B D Greenstein

The University of Arizona, Tucson, AZ, United States

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Publications (16)44.32 Total impact

  • B D Greenstein
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    ABSTRACT: Estrogens are believed to play a role in the etiology of both human and murine systemic lupus erythematosus (lupus, SLE), presumably through the agency of their cellular receptor proteins. There is now considerable interest in the molecular mechanism of action of estrogens in immune tissues, particularly with regard to autoimmune disorders, which are generally more prevalent in women. In this laboratory, an attempt is being made to characterize estrogen receptors in murine models of SLE, namely NZB/W and MRL/MP-lpr/lpr mice, and to try to relate this to estrogen receptor function in vivo. It is hypothesized that estradiol (E(2)), through its receptors, mediates the progression of murine SLE and that in autoimmune disease, the estrogen receptor is functionally or structurally changed, or both. Initial studies suggest there are differences in estrogen receptors between BALB/c mice, which do not get autoimmune disease, and two strains that do, MRL/MP-lpr/lpr and NZB/W mice. There is evidence that in at least one model of SLE, the normal regulation of estrogen action by progesterone may be impaired. In several laboratories, attempts are being made to relate estrogen action to immune function and to autoimmune diseases. The study of estrogen action on the immune system may lead to the development of treatments that attenuate the immunostimulant effects of E(2) in autoimmune diseases such as SLE.
    Journal of Women s Health &amp Gender-Based Medicine 05/2001; 10(3):233-9.
  • Y Y Dhaher, B D Greenstein, M A Khamashta, G R Hughes
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    ABSTRACT: The effects of oestradiol and the oestrogen receptor antagonist ICI 182,780 were investigated on the DTH index, serum IgG and IgM levels and spleen weight in female BALB/c and MRLL/MP-lpr/lpr mice. At six weeks, the mice were ovariectomised, and one week later, over a four-week period, given biweekly s.c. doses of (i) 5 microl of olive oil, or (ii) 5 microl of oil containing 3.2 microg of 17beta-oestradiol (E2), or (iii) 5 microl of oil containing (3.2 microl of E2 + ICI 182,780, at a dose of 30 mg/kg), or (iv) the same dose of ICI 182,780 alone, E2 significantly raised the DTH index in BALB/c mice; this effect was prevented if ICI 182,780 was included in the injection. The DTH index in MRL mice was unaffected by any of the treatments. All steroid treatments raised serum IgG levels in BALB/c mice, but those in sera of MRL were unaffected and were significantly higher than those measured in BALB/c mice. ICI 182,780 depressed IgM in BALB/c mice, while all steroid treatments increased IgM in MRL mice. ICI 182,780 depressed spleen weights in both strains. Oestrogen may influence B cell function through ICI 182,780-sensitive receptors, and ICI 182,780 may have agonist actions on the immune system. (200 words).
    Autoimmunity 02/2001; 33(4):237-43. · 2.77 Impact Factor
  • R Roa, B D Greenstein
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    ABSTRACT: Binding properties of estrogen receptors in liver, thymus and uterus of BALB/c and (NZBxNZW) F1 mice were compared. (NZBxNZW) F1 mice spontaneously develop an autoimmune disease resembling human systemic lupus erythematosus (SLE). It is hypothesized that estradiol, through its receptors, mediates the progression of murine SLE. High-speed cytosols were prepared from liver, thymus and uterus and incubated with the synthetic estrogen 3H-moxestrol (NEN). Scatchard plots were derived from binding isotherms obtained. Rectilinear Scatchard plots were obtained from all tissues, which is consistent with the presence of only one class of binding sites, or of more than one class but with the same affinity. There were, however, strain differences in that the affinity of the binding reaction was significantly higher in cytosols from BALB/c liver in males and females. In thymus, the situation was reversed in that the affinity was significantly higher in cytosols from (NZBxNZW) F1 mice. Thymic cytosols from BALB/c mice contained significantly more estrogen receptors per mg cytosol protein than did those from (NZBxNZW) F1 mice. The exacerbation of murine SLE may be due, at least in part, to these properties of estrogen receptors in liver and thymus.
    International Journal of Immunopharmacology 12/2000; 22(11):897-903.
