7-Hydroxydehydroepiandrosterone--a natural antiglucocorticoid and a candidate for steroid replacement therapy?

7-Hydroxylated metabolites of dehydroepiandrosterone (DHEA) are believed to be responsible for at least some immunomodulatory and antiglucocorticoid effects of DHEA and hence are considered candidates for hormone replacement therapy. Our experiments in vitro brought the evidence that 3beta, 7beta-dihydroxy-5-androsten-3-one (7beta-OH-DHEA), but not DHEA and its 7alpha-hydroxyisomer, could counteract the immunosuppressive effect of dexamethasone on the formation of plaques in culture of murine spleen lymphocytes. In another experiment, DHEA and after a 3-weeks pause 3beta-hydroxy-5-androstene-7,17-dione (7-oxo-DHEA) were applied transdermally to 6 male volunteers on 5 consecutive days. Blood levels of DHEA, its 7-hydroxylated metabolites, and in the first case also dehydroepiandrosterone sulphate (DHEAS), were measured before, during and one day after the end of treatment. Application of DHEA increased significantly not only DHEA and DHEAS, but also its both 7-hydroxyisomers. Application of 7-oxo-DHEA also led to a significant increase of both 7-hydroxyisomers of DHEA, with 7beta-OH-DHEA being the preferred metabolite the concentration of which was increased more than three times.

7Beta-OH-DHEA counteracts dexamethasone induced suppression of primary immune response in murine spleenocytes.

The effect of dexamethasone and of three potential antiglucocorticoids, namely dehydroepiandrosterone (DHEA) and its 7alpha-and 7beta-hydroxylated metabolites, on primary immune response has been studied by measuring the number of plaque forming cells (NPFC) and their viability in a cell culture of murine spleenocytes. As expected, dexamethasone suppressed considerably the NPFC as well as their viability. Surprisingly, DHEA as well as its 7alpha-hydroxylated metabolite decreased significantly the NPFC, while the effect of 7beta-hydroxy-DHEA was different: at low doses it decreased the NPFC, but this effect was less pronounced at higher concentrations. In addition, 7beta-hydroxy-DHEA was able to counteract the effect of dexamethasone on the NPFC. None of the natural steroids affected the cell viability.

Effect of protein kinase inhibitors on primary antibody induction in tissue cultures.

The effects of protein-kinase-inhibitors (PKIs) on protein kinase C (PKCs) i.e., staurosporin, calphostin C, H-7, H-8, H-9, on phosphatidyl inositol 3-proteinkinase (PI3-K) i.e., wortmannin, and on protein tyrosine kinase (PTKs) i.e., genistein, herbimycin A, sanguinarin, lavendustin A and B were tested on the induction phase of the primary Ab-response in vitro. The inhibitory action of PKIs was the highest with herbimycin A, sanguinarin, H-9 and wortmannin. Although wortmannin inhibits the function of T-lymphocytes (Taub et al., 1997, Shi et al., 1997), we believe that this communication is the first report of PKIs immunosuppressive action on the inductive steps of Ab-formation.

Cellular and molecular basis of immunodeficiencies: their consequences for the development and induction of the immune response.

This review lists primary immunodeficiencies which essentially involve mutations in genes coding for functionally important molecules, membrane antigens (e.g., MHC), chains of lymphokine receptors, protein kinases of the signal cascade, transcription factors, and important regulators of cellular metabolism. Mutations and subsequent immunodeficiencies occur as early as during embryogenesis (lymphopoiesis-I) as well as during induction of the immune response by antigen (ligand) binding to cell receptors, TCR and BCR (immunopoiesis-II). Immunodeficiencies are classified according the developmental stages in which they occur most markedly, even in clinical terms. Some early mutations are immediately lethal, some express themselves by blocking embryonic lymphopoiesis, while other mutations do not become demonstrable until after cell stimulation by antigens (see the Tables).

The genetic basis of primary, predominantly specific immunodeficiencies.

The presented review lists primary immunodeficiencies which essentially involve a mutation in genes coding for functionally important molecules, membrane antigens (e.g., MHC), chains of lymphokine receptors, protein kinases of the signal cascade, transcription factors, and some important regulators of cellular metabolism. Mutations are expressed as early as during embryogenesis (lymphopoiesis-I) as well as during induction of the immune response by antigen ligand binding to cell receptors, TCR, BCR (immunopoiesis-II). Immunodeficiencies are classified by the stage of development (I) or immune response induction (II) in which they occur most markedly, even in clinical terms. It has been pointed out that the same autoactivation stimuli and mechanisms, allowing differentiation-maturation of cells during embryogenesis (action of stem cell factor (SFC), IL-3, IL-7, and activation cascade), serve even later as a functional prerequisite for an adaptive immune response to antigen. As a result, this attempt to classify primary immunodeficiencies by differentiation periods (when they become evident most markedly in terms of their function) has an inherent logical limitation. Some early mutations turn immediately lethal, some express themselves by blocking embryonic lymphopoiesis while other mutations do not become demonstrable until after cell stimulation by antigens. This explains why the developmental differentiation scheme is bound to turn, in the future, into an immunodeficiency classification by localization of gene mutations and their incidence in time, e.g., increased mutation incidence during proliferation following cell stimulation by antigen stimuli.

Development of immune responses in early pig ontogeny.

