Evaluation of a novel radioimmunoassay using125I-labelled human recombinant GAD65 for the determination of glutamic acid decarboxylase (GAD65) autoantibodies
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Lühder F, Woltanski K-P, Mauch L, Haubruck H, Kohnert K-D, Rjasanowski I, Michaelis D, Ziegler M. Detection of autoantibodies to the 65-kD isoform of glutamate decarboxylase by radioimmunoassay. Eur J Endocrinol 1994:130:575–80. ISSN 0804–4643 Autoantibodies (AAb) to glutamate decarboxylase (GAD) occur with a high prevalence in sera of newly diagnosed type I (insulin-dependent) diabetic patients. The aim of this study was to establish a GAD-AAb radioimmunoassay using 125 I-labelled GAD 65 and to evaluate this assay in a cross-sectional study with newly diagnosed type I diabetic patients (diabetes duration < 6 weeks). Furthermore, subjects at high risk of developing type I diabetes and individuals suffering from other autoimmune diseases were examined in this assay. For GAD-AAb detection, 125 I-labelled GAD 65 was incubated with 10 μl of human serum overnight on ice. Thirty of 51 (59%) type I diabetic patients but none of the 54 healthy blood donors tested were found to be positive. A displacement step using 100 000 g supernatant from rat brain containing or not containing GAD showed the specificity of the binding of 125 I-GAD 65 . Concerning the individuals at high risk of developing diabetes, 9/12 (75%) islet cell antibody (ICA)-positive non-diabetic and 4/34 (12%) ICA-negative subjects with metabolic abnormalities were GAD-AAb positive. These results show the association between type I (insulin-dependent) diabetes mellitus and the occurrence of GAD 65 -AAb, which possibly predicts a risk of developing the disease. F Lühder, Department of Immunochemistry, Institute of Diabetes "Gerhardt Katsch", Greifswalder Str. I la, D-17495 Karlsburg, Germany
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In 3 separate studies, each spanning 32–50 hr, serum and pituitary LH levels of male rats killed at 1½ to 6½ hr intervals were studied by radioimmunoassay. In 2 of these studies orchidectomized rats also were examined and in one of them (spanning 50 hr) ovariectomized rats were studied as well. In no case was evidence obtained for any rhythmic diurnal variation of either serum or pituitary LH levels. In the third study, on male rats only, serum and pituitary FSH were examined by 3 different radioimmunoassay systems. Serum corticosterone and pineal serotonin levels also were measured. The animals displayed pineal serotonin and serum corticosterone rhythms very similar to those other male rats have been reported to exhibit, but they showed no sign of any rhythmic variation in pituitary or serum FSH levels. These findings lead us to conclude that: 1) the rates of LH and FSH secretion remain fairly constant throughout the day and night in intact rats, even though such rats do display striking diurnal rhythms of pineal and adrenal activity; 2) LH secretion is also fairly stable in castrated rats of both sexes; 3) any rhythmic variations which may exist in the serum and pituitary hormone levels we studied are of such small amplitude that they were not detectable by the methods we used. (Endocrinology87: 798, 1970)
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SUMMARY A radioreceptor assay (RRA) for GH, using pregnant rabbit liver membranes, was used to evaluate the receptor‐hormone interaction of circulating (serum) GH in diabetics. Multiple morning blood samples were taken from 18 fasted insulin‐dependent diabetics on three occasions and assayed for GH by the RRA and radioimmunoassay (RIA). Samples from 12 normal subjects (GH secretion stimulated by exercise and hypoglycaemia) and 11 acromegalics were also studied. The RRA/RIA ratio was higher in samples from the acromegalics than in normal subjects (0.85 vs 0.65, P < 0.01) and only a few samples had RRA values greater than RIA. All the samples from 14 of the diabetics had lower RRA values than RIA, but 40% (39 of 96) of samples from the remaining four diabetics had RRA concentrations markedly in excess of the levels measured by RIA. Sephadex chromatography (G100) of serum samples revealed similar proportions of immunoreactive GH forms in the diabetics with high RRA values compared with the diabetics with low RRA values. These findings may indicate intermittent secretion of highly bioactive GH in some insulin‐dependent diabetics. Further studies on the biological properties of circulating GH are needed to clarify the role of GH secretion in the development of diabetic microvascular complications.