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    ABSTRACT: Estrogens exacerbate the autoimmune disease SLE and progesterone is immunoprotective. Estrogens increase synthesis of progesterone receptors (PR) and it is hypothesized that this physiological balance may be impaired in SLE. To test this, cytosolic PR were measured in hypothalamus, thymus and uterus from 6-week-old female ovariectomized BALB/c and MRL/MP-lpr/lpr mice 48 h after s.c. injection of estradiol benzoate (3.2 microg/0.1 ml; OB) in peanut oil or 0.1 ml peanut oil alone. PR were measured using [(3)H]ORG 2058, which does not bind to corticosteroid-binding globulin (CBG), and bound and free ligand were separated using minicolumns of Sephadex LH20 at 0 degrees C. PR were measured in cytosols from hypothalamus and uterus of oil-treated BALB/c mice, but were undetectable in thymus, whereas receptors were measurable in all three tissues of MRL mice. There was a significantly greater priming effect of OB on PR in uterus of BALB/c mice, but not in hypothalamus, and PR became detectable in thymus cytosols from BALB/c mice. Also, the apparent affinity of the binding reaction between [(3)H]ORG 2058 and PR was significantly higher than those measured in other tissues in hypothalamic cytosols of both strains. These results suggest that there is an impairment of estrogen priming of progesterone receptors in uterus and perhaps thymus of MRL mice.
    International Journal of Immunopharmacology 08/2000; 22(7):537-45.
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    ABSTRACT: Estrogens exert their effects in reproductive tissues through a primary binding reaction with specific receptor proteins which can be detected and characterized in high speed cytosols. Administered estrogens have profound atrophic effects on the developing thymus and alter thymocyte responsiveness to mitogens in vitro. Estrogen-binding macromolecules have been described in rat thymus cytosol, and an attempt has been made to characterize thoroughly the ligand specificity of these moieties, and compare this with the specificity of the uterus estrogen receptor. Cytosols were prepared from immature rat thymus and uterus and incubated with [3H]estradiol alone or with one of a range of unlabeled steroids: estradiol-17-alpha (E2-17 alpha), estradiol-17-beta (E2-17 beta), diethylstilbestrol (DES), estriol (E3), estrone (E1), corticosterone (C), testosterone (T) or progesterone (P). Scatchard plots were derived from the binding isotherms obtained and the molar dissociation constant (Kdi) for each inhibitor measured. In both thymus and uterus, binding of [3H]estradiol was inhibited most potently by substances possessing estrogen agonist activity, and the rankings of potencies in both tissues were broadly similar, namely: DES > E2-17 beta (thymus); DES = E2-17 beta (uterus). In both tissues, E2-17 beta > E2-17 alpha and E3 > E1 > C > T > P. There were significant differences between thymus and uterus, in that the estrogen receptor in the former tissue exhibited a significantly higher selectivity for some estrogens, including DES and for corticosterone. These differences may underlie differential responsiveness of the two tissues to steroids, and may reflect structural differences between thymus and uterus estrogen receptors.
    International Journal of Immunopharmacology 12/1993; 15(8):927-32.
