Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus.

Human endometrial fibroblasts have been immortalized by infection with simian virus 40 large T antigen and established as a permanent cell line, St-2. Biochemical differentiation of this cell line has been demonstrated by the ability of a decidualizing stimulus, 8-bromo-cAMP plus medroxyprogesterone acetate (MPA), to induce PRL secretion and increase the enzymatic activity of estrone sulfatase. MPA, alone or in combination with estradiol, was unable to elicit this response, but potentiated the effect of 8-bromo-cAMP on PRL production and estrone sulfatase activity. The increase in PRL protein was accompanied by an increase in PRL messenger RNA and increased expression of the insulin-like growth factor-binding protein-1 messenger RNA. The St-2 cell PRL transcript was larger than the pituitary PRL transcript, suggesting its initiation from the distal, nonpituitary, PRL promoter. This was confirmed by reverse transcription-PCR analysis of PRL transcripts using primers specific for the additional sequences present only in the 5'-untranslated region of RNA initiated from the distal promoter. Transient transfection of a reporter construct containing 3000 bp of DNA 5' to the decidual-specific promoter of the human PRL gene demonstrated that cAMP was capable of activating this distal promoter in St-2 cells. In conclusion, this novel cell line provides an interesting new model in which to pursue aspects of biochemical differentiation of human endometrium in vitro.

Uptake and retention of androgens by the rat ventral prostate and consideration of their use as site directing agents.

Elucidation of the mechanism whereby androgens are accumulated selectively by the prostate may help in the design of drugs for the treatment of prostatic cancer. The uptake and retention of [3H]testosterone, following intraperitoneal injection, by various tissues in the 24 hr castrate rat has been studied over an extended time course. The selectivity with which prostate, as compared with blood or other tissues, accumulated 3H was shown to be dose-dependent. At a low dose (0.15 microgram), selective prostatic accumulation was greater in 24 hr castrate and diethylstilboestrol-treated rats than in normal animals. Testosterone, 5 alpha-dihydrotestosterone and oestradiol, radioactively labelled, were each administered to 24 hr castrate rats by intraperitoneal injection. Specific prostatic accumulation of radioactivity was more dependent on steroid structure at a low dose than at a high dose (0.6 mg) and at the low dose (0.15 microgram) followed the order testosterone greater than 5 alpha-dihydrotestosterone greater than or equal to oestradiol. This order was surprising in view of the androgen receptor binding affinities of these steroids. It is concluded that small quantities of material could be directed with the greatest specificity to the prostate of castrate or diethylstilboestrol-treated animals if attached to testosterone. Androgens would be more useful for site-directed radiopharmaceuticals than cytotoxic agents.

17 alpha-Z-[125I]iodovinyloestradiol and its 3-acetate: chemical synthesis in vivo distribution studies in the rat. Comparison of tissue accumulation and metabolic stability with 17 alpha-E-[125I]iodovinyl and 16 alpha-[125I]iodo oestradiols.

Certain oestrogens on substitution with halogens, have been shown to retain receptor binding affinity and accumulate in target tissues and therefore could, when labelled with gamma-emitting halogen isotope, act as radiopharmaceuticals for the imaging of receptor positive breast tumours. In order to evaluate putative radiopharmaceuticals, 17 alpha-Z-iodovinyloestradiol ((17 alpha,20Z)-21-iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diol) and its 3-acetate have been chemically synthesized and labelled with 125I. Tissue distribution studies in female rats have been compared to those obtained using 17 alpha-E-[125I]iodovinyloestradiol ((17 alpha,20E)-21-[125I]iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diol) and its 3-acetate and to 16 alpha-[125I]iodo oestradiol (1,3,5(10)-estratien-16 alpha-[125I]iodo-3,17 beta-diol). All compounds showed a similar tissue distribution with the highest concentrations (cpm/g tissue) in the thyroid greater than liver greater than uterus greater than kidney greater than lung greater than blood greater than heart, spleen. The concentration in the uterus was highest after injection of 17 alpha-Z-iodovinyloestradiols or 16 alpha-iodo oestradiol. Target (uterus) to background (blood) tissue ratios were highest after injection of 17 alpha-Z-iodovinyloestradiol-3-acetate and 16 alpha-iodo oestradiol (6:1). Deiodination in vivo took place to a small degree, amounting to 1.6%, 0.9% and 0.35% of the injected dose after 4 hr with the 17 alpha-Z, 17 alpha-E and 16 alpha compounds, respectively. For reasons of chemical expediency and consideration of the biological results 17 alpha-Z-iodovinyloestradiol-3-acetate is the compound of choice for the detection of oestrogen receptor positive tissues.

