The Hypothalamic-Pituitary Axis and Autoantibody Related Disorders.

This review summarized different studies reporting the presence of autoantibodies reacting against cells of the pituitary (APAs) and/or hypothalamus (AHAs). Both APAs and AHAs have been revealed through immunofluorescence using different kinds of substrates. Autoantibodies against gonadotropic cells were mainly found in patients affected by cryptorchidism and hypogonadotropic hypogonadism while those against prolactin cells were found in different kinds of patients, the majority without pituitary abnormalities. APAs to growth hormone (GH) cells have been associated with GH deficiency while those against the adrenocorticotropic cells have distinguished central Cushing's disease patients at risk of incomplete cure after surgical adenoma removal. AHAs to vasopressin cells have identified patients at risk of developing diabetes insipidus. APAs have been also found together with AHAs in patients affected by idiopathic hypopituitarism, but both were also present in different kinds of patients without abnormalities of the hypothalamic-pituitary axis. Despite some data being promising, the clinical use of pituitary and hypothalamus autoantibodies is still limited by the low diagnostic sensitivity, irreproducibility of the results, and the absence of autoantigen/s able to discriminate the autoimmune reaction involving the pituitary or the hypothalamus from the other autoimmune states.

Silicone Oil Microdroplets Can Induce Antibody Responses Against Recombinant Murine Growth Hormone in Mice.

Therapeutic protein products can cause adverse immune responses in patients. The presence of subvisible particles is a potential contributing factor to the immunogenicity of parenterally administered therapeutic protein formulations. Silicone oil microdroplets, which derive from silicone oil used as a lubricating coating on barrels of prefilled glass syringes, are often found in formulations. In this study, we investigated the potential of silicone oil microdroplets to act as adjuvants to induce an immune response in mice against a recombinant murine protein. Antibody responses in mice to subcutaneous injections of formulations of recombinant murine growth hormone (rmGH) that contained silicone oil microdroplets were measured and compared to responses to oil-free rmGH formulations. When rmGH formulations containing silicone oil microdroplets were administered once every other week, anti-rmGH antibodies were not detected. In contrast, mice exhibited a small IgG1 response against rmGH when silicone oil-containing rmGH formulations were administered daily, and an anti-rmGH IgM response was observed at later time points. Our findings showed that silicone oil microdroplets can act as an adjuvant to promote a break in immunological tolerance and induce antibody responses against a recombinant self-protein.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand-receptor fusions.

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GH-LRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GH-LRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans.

Identification of growth-hormone- and prolactin-containing neurons within the avian brain.

Prolactin (PRL)- and growth-hormone (GH)-containing perikarya and fibers independent of the anterior pituitary gland have been reported to exist in the central nervous system of several mammalian species. The specific distributions of PRL- or GH-like neurons in the avian forebrain and midbrain, however, have not been reported. The objective of the study was to identify GH- and PRL-containing neurons in the hypothalamus and a few extrahypothalamic areas of two avian species. Brain and peripheral blood samples were collected from laying and broody turkey hens and ring doves. Broody turkey hens and doves had significantly higher plasma PRL concentrations compared with laying hens. Coronal brain sections were prepared and immunostained using anti-turkey GH and anti-chicken synthetic PRL antibodies. In turkey hens, the most dense GH-immunoreactive (ir) perikarya and fibers were found in hippocampus (Hp), periventricular hypothalamic nucleus, paraventricular nucleus, inferior hypothalamic nucleus, infundibular hypothalamic nucleus, medial and lateral septal area, and external zone of the median eminence (ME). In the ring dove, a similar pattern of distribution of GH-ir neurons was noticed at the brain sites listed above except that GH-ir fibers and granules were found only in the internal zone of ME and not in the external zone. In both turkeys and doves, the most immunoreactive PRL-ir perikarya and fibers were found in the medial and lateral septal area, Hp (turkey only), and bed nucleus of the stria terminalis pars magnocellularis. There were no apparent differences in the staining pattern of GH- or PRL-ir neurons between the laying and broody states in either species. However, the presence of GH-ir- and PRL-ir perikarya and fibers in several hypothalamic nuclei indicates that GH and PRL may influence parental behavior, food intake, autonomic nervous system function, and/or reproduction.

Cellular signaling mechanisms for stimulation of growth hormone secretion and growth hormone primary transcripts by immunosuppressant agents, FK506 and cyclosporin A, in cultured rat pituitary cells.