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    ABSTRACT: Systemic lupus erythematosus (SLE) is a rare disease in males. There is evidence that a functional state of hypoandrogenism is important in the pathogenesis of the disease. We analysed the levels of several hormones (follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2), free estradiol (FE2) and prolactin (PRL)) in 17 male SLE patients and 17 male healthy controls with similar age distribution. Three lupus patients were excluded from the analysis due to previous cyclophosphamide therapy or pre-puberty. Thus 14 male lupus patients were eligible for the study. Six of the 14 SLE patients (43%) showed at least one abnormal level of FSH, LH or T. There were no abnormalities in these hormones in the 17 controls. This difference was significant (P < 0.01). In five of these 6 male patients (36% of all lupus patients) the hormonal profile was compatible with a functional state of hypoandrogenism (high LH and/or low T). The ratio E2/T (estradiol/testosterone:pmol/nmol) was also significantly higher in the SLE group (average = 6.5; SD 4.3) when compared with that of the control group (average 4.2; SD 1.2; Mann-Whitney rank sum test: P < 0.03). There were no significant differences in E2, FE2 or PRL between lupus patients and controls. We did not confirm the notion that left-handedness is frequent in male lupus as all our patients were right-handed. We found a significantly higher prevalence of sex hormone abnormalities in male lupus patients when compared with healthy controls with a similar age distribution.(ABSTRACT TRUNCATED AT 250 WORDS)
    Lupus 10/1993; 2(5):315-7. · 2.78 Impact Factor
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    ABSTRACT: The effects of an aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), which blocks oestrogen formation, have been studied in female MRL/MP-lpr/lpr mice which are a model of SLE. At 11.5 weeks, mice were implanted subcutaneously either with empty Silastic implants or with implants containing 25 mg 4-OHA. At 15 weeks, they were sacrificed by decapitation and liver, thymus, kidneys and uterus taken for wet weight, histology and measurement of cytosolic and nuclear oestrogen receptors. Thymus weights were significantly lower in 4-OHA-treated mice although uterus weights were similar in both groups. Also, whereas thymuses from control-treated mice were packed with plasma cells with abundant cytoplasm, those from 4-OHA-treated mice contained T cells with large nuclei. Relative oestrogen receptor abundances were: uterus > liver > thymus, although cytosolic receptors could not be detected in thymus cytosols of MRL mice unless they were treated with the aromatase inhibitor. In kidney, there was histological evidence that inflammation was limited to mesangium in 4-OHA-treated mice. These results support the hypothesis that oestrogens may be involved in the aetiology of murine SLE and provide data suggesting that substances which block oestrogen production in vivo may be useful to treat certain forms of SLE.
    Lupus 08/1993; 2(4):221-5. · 2.78 Impact Factor
  • B Ansari, P J Coates, B D Greenstein, P A Hall
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    ABSTRACT: We have investigated the use of a novel technique, in situ end-labelling, as a means of the specific identification of apoptotic cells in formalin-fixed, paraffin-processed tissue sections. The technique relies on the presence of DNA strand breaks in apoptotic cells, caused by activation of endogenous nuclease activity during the process of cell death. These strands are labelled with a non-isotopic reporter molecule in the presence of a DNA polymerase, and labelled DNA is identified immunohistochemically. We show that in situ end-labelling stains cells with the morphological characteristics of apoptosis, and greatly simplifies their identification. Furthermore, in two model systems, the number of labelled cells parallels the number of cells undergoing apoptosis as measured by alternative techniques. The ability of the Klenow fragment of DNA polymerase to label apoptotic nuclei suggests that the characteristic DNA fragmentation seen during this process involves the formation of DNA breaks with a 5' overhang. In situ end-labelling will be valuable for the identification and quantitation of apoptosis in a range of normal tissues and in a variety of pathological states. However, the technique is not specific for programmed cell death, and results must be interpreted with caution and correlated with morphological criteria of apoptosis.
    The Journal of Pathology 06/1993; 170(1):1-8. · 7.59 Impact Factor
  • B D Greenstein, E F de Bridges, F T Fitzpatrick
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    ABSTRACT: The thymus can be regenerated in aging rats by surgical or chemical castration and regeneration is inhibited by testosterone, which may exert this effect, at least in part, through its conversion to estradiol. An attempt has been made to regenerate the thymus in intact aging rats using inhibitors of the aromatase system, in the hope that this maneuver could lead to the use of such chemical intervention in the treatment of immunodeficiency syndromes. Young adult and aging (18-month-old) male rats were orchidectomized under ether anesthesia and 7 days later given s.c. implants of testosterone in silicone elastomer (SILASTIC) tubing. Some rats received testosterone together with a five-fold excess of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). One group of young intact rats received implants containing 25 mg ATD and a group of 18-month-old intact rats received 125 mg ATD or 25 mg of another, more powerful aromatase inhibitor 4-hydroxyandrostenedione (4-OH). On the 28th day after implanting, rats were killed and the thymus, spleen, prostate gland and seminal vesicles removed for weighing and histology. In addition, estrogen receptors were measured in the thymus. The thymus was enlarged after orchidectomy and greatly restored in aging rats. In aging rats, both aromatase inhibitors restored the thymus, which appeared normal histologically. In addition, ATD enlarged the thymus in young intact animals. Doses of testosterone which restored the accessory sex organs to weights measured in intact rats prevented the effects of orchidectomy on the thymus, and in old rats the effects of testosterone were blocked by ATD in both thymus and spleen. Available cytosolic estrogen receptors were reduced in thymus of testosterone-treated orchidectomized rats, and this effect blocked by ATD, which itself was apparently able to induce estrogen receptors. Receptors could not be detected in thymus from aging rats, but were measureable in cytosols from thymus of orchidectomized or ATD-treated old rats. It is therefore possible to restore the thymus in intact aging rats without recourse to surgical or chemical castration, and such a maneuver may possibly be of use to enhance an immune system weakened by aging or disease.