16 alpha-Iodo-testosterone: chemical synthesis and evaluation as a potential radiopharmaceutical.

The chemical synthesis and characterization, including 1H NMR, of 16 alpha-iodo-androstenedione and 16 alpha-iodo-testosterone are described. Each has been synthesized with 125I and tested in rats in vivo for accumulation in androgen dependent tissues over a 24 hr time course. Neither compound was accumulated in prostate against the blood gradient of normal or 24 hr castrate animals. The metabolism, subcellular distribution and binding of 16 alpha-[125I]iodo-testosterone to protein in prostate has also been examined. By comparison with data obtained after the administration of [3H]testosterone we conclude that the failure of this iodinated androgen to accumulate in androgen dependent tissues arises because of its low binding affinity for receptor protein.

Reduced neuropsychological dysfunction using epiaortic echocardiography and the exclusive Y graft.

BACKGROUND: To examine the effect of screening the aorta for atheroma before aortic manipulation and use of exclusive Y graft revascularization on the incidence of neuropsychological dysfunction after coronary artery bypass. METHODS: Aortic atheroma was detected using epiaortic and transesophageal echocardiography. Atheroma avoidance was facilitated by use of the exclusive Y graft technique, which has no aortic coronary anastomoses. In the control group aortic atheroma was assessed by manual palpation, and we attempted to avoid any atheroma detected. In this group we also used aorta-coronary grafts. Transcranial Doppler imaging of the right middle cerebral artery was used to detect cerebral microemboli. Neuropsychological dysfunction was defined as a 20% or more decline in score for at least 20% of a neuropsychometric battery of ten tests for each patient. RESULTS: Late dysfunction at 57 +/- 2 days postoperatively in the control group was 38.1% and in the echo/Y group was 3.8% (p' = 0.012). Microemboli detected by transcranial Doppler imaging during periods of aortic manipulation was greater for those with late dysfunction (5.2 +/- 3.0 compared with 0.5 +/- 0.2) (p' = 0.018). No clinical strokes occurred in either group. CONCLUSIONS: The combined techniques of epiaortic screening and exclusive Y graft for coronary artery bypass operations resulted in a low incidence of late neuropsychological dysfunction.

Lack of correlation of free deoxypyridinoline excretion with Taq1 restriction length polymorphisms in the vitamin D receptor gene in males.

An association between allelic variants in the vitamin D receptor gene and bone mineral density has been previously described. A bimodal variation in the rate of bone resorption (as measured by urinary deoxypyridinoline excretion rate) has also been reported. We have recruited male volunteers, to minimise variation associated with ovarian function, to investigate a possible connection between these observations. Allelic variants in the vitamin D receptor gene were identified as Taq1 restriction fragment length polymorphisms. The ratio of variants TT:Tt:tt occurred with a frequency of 34%:47%:17%. Excretion rates of urinary free deoxypyridinoline, measured by immunoassay, were compared in age-matched males from each genetic group. There were no significant differences based on the paired Student's t-test. Excretion rates declined with age (P = 0.04) and the best fit model fits the same regression line to each group. Genetic variation in the vitamin D receptor is not linked with differences in bone resorption rates.

