Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies.

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor.

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to "rescue" the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.

Localisation of Ca and HMFG2 antigens in breast tissue by immunoperoxidase, immunofluorescence, and immunoelectron microscopy.

The reactivities of Ca1 and HMFG2 monoclonal antibodies were compared on paraffin wax embedded breast tissues using indirect immunoperoxidase. The expression of Ca antigen, like HMFG2, is not exclusive to malignancy: Ca was present in 41/53 (77%) and HMFG2 in 42/53 (79.2%) non-malignant conditions and both were present in 33/35 (94%) carcinomas. Similar results were obtained when cryostat sections were used. Both antigens showed striking similarities in their topographical distributions, although quantitative differences were seen. Their cellular and sub-cellular localisations were investigated by double labelling immunofluorescence and immunogold electron microscopy, which showed that the expression of Ca and HMFG2 antigens was closely associated on cell membranes but that the epitopes were distinct.

Corticotrophin, cortisol, prolactin and growth hormone responses to insulin-induced hypoglycaemia in normal subjects given sodium valproate.

Plasma corticotrophin (ACTH), cortisol, prolactin and growth hormone (GH) responses to insulin-induced hypoglycaemia were measured in normal healthy subjects of both sexes before and after three weeks' treatment with sodium valproate (Epilim, 200 mg three times a day). The drug had no effect on fasting plasma glucose levels, or the extent of hypoglycaemia induced by insulin (0.15 U/kg). There was no significant difference between pre- and post-treatment values for basal or stress-induced concentrations of ACTH and cortisol (n = 12), prolactin (n = 7) or GH (n = 9). The results suggest that treatment of normal subjects with sodium valproate has no effect on the response of the hypothalamo-pituitary-adrenocortical axis to hypoglycaemia, which is in contrast to its inhibitory effects on ACTH secretion in patients suffering from Nelson's syndrome. This implies that in the disease state, there may be a unique sensitivity to GABA-ergic manipulation.

Relationship between adrenocorticotrophin bioactivity in blood and cerebrospinal fluid of rhesus monkeys.

Adrenocorticotrophin levels, measured by a cytochemical bioassay, were determined in the plasma and cerebrospinal fluid (CSF) of adult female rhesus monkeys which were ovariectomized and receiving oestrogen replacement therapy. In control monkeys, ACTH bioactivity was found in both CSF (10.2 +/- 1.8 ng/l) and plasma (186 +/- 51 ng/l) in samples taken at 14.00 h (lights on: 07.00-19.00 h). Dexamethasone treatment (0.2 mg/kg) twice daily for 4 days suppressed plasma ACTH levels (52.8 +/- 25.2 ng/l) but had no effect on CSF levels (7.6 +/- 2.7 ng/l). Raising plasma ACTH, either by daily injections of a long-acting preparation of ACTH (1-24) for 6 days or by bilateral adrenalectomy (and subsequently with-drawing cortisol replacement therapy) also resulted in no detectable changes in ACTH levels in the CSF. A regression analysis between ACTH in the plasma and CSF from samples taken throughout the experiments showed no correlation. In contrast, measurement of ACTH by radioimmunoassay, whilst satisfactory for determination of this peptide in plasma, could not identify authentic ACTH in the CSF. It is concluded that bioactive ACTH does not enter the CSF in detectable quantities from either the peripheral vascular compartment or from the animal's own pituitary gland, and that reducing ACTH secretion from the pituitary also has no effect on levels of ACTH in the CSF. This is in marked contrast to other pituitary peptide hormones, including prolactin, which is secreted together with ACTH during 'stress' but which, unlike ACTH, enters the CSF relatively easily.

Recovery of the hypothalamo-pituitary-adrenocortical axis in the rat after long-term dexamethasone treatment.

Male rats were treated for 14 days with dexamethasone (2.6 mumol/l in the drinking water) and killed at various times after withdrawal of the drug. Some animals were subjected to stress (ether or sham adrenalectomy) just before killing. The recovery of responsiveness of the components of the hypothalamo-pituitary-adrenocortical axis was assessed by measuring plasma and tissue concentrations of hormones, and the response of the tissue in vitro to appropriate stimuli. In vitro, bioactive corticotrophin-releasing factor (CRF) release in response to acetylcholine and adrenal corticosterone release in response to adrenocorticotrophin (ACTH) were significantly suppressed until 3 days after withdrawal. However, release of immunoreactive or bioactive ACTH in response to ovine CRF or hypothalamic extract did not return to normal until day 5. This was correlated with a reduction in pituitary immunoreactive ACTH content and bioactive plasma ACTH, which were suppressed until days 5 and 4, respectively. No change in hypothalamic immunoreactive CRF content could be detected after treatment, or after stress (ether or sham adrenalectomy) in either treated or control animals. Stress (ether) had no effect on the subsequent response of the anterior pituitary gland in vitro to ovine CRF. The large rises in plasma ACTH and adrenal corticosterone measured after stress (ether) in control animals were completely abolished after dexamethasone treatment and did not return to control values until 5 days after withdrawal. Therefore, it appears that after cessation of chronic dexamethasone treatment in the rat, the responsiveness of the hypothalamus and adrenal gland return to normal before that of the pituitary gland.

Biphasic inhibition of bioactive hypothalamic corticotrophin releasing factor secretion in vitro by corticosterone and prevention of the second phase by various steroids.

