Dehydroepiandosterone induction of increased resistance to vancomycin in Staphylococcus aureus clinical isolates (MSSA, MRSA).

AIMS: To investigate whether dehydroepiandosterone (DHEA), an androgen present throughout life, alters the response of Staphylococcus aureus clinical isolates to vancomycin. METHODS AND RESULTS: DHEA in physiologically relevant concentrations (0.1, 0.5, 1.0 and 5.0 micromol l(-1)) was tested for its effect on methicillin-sensitive S. aureus (MSSA, n = 53) and methicillin-resistant S. aureus (MRSA, n = 73) response to vancomycin using standard protocols. Mutant selection was determined by serial transfer of selected isolates (n = 5). DHEA-mediated at least a fourfold increase in vancomycin MIC for 42% of MSSA and 21% of MRSA. For five of the isolates (0.1 and 0.5 micromol l(-1) DHEA) the MIC was increased to levels (8 microg ml(-1)) defined as vancomycin-intermediate resistance. CONCLUSION: Resistance was detected only in the presence of DHEA, and was not related to altered generation time, indicating induction of phenotypic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings require further investigation to determine what role DHEA plays in clinical vancomycin treatment failure that has been reported in the absence of vancomycin genotypic resistance or heteroresistance.

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development.

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.

Effect of insulin on microbial growth.

The ability of insulin to affect the growth kinetics of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa was measured. For all organisms, insulin, in the absence of a metabolizable sugar source, i.e., glucose or starch in Mueller-Hinton medium, had no effect on generation time as compared with a homologous control. Response to insulin, in the form of increased or decreased generation times, for both Gram-positive and Gram-negative bacteria, was dependent on the concentration of insulin, the concentration of glucose present, and the initial concentration of bacteria exposed to the glucose and insulin.

Sex-related immune changes in young mice.

Here we describe changes in selected immune parameters related to age and sex in young mice. We focused on the T cell compartment and studied thymuses and spleens from mice 3 to 9 weeks of age in order to bracket the time period around murine puberty. With regard to distribution of immune cells, no significant sex-related changes were seen in thymocyte expression of CD3, CD4, CD8, or CD4/CD8 or splenocyte expression of CD3, CD4, CD8, or CD45R/B220, a pan B cell marker. For splenocytes, significantly more cells were positive for CD3 in older (6-9 week old) compared with younger (3-4 week old) mice. Splenocyte and thymocyte cell proliferation as measured by DNA synthesis in response to in vitro mitogens was compared for cells from male and female mice over the ages studied. Thymocyte proliferation was not related to age or sex of the mice. For splenocytes of the youngest mice (3 weeks old), the response to a cell surface-receptor-independent mitogenic combination of phorbol ester and ionomycin induced a significantly greater response in cells from female mice compared with male mice. This trend was reversed for mice of 4-6 weeks of age, where the response by splenocytes from males was significantly greater than that by cells from females. For mice 7-8 weeks of age, splenocytes from female mice responded significantly less to stimulation by antibody to CD3, a component of the T-cell receptor. Our results demonstrate that depending on the assays employed, sexual dimorphism in the immune system may be demonstrated prior to puberty.

Androgens accelerate thymocyte apoptosis.

Mechanisms of androgen-induced thymic involution are largely undefined. We have found that significant decreases in thymic size occur 2-4 h after a dose of testosterone is administered to castrated male mice. This rapid rate of change suggests a role for androgen-induced apoptosis in modulating the size and composition of the thymus. Using thymic organ cultures to define these effects of androgens, we found that dihydrotestosterone treatment of thymus tissues from females or from castrated males results in enhancement of thymocyte apoptosis. Intact (androgen-replete) or testicular feminization, Tfm/Y (androgen-resistant) mice failed to show apoptotic change with androgen treatment, although the apoptotic response to glucocorticoids was present, suggesting a requirement for a functional androgen receptor. Acceleration of thymocyte apoptosis by androgens may mediate processes of thymocyte selection, with the potential to impart gender-specific characteristics on the peripheral T cell repertoire.

Androgens alter B cell development in normal male mice.

Castration of normal male mice leads to splenic enlargement and expansion of the B cell population. Since the spleen does not express receptors for androgens, these changes are most likely mediated by effects of androgens on other target organs. Two potential sites of androgen-mediated effects on B cells are evaluated in these studies: thymus and bone marrow. We first confirmed other findings indicating that castration of normal male mice results in expansion in the numbers of bone marrow B cells and then extended these observations by showing that these changes were reversible following androgen replacement. B cell expansion in castrate marrow and spleen was not altered by prior thymectomy, suggesting that thymic androgen receptors are not involved in the observed effects. Androgen receptors were found to be present in both immature B cells and marrow stromal cells by immunoblotting and ligand binding assays. The results suggest a direct modulatory role for androgens on B cells within the bone marrow compartment.

Immunochemical and flow cytometric analysis of androgen receptor expression in thymocytes.

A variety of evidence suggests that the cells of the immune system are targets for the actions of gonadal steroids. Experiments in both normal animals and in autoimmune disease models have established that androgens exert immunomodulatory effects at the level of the thymus. We have attempted to define precisely the potential target cells for androgen action in the thymus using recently developed antibodies to the androgen receptor. We report here that these antibodies reveal AR expression in all classes of thymocytes defined by surface markers CD4 and CD8. The highest levels of AR expression were observed in the CD4-CD8+ and CD4-CD8- subsets that include the most immature cells. These experiments establish that thymocytes are potential targets for direct actions of androgens. The data further suggest AR expression in thymocytes may be developmentally regulated in these cells, and that androgen effects early in the process of thymocyte selection may contribute to the sexual dimorphism of immune responsiveness.