Measuring tidal breathing parameters using a volumetric vest in neonates with and without lung disease.

Lung function measurement is difficult in unsedated infants; tidal breathing parameters are a useful non-invasive surrogate, but even these measurements cause disturbance from applying a facemask. We investigated a novel volumetric vest system (FloRight), which measures volume changes of the respiratory system from changes in the magnetic fields induced by current-carrying coils around the entire chest and abdomen. Using a facemask and ultrasonic flowmeter as comparator, we assessed the validity and repeatability of tidal breathing parameters measured by FloRight in 10 healthy newborn infants during natural sleep. We also assessed the effect of a facemask on tidal volume and tidal expiratory flow parameters. To assess the ability of the FloRight system to detect disease, we compared the healthy infants with 11 infants suffering from bronchopulmonary dysplasia. Tidal parameters with the FloRight vest corresponded closely with facemask measurements. Mean difference, mask minus vest, for tidal volume was 0.096 ml (P < 0.05), with limits of agreement +4.5 to -4.3 ml. Coefficient of repeatability was similar for mask and vest measurements. Tidal volume measured by FloRight with mask in place (20.6 ml) was significantly higher than without mask (16.1 ml), but tidal expiratory flow parameters were not altered. FloRight measurements of tidal parameters were markedly different between the two groups of infants, with tidal volume per Kg significantly higher and tidal expiratory flow parameters significantly lower. Our findings suggest that the FloRight system is able to measure tidal breathing parameters accurately, in healthy newborn infants, without prior calibration on the infant. It appears to have at least sufficient sensitivity to detect severe respiratory disease.

An experimental approach to the optimum oestrogen dosage in prostatic carcinoma.

The action of diethylstilboestrol on the androgen response mechanism of the human prostate has been examined. A simple in vitro test system that assesses effects on hormone uptake, receptor binding and translocation to the nucleus has been employed. The test has also been modified to study the effect of diethylstilboestrol on the activity of 5 alpha-reductase, the enzyme responsible for the conversion of testosterone to 5 alpha-dihydrotestosterone. These results are discussed in relation to the dosage of diethylstilboestrol for treatment of carcinoma of the prostate.

Method for the measurement of plasma dehydroepiandrosterone by gas-liquid chromatography with electron capture detection.

A method is described for the measurement of plasma dehydroepiandrosterone, as the iodomethyldimethylsilyl ether derivative, by gas-liquid chromatography and electron capture detection using the relatively new and highly stable stationary phase Dexsil 300. Preliminary purification of the plasma extract was required and alumina column chromatography was utilised, both before and after derivatization of the steroid extract. Specificity, precision, sensitivity and accuracy were all satisfactory. The method was used to study the relationship between age and the level of plasma dehydroepiandrosterone in a group of normal women. A significant negative correlation was observed.

The synthesis of 3H labelled steroid-nitrogen mustard derivatives and studies on their action in rat and human prostate.

Tissue receptor proteins are thought to be a key factor in selective retention and nuclear translocation of steroid hormones in their target tissues. Steroid-cytotoxic derivatives with high affinity for these receptor proteins may, therefore, be useful as site directed agents in the treatment of hormone dependent cancers. The prostatic retention and subcellular localisation of estramustine and a 17 beta nitrogen mustard derivative of 5 alpha-dihydrotestosterone have been compared since the latter compound has higher androgen receptor binding affinity than the former. The 5 alpha-dihydrotestosterone derivative was not retained more avidly than estramustine by either human or rat ventral prostatic tissue. Nuclear localisation could not be detected with either compound. The cytotoxic action of these compounds requires the liberation of the nitrogen mustard moiety. The cleavage rate of both compounds was low but similar in both benign and malignant prostatic tissues. We conclude that steroids are unlikely to be suitable agents for directing cytotoxic compounds to prostatic tissue particularly if the steroid-cytotoxic derivative requires metabolism for the expression of the cytotoxic activity.

