Mouse splenocyte proliferation and its modulation by sex steroids

Concanavalin A, a T cell mitogen enhanced DNA synthesis in murine splenocytes. Amongst the early signals prior to this event was an increase in cytosolic calcium derived from both intra- and extracellular sources. The requirements for extracellular calcium persisted for four hours after the lectin administration which itself was needed for six hours. Putative calcium channel antagonists and calmodulin inhibitors blocked ihe increase in DNA synthesis. The calcium signal was mimicked by application of the ionophore, A23187, although no increase in DNA synthesis occurred. An activator of protein kinase C, 12-0- tetradecanoylphorbol 13-acetate, had little effect in isolation but the combined application of these two agents greatly enhanced DNA synthesis. The natural mediators of these events are presumed to be inositol trisphosphate and diacylglycerol derived from phosphatidylinositol bisphosphate hydrolysis. Lectin application and protein kinase C activation both increased intracellular pH possibly as a result of Na'l'/H"'' exchange since amiloride an inhibitor of this antiporter inhibited lectin induced DNA synthesis. The calcium and hydrogen ionic changes occur within minutes of lectin application; the protracted requirement for this mitogen suggests further signalling mechanisms occur to elicit maximum DNA synthesis in these cells. Gonadectomy caused an increase in thymic and splenic weight. Spleno-cytes derived from castrated mice showed no change in mitogen response whereas those from ovariectomised mice demonstrated a reduced lectin sensitivity. Testosterone, 5 a dihydrotestosterone, a and 0 oestradiol all inhibited lectin induced DNA synthesis but only at pharmacological concentrations. Testosterone glucuronide and cholesterol were without effect Studies with mouse serum fractions of differing steroidal status were unable to confirm the presence or absence of serum factors which might mediate the effects of steroid on lymphoid cells, all fractions tested inhibited lymphocyte transformation. Both interleukin-2 and lipopolysaccharide induced splenocyte mitogene-sis was also impaired by high steroid concentrations in vitro, suggesting that steroids mediate their effect by a non-specific, non-receptor-mediated event.

Sex steroids and intestinal amino acid transport in rainbow trout, Salmo airdneri Richardson

The effects of sane anabolic and naturally-occuring sex steroids on intestinal transport of leucine have been studied in rainbow trout (Sallno gairdneri), in vivo (gut perfusion), and in vitro (everted gut sacs or intestinal strips). Administration of 17a-methyltestosterone (Mr) by injection for a prolo03ed period of time, enhanced intestinal transport and accumulation of leucine. 11-ketotestosterone (KT) or MT treatment in vitro, by direct addition to incubation media, elicited significant short-term increases in active transport of leucine, without effecting intestinal accumulation. Luminal administration of Mr in vivo similarly elicited short-term responses, without effecting leucine accumulation in the intestine or other peripheral tissues. However; neither MT nor KT significantly affected intestinal transport of water in trout. Although long term injection of oestradiol (E2) enhanced intestinal transport and accumulation of leucine, E2 treatment in vitro was without effect. Addition of ouabain or 2,4,dinitrophenol in the presence of MT abolished steroid-stimulated leucine transform, in vitro. No significant differences were observed between immature male or female trout with respect to either transport of leucine and water, or intestinal granular cell density. However, 'apparent' Na+ absorption and percentage fold height were higher in females, while total intestinal thickness and enterocyte heights were greater in males. These sex differences were essentially abolished. after gonadectany. It is suggested that the short-term effects of the androgenic steroids might be partly mediated through increased activity of Na+,K+,ATPase, and that steroid-induced growth promotion in fish may,to sane extent, be a consequence of enhanced efficiency of intestinal function.

Synthetic studies of oxysterols:methodology for introducing hydroxy groups to ring A of steroids

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Treatment of ocular allergies:nonpharmacologic, pharmacologic and immunotherapy

Ocular allergy is a significant and growing issue worldwide but for many patients, it is often not differentiated from systemic conditions, such as hay fever. Management of seasonal and perennial allergic conjunctivitis is often poor. Management is principally through avoidance measures (blocking or hygiene), nonpharmaceutical (such as artificial tears and cold compresses) and pharmaceutical (such as topical antihistamines and prophylactic mast cell stabilizers). Vernal and atopic keratoconjunctivitis are more severe and generally need treatment with NSAIDs, steroids and immunomodulators. Giant papillary conjunctivitis can be related to allergy but also is often contact lens related and in such cases can be managed by a period of abstinence and replacement of the lens or a change in lens material and/or design. Immunotherapy can be efficacious in severe, persistent cases of contact lens or allergic conjunctivitis.