    International Journal of Immunopharmacology 06/1992; 14(4):541-53.
  • B D Greenstein, I M Adcock
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    ABSTRACT: There is sexual dimorphism of specific species of mRNA in the neonatal rat brain and this sexual dimorphism may be imprinted by steroids of testicular origin during the perinatal period. According to current theories, only aromatizable androgens may cause sexual differentiation of sexual behaviour and function in the adult. The effects of oestradiol benzoate on mRNA synthesis in the neonatal female limbic system were therefore studied. In addition, cytosolic and nuclear oestrogen receptors were measured after administration of testosterone propionate, oestradiol benzoate or dihydrotestosterone (DHT). An attempt was made to distinguish between the brain oestrogen receptor and the plasma oestrogen-binding protein, alphafoetoprotein (AFP) by isoelectric focussing. After injection of 50 micrograms oestradiol benzoate s.c. to neonatal female rats, the expression of mRNA coding for sexually dimorphic proteins appeared to be changed to a male-type pattern. The overall density of labelling was noticeably greater and specific changes in labelled proteins were observed. These effects were observed within 3 h of injection. Both testosterone and oestradiol caused a marked depletion of cytosolic oestrogen receptors in the limbic system whereas DHT was ineffective in this respect. Nuclear receptors were present in equal abundance in male- and female-derived nuclei and only oestradiol was able to cause a significant (P less than 0.025) increase in nuclear oestrogen receptors. The receptor and AFP could be distinguished by isoelectric focussing, since the pI of the receptor was 7.05, while that of AFP was 4.5. These results are consistent with the possibility that oestradiol alters transcription in the neonatal rat brain and may do this through the oestrogen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
    Journal of Endocrinology 02/1989; 120(1):83-8. · 4.06 Impact Factor
  • B D Greenstein, B J Mander, F T Fitzpatrick
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    ABSTRACT: The hypothesis was tested that the inhibitory action of testosterone on thymus growth is mediated by its metabolism to oestradiol. Immature female rats were given s.c. implants of silicone elastomer tubing containing 5 or 20 mg testosterone alone, or together with 25 or 50 mg of an aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). Some rats received implants containing 5 mg oestradiol or 5 mg dihydrotestosterone (DHT). After 14 days the thymus was removed and weighed. Body weight gain was similar in animals treated with empty implants, or 5 mg testosterone or DHT, or with ATD alone. The combination of testosterone and ATD significantly increased body weight gain, and oestradiol significantly decreased it. Thymus growth was inhibited by both doses of testosterone and by oestradiol, but not by DHT. ATD alone did not inhibit thymus growth, nor did the lower dose of ATD inhibit the action of testosterone. The higher dose of ATD did, however, significantly reduce the inhibitory action of testosterone on the thymus. The inhibitory action of testosterone on the growing thymus may be due, at least in part, to its conversion to oestradiol.