Issues associated with the identification of cognitive change following coronary artery bypass grafting.

OBJECTIVE: Coronary artery bypass grafting (CABG) is a surgical procedure used to treat individuals with ischaemic heart disease and to relieve angina. Disruption to the central nervous system (CNS) has frequently been reported by patients who have undergone CABG. METHOD: The following paper is a review of the literature that has examined the effects of CABG on the CNS. RESULTS AND CONCLUSIONS: It becomes apparent that issues about the incidence and severity of post-CABG cognitive decline are still unresolved. First, the cause of post-CABG CNS change has not yet been established, although the presence of changes to brain microvasculature as a result of the presence of microemboli appears to be a likely factor. Second, while some studies have reported high rates of poor performance on neuropsychological tests postoperatively, these reports are often subject to confounds such as variability in postoperative testing intervals, the definition of decline and the neuropsychological test batteries used. Finally, improvements in surgical techniques and changes in patient characteristics may have changed the real nature and prevalence of post-CABG cognitive decline. The review finishes with a series of recommendations for the neuropsychological study of CABG.

5-alpha-Reductase deficiency causing male pseudohermaphroditism.

An infant with male pseudohermaphroditism due to deficiency of 5-alpha-reductase is described, the elder of two affected male siblings. These patients, who come from Pakistan, are the first to be described outside America.

The nuclear uptake of androgen by human benign prostate in vitro: action of antiandrogens.

An in vitro test system suitable to assess the potency of putative antiandrogens has been developed, using the human benign prostatic tissue obtained at operation. The system circumvents some problems associated with using human tissue, such as the presence of endogenous steroid and contamination with plasma proteins (particularly sex hormone binding globulin). Slices of tissue were incubated in the presence of 3H-testosterone and the uptake into nuclei was determined. The nature of the nuclear radioactivity and the steroid specificity indicates a mechanism similar to the established in the rat ventral prostate. The action of antiandrogens (cyproterone acetate, diethylstilbestrol, flutamide, hydroxylated flutamide and gestonorone capronate) has been studied at various concentrations.

Nuclear components responsible for the retention of steroid--receptor complexes, especially from the standpoint of the specifcity of hormonal responses.

1. By covalently linking nuclear components from hormone-sensitive cells to Sepharose 2B, it is possible to investigate the interaction between nuclear components and cytoplasmic receptor-steroid complexes by affinity chromatography. 2. Many factors are implicated in the specifity of nuclear-cytoplasmic interactions, including the nature of the nuclear components, the presence of the cytoplasmic receptor protein and the provision of the appropriate steroid ligand. 3. Two distinct sets of binding sites are present in nuclear extracts immobilized to Sepharose 2B, namely a small number of specific high-affinity sites and a larger number of non-specific low affinity-sites. 4. Considerable evidence supports the importance of the high-affinity binding sites in the manifestation of hormonal specificity in different tissues. Although the study has centred largely on androgenresponsive systems, the findings are germane to cytoplasmic-nuclear interactions in general. 5. The high-affinity or acceptor sites in nuclear extracts reside in the basic but non-histone protein fraction. 6. Hormonal specificity is seemingly maintained by both the cytoplasmic and nuclear components, and the results are discussed in the context of the mechanism of action of steroid hormones.

The androgenic regulation of the activities of enzymes engaged in the synthesis of deoxyribonucleic acid in rat ventral prostate gland.