The effect of various steroids on the functional activity of the rat hypothalamus in vitro was investigated. The addition of corticosterone (10(-7) mol/l) for 30 min to the incubation medium inhibited immediately the release of bioactive corticotrophin releasing factor (CRF) by tissue induced by serotonin (2.6 X 10(-8) mol/l). This was followed by a period lasting from 30 min (coincident with removal of the steroid from the medium) to 60 min when no inhibition was seen. Finally a second period of suppression of hypothalamic CRF activity in vitro was shown to be fully established 120 min after addition of the steroid. In more detailed investigations the latter inhibition was shown to occur when the tissue was exposed to the steroid (3 X 10(-7) mol/l) for 5 or 30 min, but not for 1 min, and it was dose-related. Of other steroids investigated, progesterone in high concentrations (3 X 10(-6 mol/l) suppressed to a small extent the functional activity of the hypothalamus in vitro but 17 alpha-hydroxyprogesterone, 11 alpha-hydroxyprogesterone, 11 alpha, 17 alpha-dihydroxyprogesterone and 11-epicortisol had no effect on the delayed inhibition. Progesterone (10(-7) mol/l) potentiated the ability of corticosterone (10(-8) mol/l) to induce the delayed suppression of hypothalamic CRF activity in vitro. In contrast, 17 alpha-hydroxyprogesterone, 11 alpha-hydroxyprogesterone, 11 alpha, 17 alpha-dihydroxyprogesterone and 11-epicortisol competitively antagonized this inhibitory action of corticosterone (3 X 10(-7) mol/l) in a dose-related manner (1.5 X 10(-8)-3 X 10(-8) mol/l). The action of the antagonist 11-epicortisol was similar whether it was added to the tissue in vitro before corticosterone or antagonist and agonist were added together. The functional characterization of steroid action on the hypothalamus may lead to a clearer understanding of the mechanism by which the compounds influence hormone release.

The evaluation of sodium valproate in the treatment of Nelson's syndrome.

It has previously been reported that sodium valproate (Epilim) lowers plasma ACTH levels in Nelson's syndrome. This report describes further experience with its use. Ten patients with Nelson's syndrome were treated with sodium valproate (600-1200 mg/day) for 5-32 weeks. Plasma ACTH was measured by cytochemical methods and RIA. Initial treatment for 5-12 weeks significantly (P less than 0.005) lowered plasma ACTH from a pretreatment mean of 2460 +/- 1870 ng/liter to 480 +/- 330 ng/liter, and the ACTH circadian rhythm was restored in two patients. On discontinuing treatment, plasma ACTH levels remained suppressed for 3 weeks and rose to pretreatment values in 5-12 weeks. Two patients' plasma ACTH levels failed to show a second response to treatment, while a third patient had a favorable second response to treatment over 32 weeks. In six patients, skin pigmentation lightened with treatment, and in one patient, a reduction in size of a pituitary microadenoma, demonstrated radiographically, occurred with treatment. gamma-Aminobutyric acid and sodium valproate were shown to be ineffective in inhibiting ACTH secretion from cultured pituitary tumor cells from a patient with Nelson's syndrome. The results show that sodium valproate is effective in some cases of Nelson's syndrome. We suggest that it reduces the hypersecretion of ACTH by enhancing gamma-aminobutyric acid function in the hypothalamus, thereby inhibiting the release of corticotropin-releasing factor.

Differential penetration of three anterior pituitary peptide hormones into the cerebrospinal fluid of rhesus monkeys.

This paper demonstrates marked differences between blood levels and those in the CSF for three anterior pituitary peptide hormones, prolactin, luteinizing hormone (LH) and adrenocorticotrophin (ACTH) in the rhesus monkey. CSF levels of endogenous prolactin (measured by radioimmunoassay) are about 20% of those in the blood, and this proportion remains constant under conditions of persistent ('steady-state') hyperprolactinaemia (induced by injecting sulpiride). Acutely elevating prulactin, by either an intravenous injection of exogenous ovine prolactin, or sulpiride, resulted in similar rates of entry by prolactin into the CSF, suggesting that retrograde portal flow is not an important mechanism. LH, measured by bioassay, is also present in the CSF, but the CSF/blood ratio is 5-10 times less than for prolactin. Castration, causing blood LH levels to rise, resulted in equivalent changes in CSF, so that the ratio remains constant, though still much lower than for prolactin. There are significant correlations between individual animals in the blood and CSF content of prolactin and LH. In marked contrast, whilst ACTH is found (by cytochemical assay) in the CSF of both intact and adrenalectomized monkeys, no significant change in CSF levels occurs despite 10-fold changes in the plasma of adrenalectomized animals following withdrawal of cortisol. Nor is there any correlation between blood and CSF ACTH levels over a wide range of concentrations. These results show that each of the three peptides studied has a distinct pattern of entry into the CSF from the vascular compartment.

Effect of treatment with sodium valproate and diazepam on plasma corticotropin in Nelson's syndrome.

Six patients with Nelson's syndrome were given sodium valproate with or without diazepam for 3 or 5 weeks. Initial high plasma adrenocorticotropic hormone (ACTH) concentrations were greatly reduced by treatment and returned to high levels when treatment was stopped. Diazepam did not add significantly to the effects of sodium valproate alone. Three patients reported a decrease in the severity and frequency of headaches while on sodium valproate. In five patients abnormal skin pigmentation was reduced. Sodium valproate is a gamma-aminobutyric acid (GABA) transaminase inhibitor and it is suggested that the drug raises GABA levels in the hypothalamus and that this is responsible for the reduction in ACTH secretion. The data are consistent with the hypothesis that Nelson's syndrome is a neuroendocrine disease caused by a deficiency in the hypothalamic GABA-ergic system.