Low amounts of immunoglobulins, produced without any known cause of stimulation, can be detected in sera and cells of fetal and colostrum deprived newborn pigs. These immunoglobulins are believed to represent the preimmune antibody repertoire on the basis of their polyspecificity and reactivity against self antigens. In vitro activation of liver and spleen cells with various polyclonal B cell activators (PBA) results in pronounced immunoglobulins synthesis as measured in the culture media by enzyme-linked immunosorbent assay. Intrauterine injection of fetal and germfree pigs with PBA led to increased IgM, IgG and IgA levels in sera. Specific responses during fetal development were studied after intrauterine immunization. Antibodies to the heapten and its carrier flagellin, could be detected 7 days after the immunization of 55-day-old fetuses. Fetal and colostrum germfree pigs may be useful experimental models in which developmental immunity can be studied in the absence of maternal antibodies and environmental antigens.

The secondary antibody response induced in tissue cultures.

The conditions for induction of memory cells (B-MC) and evocation of the secondary antibody (Ab) response in tissue cultures (TC) were estimated. (1) In vivo primed B-MC cells were isolated 6-150 d after priming and stimulated in TC with different doses of sheep red blood cell (SRBC) antigen. The Ab response has a strict time and dose dependence: only small doses (10(5)) evoke a secondary response, high doses (10(8), 10(9)) a state of immediate tolerance. (2) Antigen added to TC directly with B-MC rescued their Ab production for a long period. Addition of the antigen 1 or 2 d after setting the TC, follows the Ab-response decay, comparable with virgin cells (B-ICC). (3) Primed B-MC stimulated in TC responded preferentially with an IgM secondary response; the same cell suspension adoptively transferred into isologous recipients switched into IgG cells. (4) Virgin, immunocompetent, B-ICC were primarily stimulated in TC with a small dose of antigen (10(5) SRBC); after 7 d of cultivation the cells were transferred into isologous recipients, SCID mice and into TC. In all cases, the secondary response of IgM was determined, 10 times higher than in the primary controls.

Effects of essential fatty acids deficiency in milk diets on the development of germ-free and conventional rats.

Conditions allowing physiological development were determined in infant rats delivered by Caesarian section on the 22nd day of gestation and fed, from birth onwards, on various diets by stomach tube. Two new diets were prepared--K 50 A (11.7% fat, 7.7% protein) and LNaH (7.6% protein, 12% fat). The sole difference between the two diets is in the essential fatty acid (EFA) content, which is 42.6% in diet K 50 A, but only 0.7% in diet LNaH. Animals reared on the LNaH diet displayed slower growth and delayed development of the adenohypophysis and the thymus. Degenerative changes (intracytoplasmic vacuolation) were found in the somatotropin-producing cells of the adenohypophysis. The serum prolactin concentration of animals fed on the LNaH diet was lower than in the suckled controls and in animals given the K 50 A diet. The damaging effect of the LNaH diet was particularly marked on the first five days after birth, indicating that this is a critical period in the development of the newborn rat. The histological structure of the adenohypophysis and thymus of animals reared on the K 50 A diet was the same as in the suckled controls.

Failure to detect the presence of pluripotential haemopoietic stem cells in the mouse brain.

Single cell suspensions prepared from adult mouse brains were tested for the presence of pluripotential haemopoietic stem cells (colony-forming units, CFU) by transfer into an irradiated recipient and enumeration of the CFU in the recipient's spleen. In contrast to the findings of others (Bartlett, 1982), we did not detect CFU after injection of brain cell suspensions, although they were detectable after inoculation with bone marrow cells. The number of CFU in recipients after transfer of increasing numbers of brain cells was the same as that detected in the irradiated controls which had not received any transferred cells. Finally, cells from the brain, in contrast to bone marrow cells, were not able to protect recipient animals from the effects of lethal irradiation.

The use of gnotobiological models for the studies of immune mechanisms.

Germfree (GF) animals are widely used for discrimination of natural mechanisms (genetically determined) from adaptive mechanisms of immunity induced by external antigens. GF rats fed at least for first 5 days after birth with artificial diet LNa, deficient in unsaturated fatty acids, were in contrast to those fed with K 50 diet deeply suppressed in humoral and cellular immune reactivities during their life. GF piglets, deprived from maternal colostrum (CF) as a source of passively transferred maternal antibody, immunized with preformed Ag/Ab complexes (HSA anti HSA) responded according to the composition of complexes (Ab excess, equivalence, Ag excess) by low or high dose tolerance or by secondary response. Similar pictures of response were obtained in groups of presensitized GF-CF piglets by immunization with different doses of antigen. It is concluded that 3 different types of response are the result of Ag/Ab complexes acting or formed on the lymphocyte membrane and exerting stimulatory or down-regulatory signals.

Regulation of the immune response by ergot alkaloids.

We tested the effect of 33 ergot alkaloids (natural and semisynthetic) on the induction of antibody formation in isolated spleen cells by antigen in tissue cultures. The results are quantitatively expressed as the number of antibody-forming cells. The group of natural ergot alkaloids (ergopeptines) exerted an effective inhibitory action on the antibody response. Compounds derived from lysergic acid are completely inactive in this respect. A new, very active semisynthetic drug (Table 1, compounds 2) derived from inactive lysergol was described. The compounds interacting with alpha-adrenergic-receptors on lymphocytes (methoxamine, phentolamine, prazosin act synergistically with the inhibitory effect of ergopeptine alkaloids.

Long-term effect on the immune status of defined diets during the early postnatal period in the rat.

Germ-free and conventional infant rats delivered by hysterectomy were reared artificially, using one of two diets--diet LNa (with a high saturated fatty acid content) and diet K 50 (with a high unsaturated fatty acid content). Animals reared for a short time (up to 5 days) after birth on the LNa diet had a low antibody (PFC = plaque-forming lymphocyte) response and low resistance to Yoshida's sarcoma. Rats reared on the K 50 diet had the same response as the suckled controls. The first 5 days after birth were found to be the decisive period for long-term consequences of nutrition.