Castration alters peripheral immune function in normal male mice.

While it is generally recognized that females show enhanced cell-mediated and antibody responses to antigenic stimulation, the physiological basis for this observed sexual dimorphism of the immune response is not well understood. We report here studies on the effects of androgen deficiency on the peripheral immune system. Intact male mice were compared to animals castrated 3-4 months previously. Phenotypic characterization of thymocyte and lymphocyte subpopulations was carried out using dual-colour flow cytometry. In vitro production by spleen cells of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma), and levels of total immunoglobulin and autoreactive antibodies was measured by specific immunoassays. In addition to thymic hypertrophy, castrated animals showed significant splenic enlargement, which was largely owing to expansion of the B-cell population. The castrated spleens contained relatively fewer mature T cells than intact controls (P < or = 0.001), but culture supernatants from these spleen cells contained higher levels of IL-2 and IFN-gamma than control cultures (P < 0.04). Levels of in vitro antibody synthesis (IgM, IgG, IgA) were not higher in castrated animals compared to controls, but the castrate spleen cell cultures showed increased levels of production of two autoreactive antibodies, anti-IgG (rheumatoid factor) and anti-thyroglobulin. These data suggest that androgen deprivation results in a relative decrease in the number of mature peripheral T cells, but those which reach the spleen have functional characteristics suggestive of enhanced activation. Dysregulation in the B-cell compartment may be the result of altered effects of T-cell-mediated control.

Induction of immature thymocyte proliferation after castration of normal male mice.

The physiological basis and immunological significance of thymic enlargement in castrate male animals is not known. We used normal male C57 Bl/6 mice to examine the contribution of in situ thymocyte proliferation to castration-induced enlargement of the thymus. Animals castrated at 8-10 weeks of age were compared to normal intact males. Thymocytes were examined 4-120 days after castration using flow cytometry to determine DNA content and thus the number of cells in active phases of the cell cycle. These properties were examined in unseparated thymocytes and in phenotypic subpopulations defined by expression of CD3, CD4, and CD8. For thymocytes obtained from intact control glands, a mean of 11.0 +/- 1.0% were in active phases of the cell cycle. The percentage of cycling thymocytes was increased to a mean of 22.5 +/- 1.9% in the week after castration (P < 0.001). This change occurred in the absence of significant thymic enlargement. At 8-10 days after castration, thymic weight increased abruptly to a new steady state which was double that of intact controls (78.0 +/- 4.1 vs. 39.1 +/- 2.6 mg; P < 0.001). In these enlarged glands, only 9.9 +/- 0.8% of cells were cycling, which was not significantly different than controls (P > 0.3). Proliferating cells identified in fixed thymus tissue sections after in vivo administration of bromodeoxyuridine were located in the subcapsular cortex and medulla. Analyses of thymocyte subpopulations indicated that most cycling cells had immature phenotypes (CD4+CD8+, CD4-CD8+, and CD3lo or CD3-). Castrate glands studied in the steady state period 8-120 days after surgery contained significantly fewer CD3+ cells than intact controls (P < or = 0.045). The findings suggest an intrathymic role for androgens in affecting generation of the mature T cell repertoire.

Prolactin receptors are found on heterogeneous subpopulations of rat splenocytes.

An increasingly large body of evidence implicates PRL as an immunoregulatory molecule. While most of the data relate to PRL levels and immunocompetence in vivo, we have shown that PRL is mitogenic for splenocytes from ovariectomized rats and rats in certain other hormonal states. This finding suggests that these lymphocytes express PRL receptors. Here, we wished to determine whether all or only a subset of splenocytes were PRL receptor positive. By using polyclonal as well as monoclonal antibodies to PRL receptor, we determined that as many as 20% of the primary splenocytes expressed PRL receptors. In a culture of Nb2 cells, a PRL receptor-positive lymphoid line, as many as 70% were PRL receptor positive. Dual labeling for lymphoid-specific antigen surface markers and PRL receptor indicated that about one third of the PRL receptor-positive splenocytes were kappa-light chain-positive B-cells, while the others stained with antibodies to T-cell markers, CD4 or CD8. These data confirm that lymphocytes express PRL receptors and show for the first time that PRL receptor-positive lymphocytes are a heterogenous subset of total primary splenocytes. These cells may be the target for PRL-mediated immunoregulation.

Prolactin-induced mitogenesis of lymphocytes from ovariectomized rats.

PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.

Detection and quantitation of interleukin-2 from individual cells.

In this report we present the use of cell blotting for the detection of interleukin-2 (IL-2)-producing lymphocytes. This is a rapid and sensitive immunochemical method analogous to Western blotting of proteins. When combined with image analysis one can determine the percentages of IL-2 positive cells as well as quantitate the amount of IL-2 surrounding each cell. When bovine lymph node cells (bLNC) were stimulated with the combination of concanavalin A (ConA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h, 46.4 +/- 0.6% stained positive for IL-2 and, on average, each cell produced 0.92 +/- 0.6 pg of IL-2 in 24 h. Phytohemagglutinin (PHA) and TPA-stimulated human peripheral blood mononuclear cells (hPBMC) produced approximately the same amount. 0.86 +/- 0.4 pg of IL-2 per cell in 24 h; 45.6 +/- 3.6% stained positive for IL-2.