The restoration of mitosis and growth of the prostate gland of castrated animals by androgens provides a favourable experimental system for studying the hormonal regulation of enzymes engaged in DNA replication. 2. Many DNA polymerase activities were identified in the prostate gland, but only a 9S form with a particular preference for denatured DNA as template was conspicuously enhanced by androgenic stimulation. 3. Thymidine kinase also provided a sensitive indicator of the hormonal regulation of DNA replication, and on electrophoretic criteria, one discrete form of the enzyme appeared precisely with the onset of mitoris. 4. Evidence is presented to support the view that DNA ligase activity is intimately associated in the process of DNA replication in the prostate gland. 5. A spectrum of deoxyribonuclease activities is present in the prostate gland, but only one form (pI7.0) can safely be said to be implicated in the process of DNA replication. 6. Androgenic stimulation of the prostate gland leads to the appearance of a component capable of denaturing or unwinding prostate DNA. This component is seemingly distinct from RNA or DNA polymerase activities on the basis of several distince physicochemical characteristics. 7. The conspicuous feature of all the changes in enzyme activities evoked by androgens in the prostate gland is their acute tissue- and steroid-specificity. Such changes could not be mimicked in liver or spleen and the regulatory role of androgens could not be simulated by other classes of steroid hormones. Particularly on the basis of studies with the anti-androgen cyproterone acetate, it is concluded that the changes are initially mediated by the androgen-receptor system and the high-affinity binding of 5alpha-dihydrotestosterone in the prostate gland. 8. The results are discussed in the context of the mechanism of action of androgens.

The synthesis of 16 alpha-[131I]iodo-oestradiol and evaluation of its use as a radiotracer for oestrogen receptor positive breast tumours.

16 alpha-Iodo-oestradiol binds with high affinity to the oestrogen receptor and has been shown to accumulate in oestrogen sensitive tissues in many test systems. We have prepared the compound labelled with 131I at four specific activities. Using these preparations we have attempted to image human primary and metastatic breast cancer deposits at various times from 15 min to 24 h post injection by external gamma scintigraphy. Clinical studies were conducted on 10 post-menopausal patients. The receptor status was determined in seven cases, four were positive and three negative. The imaging results were very poor, in only two cases were images obtained, these were very faint and only of the primary, never of the metastatic deposits. The oestrogen receptor status was only available in one of these cases, it was positive. Dynamic studies in vivo revealed that the compound is cleared rapidly from the circulation during the first 5 min and thereafter undergoes extensive enterohepatic recycling. Studies of the radiochemical identity of the circulating species revealed that the injected compound was extensively metabolised. Neither an increase in specific activity of injected radiotracer nor imaging at shorter times after injection improved the results.

Increased uterine uptake and nuclear retention of [3H]oestradiol through the oestrous cycle and enhanced end-organ sensitivity to oestrogen stimulation in vitamin B6 deficient rats.

Vitamin B6 deficient female rats showed a significantly earlier, greater and more prolonged uptake of a tracer dose of [3H]oestradiol into the uterus, with increased nuclear accumulation, compared with vitamin B6 supplemented animals. This was most marked at oestrus, with little difference at anoestrus. The responses to low doses of ethynyl-oestradiol were greater in ovariectomized deficient animals than in those receiving the supplemented diet, with an increased uterotrophic response and greater induction of peroxidase. In the deficient animals there was virtually complete suppression of LH secretion at doses of ethynyl-oestradiol that had no effect in controls. At high doses of ethynyl-oestradiol there was no difference between the two groups of animals. The results suggest that increased uterine uptake and accumulation of [3H]oestradiol in vitamin B6 deficiency is associated with enhanced end-organ responsiveness to sub-maximal oestrogen stimulation, and that pyridoxal phosphate may have a coenzyme role in oestrogen action.

Effects of vitamin B6 nutritional status on the uptake of [3H]-oestradiol into the uterus, liver and hypothalamus of the rat.