The environmental control of reproduction in the female dace, Leuciscus leuciscus

An investigation was made into the nature and control of the annual reproductive cycle of the dace, Leuciscus leuciscus. It includes 1) a study of the natural reproductive cycle, 2) the use of Carp Pituitary Extract (CPE) to induce final maturation and ovulation in captive fish, 3) the effect of artificial light treatments on ovarian development and 4) the measurement of serum melatonin levels under different photoperiod regimes. Ovarian development was monitored by endocrinological data, notably serum cycles of 17-oestradiol (E2), testosterone (T), and calcium (as an index of vitellogenin), oocyte diameter, the gonadosomatic index and histological studies of the ovary. Under natural conditions, ovarian development can broadly be divided into 4 stages: 1) oogenesis which occurs immediately after spawning; 2) a primary growth phase (previtellogenic growth) prevalent between spawning and June; 3) a secondary growth phase (yolk vesicle plus vitellogenic growth) occurring between June and December and 4) final maturation and ovulation which occurs in mid-March. During the annual ovarian cycle, the sex steroids E2 and T showed two clear elevations. The first occurred initially in April followed by a rise in serum calcium levels. This subsequently initiated the appearance of yolk granules in the oocytes in June. The second rise occurred in September and levels were maintained until December, after which there was a decline in serum E2 levels. It is proposed that in the dace, high serum E2 levels between September and December were required to maintain vitellogenin production and therefore its uptake into the developing oocytes which occurred during this time, albeit at a slower rate than in the summer months. After December, prior to final maturation, whereas serum E2 and calcium levels declined, serum T levels remained elevated. In captivity, final maturation beyond the germinal vesicle migration stage failed to occur suggesting that the stimuli required for these events were absent. However ovulation could be induced by a single injection of CPE, which induced ovulation between 6 and 14 hours after treatment. Endocrine events associated with the artificial induction of spawning included a rise in serum levels of E2, T and the maturation inducing steroid 1720-dihydroxy progesterone. Photoperiodic manipulation demonstrated that whereas short or increasing daylengths were stimulatory to ovarian development, long days delayed development. Changes from long to short and constant short daylengths early in the reproductive cycle advanced maturation (up to 5 months), suggesting that the stimulus for ovarian development and maturation was a short day. However, experiments conducted later in the reproductive cycle demonstrated that only a simulated ambient photoperiod could induce final maturation. It is proposed therefore that under natural conditions the environmental stimulus for ovarian development and final maturation are short and increasing daylengths respectively. Further support that photoperiod is the dominant timing cue in this species was provided by the pattern of serum melatonin levels. Under different photoperiod treatments, serum melatonin, which is believed to be the chemical transducer of photoperiodic information (similar to other photoperiodic species) was elevated for the duration of the dark phase, indicating that the dace at least has the ability to `measure' changes in daylength.

The isolation and identification of dermatologically active constituents of coal tar

The production and uses of coal tar are reviewed as are the uses of steroids and cytotoxic agents in the treatment of psoriasis with a review of the condition also. An attempt was made to improve the efficaciousness and cosmetic acceptability of a low temperature tar, by screening fractions of this tar, derived from a variety of separation procedures. The most efficacious fraction was the highest boiling acid fraction, which is believed to consist mainly of mono- and di-hydric phenols. A time and concentration study showed that the optimum regime was the application of a 10% concentration in 5% wool fat in soft, yellow paraffin daily for 21 days. The mouse tail skin was selected as an experimental model, to ascertain the efficaciousness of fractions, because of the similarities between this skin and the psoriatic lesion. The activity of a fraction was monitored by the inducement of a granular layer in the mouse tail epidermis. Because coal tar is not an easy medium to work with, and the active fractions showed no increase in cosmetic acceptability over the parent coal tar, likely coal tar constituents were selected for screening on the basis of phenolic character, and the molecular weight range elucidated by mass spectroscopy. 32 potential anti-psoriatic agents were screened on mouse tail. Two catechols, 3,5-di-t-butyl and 4-t-butyl catechols were active. Other structures showed little or no activity. 24 catechols were screened and two extremely active catechols were discovered, 3-methyl-5-t-octyl and 5-methyl-3-t-octyl catechols. The screening of catechol-rich coal tar fractions and a coal tar fraction which had had the catechols removed by oxidation, showed that some anti-psoriatic activity was contained in the catechol fraction of coal tar. Attempts to elucidate the mode of action of these two compounds met with little success, but two modes of action are suggested.

A study of the effects of orally administered female hormones on the volume and composition of lacrimal fluid related to the toleration of corneal contact lenses

This study was designed to evaluate the effects of certain orally active contraceptive steroids on the eye, related to the tolerance of a corneal contact lens. An oestrogen, ethinyloestradiol BP. 0.05 mg, a progestogen, norethisterone acetate BP. 2.50 mg and a control tablet (vitamin C, 50 mg) were utilised. The effect of these preparations on corneal curvature, lacrimal fluid volume and protein composition and directly on corneal lens tolerance was monitored in a group of 23 volunteer patients. The progestogen was found to produce a significant (P≥ 0.05) decrease in tear volume as measured by a 3 minute Schirmer test. A smaller volume reduction was observed with ethinyloestradiol. A normal cornea appears unaffected, within the measurement limits available, by the use of either hormone. However, in the presence of a corneal lens, oestrogen was found to induce substantial corneal steepening, indicative of tissue oedema, during the initial 2-3 weeks of medication. Progestogen occasionally produced a similar effect, which could recur with either hormone shortly after the end of the treatment period. A new method of acrylamide gel electrophoresis was developed for examination of the protein concentration and composition of lacrimal fluid. This allowed much greater resolution of microquantities of unconcentrated fluid than anything previously reported. Quantitation by densitometry has permitted the recording of medication and lens-induced changes in the protein pattern. Tear albumin has been shown to differ from serum albumin and to consist of up to 3 subfractions, 7 further protein fractions may also be resolved. The concentration and probable origin of these proteins have been established and the overall effects of hormone administration described. Individual idiosyncratic responses are also discussed. The study has established tbenature of some effects of contraceptive steroids on the anterior eye, and the probable reasons for resultant corneal lens intolerance.