Although an immunosuppressant, FK506, has been known to stimulate growth hormone (GH) release from rat somatotropes, the cellular signaling mechanism is unknown. In the present study, intracellular signaling pathways were investigated for FK506- and cyclosporin A (CsA)-induced GH release in cultured rat anterior pituitary cells. Northern and Western blot analysis revealed that the FK506-binding protein (FKBP12) and the CsA-binding protein (cyclophilin A) exist at the mRNA and protein level in the rat anterior pituitary tissue. FK506 and CsA increased GH release in a dose-dependent manner and inhibited calcineurin (CaN) activity in the cultured pituitary cells. The third immunosuppressant, rapamycin (RP), inhibited the FK506-induced GH release, although RP alone had no effect. Protein kinase A (PKA) inhibitors, H-89 and HA-1004 and EGTA blocked FK506- and CsA-induced GH release. TGF-beta did not alter basal GH release, but inhibited FK506-induced GH release. GH primary transcripts were increased by FK506, and the effects were blocked by H-89 and HA-1004. These results suggest that the immunosuppressants, FK506 and CsA, stimulate GH release by inhibiting CaN activity which results in the activation of the PKA system in the rat somatotropes. TGF-beta receptors might be involved in FK506-induced GH release as a separate pathway. FK506 also stimulates GH primary transcripts via a PKA-dependent mechanism in a manner similar to its effects on GH release.

Growth hormone improves the resistance of thermally injured mice infected with herpes simplex virus type 1.

BACKGROUND: Growth hormone (GH) has been shown to promote wound healing and to improve protein metabolism in burned patients. Through immunomodulation, GH has also protected rats infected with Salmonella typhimurium and mice infected with Escherichia coli. In spite of advances in the management of patient care for those with thermal injuries, high mortality rates of burned patients as a result of infections are of special concern. An improvement in the resistance of burned patients to certain infections will make the beneficial role of GH very clear. In this study, therefore, the immunomodulating effects of recombinant human GH (rhGH) in thermally injured mice exposed to opportunistic herpesvirus infections were investigated. METHODS: (1) Burned mice, exposed to herpes simplex virus type 1 (HSV-1), were treated subcutaneously with rhGH (4 mg/kg) and observed for 21 days to determine the protective antiviral effect of rhGH. (2) Because of reports describing a lack of interferon-gamma (IFN-gamma) responsiveness in burned mice, the IFN-gamma-producing ability of the splenic mononuclear cells (SMNC) from burned mice treated with rhGH was examined. (3) Because the generation of burn-associated suppressor macrophages that can inhibit the IFN-gamma production by SMNC has been previously described, the suppressor cell activities of macrophages from burned mice treated with rhGH were examined. RESULTS: After exposure to lethal amounts of HSV-1, mice treated with rhGH displayed a reduced mortality rate compared with control mice treated with saline. SMNC from burned mice treated with rhGH produced IFN-gamma, whereas this cytokine was not produced by SMNC from burned mice treated with saline. Also, an inhibition of the generation of burn-associated suppressor macrophages was displayed in burned mice treated with rhGH. CONCLUSION: Exogenous administration of rhGH caused an improvement in the resistance of burned mice to HSV-1 infection. In burned mice treated with rhGH, the impaired IFN-gamma responsiveness was restored and the generation of burn-associated suppressor macrophages was inhibited. IFN-gamma, a typical antiviral cytokine induced by rhGH through the regulation of the suppressor macrophage generation, may therefore play a role in the protection of burned mice infected with a lethal amount of HSV-1.

Growth hormone-releasing factor regulation by somatostatin, growth hormone and insulin-like growth factor I in fetal rat hypothalamic-brain stem cell cocultures.