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The effects of thyroid hormone deprivation and restitution on growth hormone (GH) economy have been studied in the rat by means of a specific radioimmunoassay. The pituitary GH content and the plasma GH levels before and during stimulation with pentobarbital ("PB-test") were studied in male rats at different intervals after surgical thyroidectomy (T), and in T rats at different time intervals after the ip injection of 0.20, 1.75, and 5.0 mug thyroxine (T4) or 0.05, 0.10, 0.20 and 1.0 mug triiodothyronine (T3), all doses being referred to 100 g body wt. Pituitary GH content decreased very rapidly after T, a difference being shown at the end of the shortest time interval studied (24 h); 24 days after T, pituitary GH content was 0.3 percent or less of the pre-T level, the basal plasma GH was lower than in intact controls and an increase in plasma GH during PB-stimulation was no longer observed. When rats T for 30 days or longer were injected once with T4 or T3, pituitary GH content increased; basal plasma GH levels increased also and a positive response to PB was observed. An effect on pituitary GH content could be observed as soon as 2 h after the ip injection of 1.0 mug T3, or 6 h after 5.0 mug T4. The "latent period" was somewhat longer when lower doses of the hormones were used. Effects of a single 0.10 mug T3 dose could be detected within 12 h L-T3 appeared to be at least 9 times more potent in vivo tha T4, as assessed from the effect on pituitary GH. The mea-urement by RIA of changes in GH content of the rat pituitary may thus provide the most adequate parameter available at present (other than suppression of TRH-induced TSH release) for a biological effect in vivo of single doses of the thyroid hormones, a measurement clearly related to an important physiological role.
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In the present study the influence of dexamethasone treatment of rats on the basal values of thyrotrophin (TSH) and prolactin (PRL) and the response of both hormones to thyrotrophin releasing hormone (TRH) has been investigated. Male rats were given 100 μg of dexamethasone/rat for 8 days at the same time of day. Four hours after the last administration of dexamethasone 200 ng or 100 μg of TRH/rat was injected ip. Blood was collected 10 min later by decapitation. TSH and PRL were estimated by radioimmunoassay (RIA) using the NIAMD kits. The basal and TRH stimulated values of PRL in plasma were significantly lower in dexamethasone treated rats than in controls (P < 0.01). The basal TSH levels in the treated animals were also lowered (P < 0.05). After 200 ng TRH/rat the increase in TSH was not as high in both groups than after the administration of 100 μg/rat. There was no significant difference between the response of TSH to TRH in the dexamethasone treated and the control rats. The different effects of dexamethasone on PRL and TSH release after TRH may give a further insight into the different regulating mechanisms of both hormones in rats.
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TRH stimulation test
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Autoantibodies against glutamate decarboxylase-65 (GAD65Abs) are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD65Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD65Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD65 in these three different GAD65Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD65 (ROS-GAD65) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD65 (ROS-GAD65Abs) and quantitative assays in T1D associated complications. From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD65 as compared to native GAD65 (N-GAD65). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD65. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10-6 M) followed by nephropathic (1.81 × 10-6 M) and uncomplicated (3.11 × 10-7 M) T1D patients for ROS-GAD65 compared to N-GAD65. Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD65 that induce increased production of ROS-GAD65Abs. Hence regulation of ROS-GAD65Abs could offer novel tools for analysing and possibly treating T1D complications.