Identification of putative steroid-binding sites in human ABCB1 and ABCG2

Homology modelling was used to generate three-dimensional structures of the nucleotide-binding domains (NBDs) of human ABCB1 and ABCG2. Interactions between a series of steroidal ligands and transporter NBDs were investigated using an in silico docking approach. C-terminal ABCB1 NBD (ABCB1 NBD2) was predicted to bind steroids within a cavity formed partly by the P-Loop, Tyr1044 and Ile1050. The P-Loop within ABCG2 NBD was also predicted to be involved in steroid binding. No overlap between ATP- and RU-486-binding sites was predicted in either NBD, though overlaps between ATP- and steroid-binding sites were predicted in the vicinity of the P-Loop in both nucleotide-binding domains.

Antenatal corticosteroids impact the inflammatory rather than the antiangiogenic profile of women with preeclampsia

Circulating antiangiogenic factors and proinflammatory cytokines are implicated in the pathogenesis of preeclampsia. This study was performed to test the hypothesis that steroids modify the balance of inflammatory and proangiogenic and antiangiogenic factors that potentially contribute to the patient’s evolving clinical state. Seventy singleton women, admitted for antenatal corticosteroid treatment, were enrolled prospectively. The study group consisted of 45 hypertensive women: chronic hypertension (n=6), severe preeclampsia (n=32), and superimposed preeclampsia (n=7). Normotensive women with shortened cervix (<2.5 cm) served as controls (n=25). Maternal blood samples of preeclampsia cases were obtained before steroids and then serially up until delivery. A clinical severity score was designed to clinically monitor disease progression. Serum levels of angiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], placental growth factor [PlGF], soluble endoglin [sEng]), endothelin-1 (ET-1), and proinflammatory markers (IL-6, C-reactive protein [CRP]) were assessed before and after steroids. Soluble IL-2 receptor (sIL-2R) and total immunoglobulins (IgG) were measured as markers of T- and B-cell activation, respectively. Steroid treatment coincided with a transient improvement in clinical manifestations of preeclampsia. A significant decrease in IL-6 and CRP was observed although levels of sIL-2R and IgG remained unchanged. Antenatal corticosteroids did not influence the levels of angiogenic factors but ET-1 levels registered a short-lived increase poststeroids. Although a reduction in specific inflammatory mediators in response to antenatal steroids may account for the transient improvement in clinical signs of preeclampsia, inflammation is unlikely to be the major contributor to severe preeclampsia or useful for therapeutic targeting.

Analysis of secretory, immunostaining and clinical characteristics of human "functionless" pituitary adenomas:transdifferentiation or gonadotropinomas?

In this study, the central technique of in vitro culture has been used to further investigate whether LH/FSH-expressing, but clinically "functionless" pituitary adenomas are gonadotropinomas or whether their hormone secretion is due to transdifferentiation events. 664 "functionless" pituitary adenomas were examined for hormone secretion by in vitro culture and for hormone content by immunostaining. The results were correlated with the clinical findings. 40% of the tumours (n = 263) secreted at least one of the gonadotropins alone, 8% (n = 53) exhibited various patterns of anterior pituitary hormones, whilst the remaining 52% of tumours were not associated with any hormone. In the secretory tumours, immunostaining revealed only a few scattered hormone-containing cells (5 to 15%). Mild hyperprolactinaemia was observed in some cases, presumably because of pressure effects of the tumours. The majority of the patients suffered clear cut hypopituitarism (p < 0.05). Pre-operatively, gonadotropin hypersecretion was observed in 3 cases, but only one of these secreted hormones in culture. Interestingly, a higher proportion of tumours removed from patients with hypopituitarism showed secretory activity in vitro than those tumours removed from patients showing no hormonal dysfunction or hyperprolactinaemia. We conclude that the term "gonadotropinoma" to describe functionless pituitary tumours associated with LH and/or FSH secretion is a misnomer, because the presence of LH and/or FSH confirmed by in vitro methods in the present series is a result of only a few scattered cells. We suggest that primary pituitary tumour cells differentiate into a secretory type (transdifferentiation), possibly in response to altered serum hormone levels such as decreased steroids. Further work is required to identify the factors which trigger the altered cells' characteristics. © J. A. Barth Verlag in Georg Thieme Verlag KG.