Information about growth hormone-releasing factor (GRF) regulation by somatostatin, GH and IGF-I is scarce and controversial. This could be due to the in vivo interactions among these signals and the lack of models for individualizing the action of one of them from the others upon GRF regulation. The aim of the present work was to study GRF regulation by these signals, using primary fetal rat hypothalamic-brain stem cell cocultures. Coculturing of these two cytotypes increases hypothalamic immunoreactive rat GRF (IR-rGRF) content in cells by 45% and in media by 36%. The effect of SS on GRF in cocultures was examined by using a multiple approach: (1) depleting endogenous SS by adding 1 mM cysteamine (CSH); (2) blocking endogenous SS by incubation with SS antiserum, and (3) incubating with synthetic SS14 at different concentrations and exposure periods. 1 mM CSH depleted IR-SS content (pg/plate, mean +/- SE) in cells (CSH-treated: 68 +/- 8 vs. control: 322 +/- 10, p < 0.01) and media (CSH-treated: 211 +/- 15 vs. control: 880 +/- 70; p < 0.01). In the CSH-induced SS-depleted cultures, a slight reduction in the IR-rGRF content in cells was observed (CSH-treated: 93.5 +/- 4.5 vs. control: 111 +/- 6; p < 0.05), with no effect on media content. When SS antiserum was added to plates, there was a slight reduction in the IR-rGRF content in cells and media, but it was not significantly different from the controls. However, SS14 (10(-10)-10(-8) M) could not modify IR-rGRF content in media and cells. The GH effect on IR-rGRF was studied in the absence of CSH and in CSH-induced SS-depleted cultures. GH (5 microM, 24 h) decreased (52%) the IR-rGRF content in media (GH-treated: 28.7 +/- 4.6 vs. control: 60.2 +/- 7; p < 0.01) without causing changes in cell content. In SS-depleted cultures, the inhibitory action of GH on media IR-rGRF was greater (62% decrease) (GH-treated: not detected, control 56 +/- 10; p < 0.01) and also affected IR-rGRF cell content (GH-treated: 64.3 +/- 7.3 vs. control: 160 +/- 9.6; p < 0.01). In the same experiments, GH increased IR-SS content in cells (GH-treated: 31.8 +/- 4.6 vs. control 20.9 +/- 0.5; p < 0.01) and in media (GH-treated: 413 +/- 7 vs. control: 286 +/- 9; p < 0.01). 1 mM CSH again depleted IR-SS content and abolished the GH stimulatory effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrical stimulation of hypothalamic periventricular nucleus is followed by a large rebound secretion of growth hormone in unanesthetized rats.

The effect of electrical stimulation of the hypothalamic periventricular nucleus (PVN) on plasma GH profile was studied in unanesthetized female Wistar rats. A bipolar concentric electrode was implanted into the PVN, hypothalamic ventromedial nucleus (VMH), or intervening area between the PVN and VMH. Serial blood specimens were collected from an indwelling right atrial cannula. Plasma GH levels were reduced significantly during electrical stimulation of PVN, and a large rise of plasma GH levels followed after cessation of stimulation. An identical plasma GH profile was observed in response to the repeated stimulation. This rebound secretion of GH was completely inhibited by the administration of rat GRF antiserum. The effect of electrical stimulation of VMH on plasma GH levels was similar to that of PVN stimulation. However, the stimulation of hypothalamic area intervening between PVN and VMH was not followed by a surge of GH secretion. Since a continuous exposure of somatotrophs to GRF even in a concurrent presence of somatostatin (SS) is known to induce attenuation of the GH response to GRF through receptor effect, the results suggest that the release of endogenous GRF is augmented following the cessation of electrical stimulation of neurons providing hypophysiotropic SS.

Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys.

The relative concentrations of antibodies produced in monkeys against three forms of human growth hormone (hGH) were determined using an antigen-specific avidin/biotin ELISA assay. Monkeys were treated in two separate 90-day studies with recombinant methionyl-hGH (met-hGH) and pituitary-derived hGH (pit-hGH) (Study 1) and recombinant natural sequence hGH (Study 2). The lowest dose was equal to the expected therapeutic dose of 0.1 IU/kg. Sixty-nine percent of monkeys treated with pit-hGH and 81% of those treated with met-hGH developed detectable anti-hGH responses. The magnitudes of the responses exhibited wide animal to animal variability, were not markedly related to dose or sex, and were lower than levels obtained in monkeys immunized with hGH in Freund's adjuvant. In contrast, the incidence of antibody responses in monkeys treated with natural sequence hGH was lower (23% in one experiment and 5% in a replicate experiment) and took longer to develop. Antibody concentrations were lower, on average, than in those animals treated with met- or pit-hGH. These results are in accord with those observed clinically, thus supporting the use of the monkey model to predict the relative immunogenicity of some proteins in humans.

Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H]estradiol in GRF neurons.

