higher specificity of the antibody used (p less than 0.005).

Influence of the adrenal cortex on allograft rejections in rats.

I. The bibliography about corticosteroid influence on immune response is briefly reviewed. Generally, it is admitted that corticosteroids are immunosuppressive when administered in large amounts. Divergent opinions are recalled. II. The aims of the experiments are summarized: A) Investigate on the influence of corticoids (given in just substitutive amounts) on skin allograft rejection. B) Investigate on possible interactions between adrenal cortex and thymus with this test. III. It was observed, that A) Adrenalectomy resulted in a significant delay of allograft rejection. B) This could be suppressed partially by administering aldosterone, corticosterone, and desoxycorticosterone and completely by administering at least two of these hormones. C) Cortisol had shown a minor inhibitory influence. D) There was no obvious difference between adrenalectomized and thymectomized rats and those adrenalectomized only. IV. These observations are replaced in the context of bibliography and their significance is discussed.

Compounds in the urinary pH 1 extract reacting with corticosteroid and tetrahydrocorticosteroid antisera. Separation of a nonpolar substance with affinity to a tetrahydroaldosterone antiserum.

In human urinary pH 1 extracts prepared for the aldosterone-18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. Aldosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made by immunological analysis of paper chromatogram eluats. Pregnancy, and administration of ACTH, dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.

Increased plasma 11-deoxycorticosterone during spironolactone medication.

The increase of plasma deoxycorticosterone (DOC) levels after administration of spironolactone is a real effect and not due to cross-interference of the drug or its metabolites with the DOC-assay. This is proved by in vitro and in vivo results. Of all the metabolites only canrenone interferes to some extent with DOC after liquid-liquid-extraction and paper chromatography. The antiserum, however, is so highly specific, that the final cross-interference of the total procedure amounts to less than 2% of the DOC plasma levels. Moreover, in patients with Addison's disease only a very small increase of DOC plasma levels can be observed after administration of spironolactone in contrast to normal subjects.

Radioiodinated derivatives for steroid radioimmunoassay. Application to the radioimmunoassay of cortisol.

Gamma-emitting steroid tracers for use in the radioimmunoassay of steroids have a number of advantages over the more common tritiated tracers. The steroid derivatives aldosterone3-(p-hydroxybenzoyl) hydrazone, aldosterone-3-(p-hydroxyphenylpropionyl)hydrazone, deoxycorticosterone-3-(p-hydroxyphenylpropionyl)hydrazone, and cortisol-3-(p-hydroxyphenylpropionyl)hydrazone were synthesized by a one-step procedure and iodinated ([125I]). To illustrate the usefulness of these derivatives, we describe the details of a cortisol radioimmunoassay. The use of the radioiodinated tracer appeared to increase the specificity of the antigen-antibody reaction when compared with [3H]cortisol. The methodology involved in the preparation of the steroid derivatives described above can be extended to other 3-oxo-4-ene-containing steroids, with the advantages of economy, simplicity, and versatility.

[Study of the radioimmunological and physicochemical properties of anti-aldosterone and anti-desoxycorticosterone antisera (author's transl)]. / Etude des propriétés radioimmunologiques et physicochimiques d'antisérums anti- aldostérone et anti-désoxycorticostérone

Study of the radioimmunological and physico-chemical properties of three anti-aldosterone antisera permitted practical conclusions to be drawn. By its high degree of specificity, anti-aldosterone-3-oxime-BSA is the most useful antiserum for the clinical assay of aldosterone. The principal advantage of this antiserum is that it allows both urinary and blood aldosterone radioimmunoassay without the necessity of including a chromatographic step. This work also includes the study of two anti-desoxy-cortico-sterone antisera.

Interference of spironolactone in 11-doexycorticosterone radioassays.

Uniformly elevated plasma 11-deoxycorticosterone (DOC) values are obtained in patients receiving spironolactone. This elevation is shown to be spurious, and is a result of cross-interference of spironolactine and its metabolite, canrenone, in both the DOC radioimmunoassay and competitive protein-binding radioassay.

Quantitation of deoxycorticosterone and its relationship to progesterone in the prepartum bovine.

A method and its validation is described for the radioimmunological measurement of deoxycorticosterone (DOC) in bovine serum. Levels of DOC and progesterone were determined in six pregnant heifers from one to three weeks before and during parturition. Levels of these steroids fluctuated widely from day to day and tended to be inversely related (r = -0.24). High levels of DOC in conjunction with low levels of progesterone at or near parturition are suggestive that DOC is involved in the parturition process.

Deoxycorticosterone excretion in normal, hypertensive and hypokalaemic subjects.

1. Radioimmunoassay has been used to detect and estimate the urinary excretion of deoxycorticosterone (DOC) in normal, hypertensive and hypokalaemic subjects. The range of excretions in ten healthy normal subjects was 41-232 pmol (13.7-76.7 ng) daily, with a mean of 124 pmol (41 ng). 2. In fourteen subjects with essential hypertension without metabolic disturbance the range found was 29-144 pmol (9.7-47.7 ng) daily, with a mean of 87 pmol (28.8 ng), which is not significantly different from that in normal subjects. 3. In twelve patients with Cushing's syndrome due to adrenal cortical hyperplasia the range found was 26-542 pmol (8.7-179 ng). Ten of these twelve patients had values within normal limits. 4. Of nine subjects showing hypokalaemia, eight had elevated excretion of deoxycorticosterone with values from 263 to 5515 pmol (87-1820 ng) daily. Seven of these were hypertensive and two were normotensive. The elevated excretion of deoxycorticosterone found in hypokalaemic subjects is thus not confined to those with hypertension. 5. No correlation has been found between excretion rates for aldosterone and deoxycorticosterone. Raised excretion of the latter provides an indicator of disturbed adrenal cortical metabolism.