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ABSTRACT Administration of pregnant mare serum gonadotrophin (PMSG) to peripubertal rats, aged 27 days, induces ovulation provided the animals weigh more than 60 g at the time of the injection. In an attempt to determine whether the apparent immaturity of the ovaries in smaller rats is associated with an inability of the pituitary gland to secrete LH, the biological and immunological properties of LH in peripubertal PMSG-treated rats were examined. A single injection of PMSG caused a marked hypersecretion of LH in rats aged 27 days. The LH in the plasma of rats weighing more than 60 g was active in both the radioimmunoassay and the cytochemical bioassay but that in smaller rats was active only in the former. Plasma from both groups of rats stimulated the release of testosterone from dispersed Leydig cells. Luteinizing hormone-releasing hormone stimulated the secretion, in vitro , of immunoreactive, cytochemically active LH by pituitary tissue from rats weighing over 60 g. The LH released in vitro from tissue from the smaller animals, like that in their plasma, was active in the radioimmunoassay but not in the cytochemical system. The results suggest that an abrupt change in the nature of LH occurs at puberty and that ovulatory cycles commence only when the pituitary gland secretes the adult form of LH with a full spectrum of biological activity. J. Endocr. (1985) 104 , 173–177
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In order to investigate the role of cortisol in the regulation of testicular function, adult male guinea pigs were challenged with ACTH (20 IU), cortisol (8 or 16 mumol), or with ACTH plus dexamethasone (DEX, 2 mumol). The amounts of cortisol, testosterone, progesterone, and androstenedione present in the plasma or secreted by incubated adrenals or testes were determined by radioimmunoassay. The plasma concentrations of LH were determined using a radioimmunoassay for rat LH. ACTH treatment elevated cortisol plasma concentrations to 999% of control values, whereas it reduced testosterone plasma levels to 43% of control values. ACTH treatment did not affect LH plasma levels. A significant negative correlation was found in ACTH-treated animals, when the cortisol and testosterone plasma concentrations in serially taken blood samples (30-240 min after treatment) were compared (rs = -0.90 and rs = -0.99, P < 0.05). In addition to cortisol, ACTH raised progesterone and androstenedione plasma concentrations. If animals were treated with 2 mumol DEX + ACTH, the plasma levels of cortisol and androstenedione but not of progesterone, testosterone or LH were changed. ACTH stimulated the in vitro secretion of cortisol, progesterone and androstenedione by the adrenals but reduced the in vitro release of androstenedione and testosterone by the testes. In summary, treatment of guinea pigs resulted in elevated cortisol and in reduced testosterone plasma concentrations. The mechanism of the cortisol-induced inhibition of testicular function was independent of the LH plasma concentrations. The in vitro experiments indicate that cortisol directly interacts with the Leydig cells, presumably by inhibiting the activity of the testicular 17 alpha-hydroxylase and/or C17,20-lyase. Taking into account the results of comparable investigations in the rat, the inhibition of the testicular 17 alpha-hydroxylase and/or C17,20-lyase takes place if the intracellular cortisol exceeds the capacity of the 11 beta-hydroxysteroid dehydrogenase to inactivate it.
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Abstract. The circulating levels of growth hormone and thyroxine during the last 2 weeks of gestation, and the effect of thyrotrophin releasing hormone on their secretion, has been examined in 13 chronically catheterised pig foetuses. Porcine growth hormone, measured by homologous radioimmunoassay, in the foetal plasma was found to average 83.5 ± 10.3 ng/ml, while thyroxine levels were 92.9 ±4.1 nmol/l. There was no change in thyroxine or growth hormone levels with gestational age. Injection of 5 μg TRH produced a marked increase in plasma growth hormone levels (+ 194 ± 51 ng/ml) but no change in thyroxine was observed. Injection of saline and subsequent blood sampling had no effect on the levels of either growth hormone or thyroxine.
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SUMMARY Plasma insulin and growth hormone were measured by radioimmunoassay in healthy adults during the latter part of 22 hr. fasts and after graded glucose loads. The insulin concentration was less than 8 μ-u./ml. in all fasting samples and in samples taken after the blood glucose had returned to fasting levels. Insulin levels increased with increasing glucose loads. During fasting, growth hormone showed intermittently raised secretion; in some subjects high values were reached at times which could not be related to external events or to stress. Growth hormone levels were consistently low during the absorption of glucose but rose immediately thereafter. This rise occurred increasingly late and reached increasing levels as the glucose loads were made progressively larger. Insulin had almost invariably returned to fasting levels before the growth hormone concentrations began to rise.
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