Rats were depleted of vitamin B6 by treatment with isoniazid, and then maintained on diets providing: no vitamin B6, an adequate amount (1.2 mg/kg diet) or a very large amount (120 mg/kg) for 5 weeks. The uptake of a tracer dose of [3H]-oestradiol into the nuclei of liver and uterus was significantly greater in deficient animals than in those receiving an adequate or greater than adequate amount of the vitamin. Similarly the accumulation of oestradiol in the region of the brain corresponding to the hypothalamus, pre-optic area and septum (the major oestradiol-sensitive regions of the central nervous system) was higher in deficient animals than in other groups. There were significant inverse correlations between the uptake of oestradiol into target tissues and vitamin B6 nutritional status as determined by the concentrations of pyridoxal phosphate in plasma and liver. Uteri from deficient animals were significantly smaller than those from animals receiving an adequate or greater amount of vitamin B6, and the induction of uterine peroxidase by oestradiol was impaired. It therefore seems likely that despite the greater net accumulation of steroid in target tissues, vitamin B6 deficiency impairs biological responsiveness to oestrogens. It is suggested that this may be due to a failure of the recycling of oestradiol receptors from the nucleus.

The synthesis of E-17 alpha-[131I]iodo-vinyl oestradiol and evaluation of its use as a radiotracer for oestrogen receptor positive breast tumours.

17 alpha-Iodo-vinyl oestradiol binds with high affinity to the oestrogen receptor, is metabolically stable and has been shown to accumulate in oestrogen sensitive tissues of rodents. We have synthesised this compound and its 3-acetate, labelled with carrier free 125I and shown the accumulation of both compounds in rat uterus. 17 alpha-Iodo-vinyl oestradiol-3-acetate was prepared labelled with carrier free 131I and administered to twelve patients with suspected breast cancer, approximately 1 h before surgical removal of the primary tumour mass. At the time of surgery a sample of the tumour and a peripheral blood sample were taken. The ratio of radioactivity in the tumour to blood was determined. All tumours accumulated radioactivity and ratios ranged from 1.5:1 to 3.7:1. There was no correlation between the degree of accumulation and either cytosolic oestrogen or progesterone receptor concentration in the tumour. Analysis of blood revealed a single circulating species, 17 alpha-iodo-vinyl oestradiol. The results show that 17 alpha-iodo-vinyl oestradiol is metabolically stable and accumulates in breast tumours though this accumulation is not sufficient to permit imaging.

Changes in concentrations of cortisol, dehydroepiandrosterone sulphate and progesterone in fetal and maternal serum during pregnancy.

OBJECTIVE: In fetuses, adrenal steroids have been implicated in organ maturation and in some species in initiation of labour. The fetal adrenal gland differs from the adult in its complement of steroid metabolizing enzymes. This study sought to examine the changes in peripheral cortisol, progesterone and dehydroepiandrosterone sulphate (DHEAS) in unstressed fetuses during pregnancy. DESIGN: Paired maternal and fetal samples were collected from 47 patients. Fetal blood samples were collected by transabdominal needling. All fetuses were appropriately grown for age which ranged from 18 to 41 weeks. MEASUREMENTS: Hormones were measured using specific, validated immunoassays. RESULTS: Fetal progesterone (mean, 822 nmol/l; 95% data intervals 196-1449 nmol/l) varied considerably between individuals but there was no significant change in serum concentration with gestational age, nor was there any difference between male and female fetuses. There was a small, but significant (y = 0.339x2 - 13.5x + 231; r = 0.72, P = 0.0001) rise in cortisol in the fetal circulation from 32 to 41 weeks gestational age, whereas the mean fetal DHEAS concentration decreased linearly with gestational age from 4.1 mumol/l at 18 weeks to 2.6 mumol/l at 41 weeks (r = -0.41; P = 0.007). Mean progesterone concentration in the maternal serum increased linearly from 98 nmol/l at 18 weeks to 783 nmol/l at 41 weeks. In the fetus there was a significant correlation between progesterone and cortisol concentrations. CONCLUSIONS: These results are compatible with the proposed role of cortisol in fetal lung maturation, confirm high levels of progesterone in the fetus from an early stage of gestation, and provide further evidence for placental progesterone being the precursor of fetal cortisol.