Schistosoma haematobium: identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells.

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of Schistosoma-infected individuals. We also show that Sh is able to interact in vitro with estrogen receptor (ER), explaining how host endocrine system can favor the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

Hormones, nevi, and melanoma: an approach to the patient

For many years, clinicians have been concerned about a potential adverse effect of pregnancy-associated hormones and exogenous hormones on melanocytic nevi and malignant melanoma. Today, these issues are more significant as women have delayed childbearing into their 30's and 40's, and the likelihood of diagnosis with melanoma during pregnancy is enhanced. More recent clinical, epidemiologic, and laboratory studies have shed some light on the relationship among hormones, nevi, and melanoma in pregnancy.

A monoclonal antibody to oestradiol potentiates the stimulation of the specific activity of the brain type creatine kinase by oestrogen in vivo and in vitro.

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E2 15 plus oestrogen (E2) potentiated the specific activity of the brain type creatine kinase (CK) response to E2 in the rat tissues or skeletal cells. The enhancing CK response of anti E2 15 plus E2 was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E2 15 blocked the stimulatory CK response to E2. Two other high affinity anti-E2 antibodies, clones 8D9 and 11B6, had no effect in augmenting the response of CK to E2 in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E2 15 plus E2 since the intact anti-E2 in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E2 15 abolished the CK response to E2 in rat tissues and not to anti-E2 15 plus E2 whereas tamoxifen completely blocked the CK response to anti E2 plus E2. Anti E2 15 may therefore serve as a specific carrier in delivering E2 to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E2 effects in vivo and in vitro.

The measurement of the isoflavone daidzein by time resolved fluorescent immunoassay: a method for assessment of dietary soya exposure.

We report a novel method for the measurement of urinary daidzein that is suitable for assessment of dietary soya exposure. The method incorporates the following features: (i) a highly specific monoclonal antibody to daidzein (clone 4E4) raised through the 7 position of daidzein and (ii) a europium labeled ovalbumin daidzein conjugate. In the present format, dilute urine samples of subjects who ingested soy milk are hydrolyzed with beta-glucuronidase for 30 min on rabbit anti-mouse coated plates. Afterwards, the specific monoclonal antibody to daidzein, clone 4E4, and europium labeled ovalbumin daidzein conjugate are added. After 1 h incubation, the wall bound fluorescence of europium is measured by time resolved fluorescence and is inversely proportional to the concentration of daidzein over the range 0.1-10 ng daidzein/well. The method demonstrates good sensitivity, precision and comparability with the chemical method GC-FID. Unlike the chemical method, the present immunoassay technique for daidzein is applicable for the measurement of large amounts of samples in epidemiological studies for the assessment and monitoring of human exposure to soya food.

Development of a hormone neutralizing vaccine, using GnRH-glycys-PPD, for use in the treatment of oestrogen-dependent disorders.

The aim of this study was to develop an effective and nontoxic vaccine, suitable for use in humans, which was capable of effectively controlling oestrogen levels. Female Sprague-Dawley rats were immunized with a conjugated analogue of gonadotrophin releasing hormone, GnRH-glycys-PPD. This resulted in high levels of neutralizing antibody which disrupted GnRH function and consequently caused a reduction in serum oestrogen. The effect of oestrogen deprivation correlated well with ovarian failure and gonadal atrophy. An examination was made of various adjuvants in conjunction with the analogue to determine the suitability of the combinations in the formulation of an effective human vaccine. This investigation included a novel adjuvant, non-ionic surfactant vesicles (NISV); the results showed that NISV are completely nontoxic and in terms of potentiating and sustaining an immune response, compare favourably with Freund's adjuvant and alum. In addition the long term effects of immunization were investigated and the data showed that immunoneutralization of GnRH effectively suppresses fertility on a long-term basis.

Hormonal regulation of immune responses.

Females have stronger immune responses than males. Female predominance in autoimmune diseases in both experimental animals and in man is also established. Sex hormones and some other steroid hormones have been shown to exert immunoregulatory effects in both in vivo and in vitro situations. Important mechanisms of hormone action on lymphocytes have gradually been revealed. In this paper the most intriguing effects of steroid hormones on immune system and present data suggesting hormonal influence on the pathogenesis of autoimmune diseases are summarized.

Purification of alpha-fetoprotein from human cord serum with demonstration of its antiestrogenic activity.

Alpha-fetoprotein (AFP) was purified from pooled human cord serum to determine whether it would be similar to purified mouse AFP in its ability to be transformed into an antiestrogen by incubation with estradiol (E2). Greater purity was attained with a three-step purification procedure of chromatofocusing, Blue-Sepharose chromatography and immunoaffinity chromatography than with a two-step procedure of polyacrylamide gel electrophoresis followed by Blue-Sepharose chromatography. Nevertheless, both procedures rendered AFP in a form that was transformable by E2 to an antiestrogen, although the product of the three-step procedure afforded more consistent biological activity. Removal of albumin from AFP was crucial for transformation of AFP to an antiestrogen. Thus, human AFP is similar to mouse AFP in being transformed to an antiestrogen upon incubation with E2, even though there is only 66% structural homology between the two proteins, and human AFP lacks the high-affinity binding site for E2 present in the mouse AFP molecule.

Immuno-neutralization of maternal plasma oestradiol stimulates fetal growth in rats.

On day 12 of pregnancy, 7 rats were injected with 2 ml of an anti-oestradiol antiserum a quantity capable of binding 2.4 micrograms oestradiol. Seven further (control) rats were injected with an antiserum raised against human serum globulin. After sacrifice on day 21 of pregnancy, the fetuses from treated rats showed an increase in both placental weight (0.851 +/- 0.023 g, n = 98 vs 0.752 +/- 0.019 g, n = 97) and fetal weight (4.446 +/- 0.426 g, n = 98 vs 3.403 +/- 0.211 g, n = 97). There was no significant difference in protein, DNA and RNA contents between the fetuses from treated mothers and those from control mothers. This experiment confirms that oestrogens may play a role in fetal weight gain.

Interaction of [3H] estradiol - and [3H] monohydroxytamoxifen-estrogen receptor complexes with a monoclonal antibody.

The aim of this study was to compare and contrast the interaction of estrogen [( 3H]17 beta-estradiol)- or antiestrogen [( 3H]monohydroxytamoxifen)-receptor complexes from human breast tumor cytosols with monoclonal antibodies raised to the human breast tumor estrogen receptor. Breast tumor cytosols containing estrogen receptor which sedimented as radiolabeled peaks in either the 8S, 8S and 4S, or 4S regions of sucrose density gradients, interacted with the monoclonal antibody D547 to produce a broad 9-10S peak, a broad 8S-10S peak, or a more discrete 8S peak, respectively. On high salt (0.4M KC1) sucrose density gradients the 4S ligand-receptor complex plus antibody produced a binding peak at approximately the 8S region of the gradient. These sedimentation studies with the monoclonal antibody D547, and similar studies with the monoclonal antibody D58, could detect no differences in the cytosolic estrogen receptor whether complexed with [3H]estradiol or with [3H]monohydroxytamoxifen. These observations were confirmed by Scatchard equilibrium saturation analysis and sucrose density gradient analysis of cytosols from the MCF-7 human breast cancer cell line. The antibody D547 interacted with 8S ER from these cytosols to produce a broad 8S-10S peak, but the antibody produced no change in the affinity or number of binding sites present in these cytosols. It seems, therefore, that the antigenic determinants recognized by these particular antibodies on the breast tumor cytosolic receptor are not significantly altered by the binding of either an estrogen or an antiestrogen to the receptor.