Formation of hollow core granules by fluid bed in situ melt granulation: modelling and experiments.

Granules with a characteristic core-shell internal structure have been formed by in situ melt fluid-bed granulation, using d-mannitol primary solid particles and poly-ethylene glycol (PEG-6000) binder. The effect of binder particle size and binder/solids ratio on granule size distribution was systematically investigated. The mean granule size was found to be directly proportional to the binder particle size. The binder amount did not measurably affect the granule size, only the fraction of un-granulated fines. The microstructure of the granules was analysed by X-ray micro-tomography; the average shell thickness in the granules was found to depend on the binder/solids ratio, and the core volume was found to be directly proportional to the binder particle size. However, for binder particle size below a certain value the core-shell structure disappeared. A mathematical model based on a layering growth mechanism has been proposed and found to be consistent with experimental data. The proposed growth mechanism was confirmed by creating granules with bi-modal size distribution using a mixture of differently sized binder seeds.

Modulation of diabetes with gonadotropin-releasing hormone antagonists in the nonobese mouse model of autoimmune diabetes.

The nonobese mouse model of autoimmune diabetes (NOD mouse) exhibits a strain-dependent preponderance of disease in females. Castration of male NOD mice leads to an increased incidence of diabetes, suggesting that testosterone directly modulates the expression of diabetes in the NOD mouse. However, castration also modulates hypothalamic and pituitary hormone production via removal of the negative feedback effects of testosterone. One hypothalamic hormone with immunomodulatory properties whose expression is increased by castration is GnRH. To test whether the increased incidence of diabetes in castrated male NOD mice is related to an increase in GnRH activity, we treated castrated male NOD mice with Antide, a GnRH receptor antagonist, to determine the effect on the incidence and timing of onset of diabetes. The prevalence of diabetes at 40 wk of age in male NOD mice was 50% in sham-operated mice, compared with an 83% prevalence in castrated males. Antide administration prevented the increased incidence of diabetes in the castrated male mice. Antide reduced total serum IgG levels, IL-6 cytokine expression in cultured splenocytes, and the lymphocytic infiltration of islets. GnRH administration exerted reciprocal effects, leading to earlier timing of onset of diabetes and increases in serum total IgG levels. We conclude that GnRH modulates the expression of diabetes in the NOD mouse independently of gonadal steroids.

Immunomodulatory actions of gonadal steroids may be mediated by gonadotropin-releasing hormone.

Estrogens are considered to be immunostimulatory, whereas androgens are considered to be immunosuppressive. We hypothesized that the divergent actions of gonadal steroids on the immune system may be mediated indirectly, via their potent divergent feedback effects on the hypothalamic hormone GnRH, which is itself immunostimulatory. We used the GnRH-deficient HPG/Bm mouse in an effort to disentangle the effects of gonadal steroids from the effects of GnRH. We randomized GnRH-deficient mice and their GnRH-sufficient littermates to receive androgens, estrogens, or GnRH. We subsequently measured B and T cell proliferative responses to mitogen and serum IgG levels. We demonstrate that estrogens exert stimulatory effects on B cell proliferation and serum IgG levels in the presence of GnRH but not in the absence of GnRH. Testosterone exerts suppressive effects on B cell function in the presence of GnRH but not in its absence. Androgens and estrogens exerted divergent actions on T cell function irrespective of the presence and absence of GnRH, although responses were markedly attenuated in GnRH-deficient mice. Our data suggest that the immunostimulatory effects of estrogen and the immunosuppressive effects of androgens on B cell function may be mediated indirectly via GnRH.

Gender differences and hormonal modulation of G proteins Galpha(q/11) expression in lymphoid organs.

Androgens and estrogens exert potent divergent feedback effects on gonadotropin-releasing hormone (GnRH) production at the level of the hypothalamus and GnRH action at the level of the pituitary. Androgens exert generally suppressive effects on GnRH production and action, whereas rising levels of estradiol increase both GnRH release and action. In addition to its known endocrine actions, GnRH possesses immunomodulatory effects. We have previously demonstrated gender differences in immune responsiveness to GnRH that parallel gender differences in endocrine responsiveness: females appear to be more immunologically responsive to GnRH than males. GnRH exerts its actions via the stimulatory G protein Galpha(q) and Galpha(11) (referred to collectively as Galpha(q/11)) as well as via Galpha(s). We have recently demonstrated that the heightened immune responsiveness to GnRH in lupus-prone female mice correlated with increased expression of Galpha(q/11) in lymphoid cells from females compared to males. We hypothesize that the hormonal milieu of females may contribute to increased expression of stimulatory G proteins and to the heightened immune and endocrine responsiveness to GnRH. In this report, we document gender differences in expression of Galpha(q/11) protein in lymphoid organs in non-autoimmune DBA/2 mice. In an effort to address the mechanisms for the gender differences in G-protein expression, we used competitive reverse transcription PCR to quantitate mRNA for stimulatory G proteins in immune cells under various hormonal conditions. We quantitated the expression of Galpha(q/11) mRNA and protein under physiologic hormonal alterations, i.e. throughout the estrous cycle in female mice. We demonstrate that expression of Galpha(q/11) mRNA and protein in lymphoid organs is significantly increased on the afternoon of proestrus compared to metestrus. Additional studies demonstrate that exposure to GnRH or to estrogens significantly increases the expression of Galpha(q/11) mRNA in immune cells. These findings support an active role for hormonal modulation of G proteins in the gender differences in endocrinologic and immunologic responsiveness to GnRH.

Administration of antisense oligonucleotides to Galpha(Q/11) reduces the severity of murine lupus.

Our principle hypothesis is that the hypothalamic hormone, gonadotropin-releasing hormone (GnRH), is an immunostimulatory hormone and plays a pivotal role in the gender differences in immunity and/or autoimmunity. As a general rule, females display heightened immune responses and heightened endocrinological responsiveness to GnRH compared to males. We have previously demonstrated that GnRH receptor antagonists are effective in ameliorating murine lupus and that GnRH receptor agonists exacerbate murine lupus. GnRH exerts its actions via stimulatory G proteins, specifically via Galpha(s) and the homologous G proteins Galpha(q) and Galpha(11) (referred to together as Galpha(q/11)). We have previously demonstrated that females express higher levels of Galpha(q/11) mRNA and protein compared to males. We hypothesized that antisense inhibition of these specific G proteins would lead to a reduction in inflammatory cytokines and to an amelioration of disease in a mouse model of lupus. We randomized gonadectomized female (NZB x NZW) F1 hybrid mice to treatment with antisense oligonucleotides to Galpha(q/11) or to missense oligonucleotides. Administration of antisense oligonucleotides to Galpha(q/11) led to significant reductions in autoantibody levels, serum IgG levels, hematuria, and proteinuria compared to missense oligos. A trend toward prolonged survival was also noted. In vitro co-culture experiments demonstrated that antisense to Galpha(q/11) significantly inhibited IL-6 production compared to control.