Sex steroids have been shown to influence the secretion of GH. There appears to be no good evidence of the effect of estradiol on the anterior pituitary, while the central site of estradiol action on the regulation of GH secretion is not known. The present investigation was carried out to determine whether some of the GH-releasing factor (GRF) neurons and somatostatin (SRIF) neurons in the hypothalamus and GH cells in the pituitary contain estradiol receptors. Colocalization of [3H]estradiol and antibodies to GRF or SRIF in brain and antibodies to GH in pituitary was studied to show interrelationships between estrogen target cells and peptidergic cells. Eight female Sprague-Dawley rats were ovariectomized, each rat was treated with colchicine, and 24-48 h later the animals were given an iv injection of [2,4,6,7,16,17-3H]estradiol (SA, 166 Ci/mM) at a dose of 0.5 micrograms/100 g BW. One hour after the injection, the rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The hypothalami from the perfused rats and the pituitaries from unperfused rats were frozen in isopentane precooled in liquid nitrogen (-190 C) and processed for autoradiography. The brain autoradiograms were immunostained for GRF, SRIF, and tyrosine hydroxylase [TH; an enzyme for the synthesis of dopamine (DA)], and the pituitary autoradiograms were immunostained for GH by the avidin-biotin peroxidase method. The majority of GRF-containing neurons were found in the arcuate nucleus, with some scattered cells in the lateral region of the ventromedial nucleus and the basal lateral hypothalamus. In the central portion of the arcuate nucleus, 20-30% of GRF-containing neurons showed nuclear concentration of [3H]estradiol. In the anterior portion of the hypothalamus, 10-15% of immunoreactive GRF-containing neurons were labeled with [3H]estradiol. In the lateral basal hypothalamus and the lateral region to the ventromedial nucleus, a few GRF neurons showed nuclear concentration of radioactivity. In contrast, a few SRIF cells in hypothalamic periventricular nucleus showed nuclear labeling with [3H]estradiol. Dual immunostaining with GRF and TH antibodies revealed that the estradiol-labeled GRF neurons did not contain TH immunoreactivity. In addition, 80-90% of GH cells in the anterior pituitary showed nuclear concentration of [3H]estradiol. The present studies demonstrate for the first time that certain populations of GRF neurons are targets for estradiol and indicate that estradiol acts directly on certain hypothalamic GRF neurons. The results suggest that estradiol may have a role in the regulation of GH secretion by modulating GRF release and acting directly on the somatotrophs.

Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay.

Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (less than 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was less than 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 X 10(-7) M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 X 10(-7) M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.

The effects of lateral hypothalamic-medial forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats: evidence for a dual regulatory mechanism.

The purpose of this investigation was to examine the role of somatostatin (SRIF) and electrical stimulation of the lateral hypothalamic-medial forebrain bundle (LH-MFB) on dynamics of pulsatile GH secretion in freely behaving, chronically cannulated male rats with implanted brain electrodes. The effects of administration of anti-SRIF serum (AS-SRIF) on pulsatile GH and on TSH and PRL secretion was also studied. The results are: 1) circulating AS-SRIF increases trough levels of GH in freely behaving rats but has no significant effect on the amplitude of GH secretory bursts or mean GH levels; 2) LH-MFB excitation can stimulate GH release if delivered when circulating GH levels are low; 3) LH-MFB stimulation inhibits secretion of GH if given at the time of a spontaneous GH burst; 4) stimulation-induced GH inhibition is prevented by pretreatment with AS-SRIF, suggesting that this response is mediated by endogenous SRIF; and 5) AS-SRIF increases TSH secretion but has no effect on PRL. These results provide evidence to support the hypothesis that pituitary GH secretion is regulated by a combination of excitatory and inhibitory influences, the inhibitory component of which is mediated by SRIF.

Immunocytochemical localization of the posterior lobe hormones and of their carrier proteins.

Serial sections of vertebrate hypothalami were stained with the immunocytochemical peroxidase-antiperoxidase method. In addition to the single staining method, our double staining method was used, which enabled us to visualize two tissue antigens in single tissue sections. In both staining methods, differentially adsorbed antineurohypophysial hormone sera, anti-somatostatin serum and anti-bovine neurophysin sera were used. The results confirm the one hormone, one neuron hypothesis.

Immunochemical staining of the rat adenohypophysis in organ culture.

Rat anterior pituitaries were cytologically studied following cultivation in organ culture, with and without the addition of hypothalamic and cortical extracts. Although five distinct cell types could be identified with classical stains in the uncultivated glands, the peroxidase-labeled antibody technique (using antibodies against STH, LTH, FSH, LH and TSH) showed that not all of the immune-specific cell types were being identified with the classical stains. This discrepancy was magnified following culture as chromophilic cells seen with classic stains decreased in number with an increase in culture time. The peroxidase technique, however, revealed that all cells remained constant in type and number regardless of time in culture. While the addition of either hypothalamic or cortical extract to the culture medium produced cytological alterations demonstrated by the classical dyes, the antibody technique showed no such alterations. Such a comparison of staining techniques emphasizes the hazards of relying solely on histological procedures to reveal the hormonal activity of the pituitary gland.