    Journal of Endocrinology 11/1988; 119(1):65-7. · 4.06 Impact Factor
  • F T Fitzpatrick, B D Greenstein
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    ABSTRACT: The effects of several steroids on the regenerating thymus in ageing male rats have been studied. Rats aged from 12 to 15 months were orchidectomized and 7 days later implanted s.c. with silicone elastomer tubing containing 25 mg testosterone, 5 alpha-dihydrotestosterone (DHT), oestradiol, progesterone or corticosterone. One group of rats received an empty implant. Thirty days later the rats were killed and the thymus, spleen, ventral prostate and seminal vesicles weighed and retained for histology. Whole blood was taken for total and differential white cell counts; plasma was prepared for radioimmunoassay of testosterone, oestradiol, progesterone and corticosterone. After orchidectomy only, a multilobular thymus was present, and histologically the tissue appeared healthy. In testosterone- and oestradiol-treated rats, thymus weight was reduced to about 50% of that in untreated animals. Histologically, much of the thymus taken at autopsy was fat and what remained was poorly organized and contained a much lower density of thymocytes. The total white cell count was significantly reduced in these animals, the effect appearing to be predominantly on lymphocytes. Although treatment with DHT also resulted in a lower mean thymus weight than that of orchidectomized animals, histologically the tissue appeared similar to that of the untreated castrated animals. In rats treated with DHT, the total white cell count was significantly higher than in testosterone-implanted rats. Both progesterone and corticosterone implants resulted in significantly smaller mean thymus weights, although these steroids were not as potent as testosterone or oestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)
    Journal of Endocrinology 05/1987; 113(1):51-5. · 4.06 Impact Factor
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    ABSTRACT: It has been shown that the thymus can be regenerated in intact old rats by implanting s.c. a stable analogue of LHRH. Old male rats were given s.c. implants of osmotic pumps containing a solution in citrate buffer of the analogue which was given at a rate of 1 microgram/h for 28 days. Some animals were given pumps containing buffer alone, and another group of rats was orchidectomized. The animals were killed after 28 days and the tissues weighed and taken for histology. Serum testosterone was measured by radioimmunoassay. Sham-treated rats had small fatty thymuses, which were poorly organized with a very narrow band of cortex. Animals treated with the analogue of LHRH and those which had been orchidectomized had relatively large thymuses which were multi-lobed in drug-treated rats, and atrophied accessory sex organs. The testes were grossly atrophied in analogue-treated rats. Histologically, the thymus looked healthy, having a wide, thymocyte-filled cortex and a clearly defined corticomedullary junction. Serum testosterone concentrations were similar in orchidectomized and analogue-treated rats. It is concluded that it is possible to regenerate the thymus in old rats treated with an analogue of LHRH, but the effect is accompanied by chemical castration. It is also clear that the old pituitary gland is susceptible to the desensitizing action of an LHRH analogue.
    Journal of Endocrinology 04/1987; 112(3):345-50. · 4.06 Impact Factor
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    ABSTRACT: The thymus is a critically important organ during development, but atrophies progressively during the ageing process after puberty and is often considered to be unimportant in adult life. We have found that the thymus, which is grossly atrophied in 12- to 15-month-old male rats, is markedly restored in size 30 days after orchidectomy. The organ then appears normal histologically, having a well-defined cortex and medulla, is vascularized and filled with thymocytes. The regeneration of the thymus after orchidectomy was inhibited in a dose-related fashion by testosterone implants which produced serum concentrations of testosterone within the physiological range. The thymus was also increased in size after orchidectomy of 10-week-old rats, and testosterone inhibited the enlargement of the thymus. These results have important implications for the possible enhancement of the immune system with associated improvement of health during ageing and disease. They also point to an important physiological link between the endocrine and immune systems.
    Journal of Endocrinology 10/1986; 110(3):417-22. · 4.06 Impact Factor
  • I M Adcock, B D Greenstein
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    ABSTRACT: The rat brain is sexually dimorphic with respect to structure and function, and there is evidence that these differences are effected in the fetus through changes in protein synthesis, some of which may result from the intervention of gonadal steroids. To investigate this, messenger RNA (mRNA) from the limbic system and cerebellum of neonatal rats was prepared, translated in a rabbit reticulocyte system in vitro and the products were analysed by two-dimensional electrophoresis and fluorography. Some of the results were further analysed using image analysis. There was a striking sexual dimorphism in the patterns of incorporation of [35S]methionine into proteins using mRNA from the limbic system, in that groups of proteins were apparently present in male-but not in female-derived fluorograms and vice versa. One protein, tentatively identified from its coordinates as alpha-tubulin, was more abundant in male-derived fluorograms. Although there were no clear-cut qualitative sex differences using mRNA derived from the cerebellum, that derived from the male cerebellum appeared to be consistently more active. These results provide direct evidence for a sexual dimorphism at the transcriptional level in the neonatal limbic system of the rat.
    Journal of Endocrinology 05/1986; 109(1):23-8. · 4.06 Impact Factor
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    ABSTRACT: There was no visible thymus in ageing rats of 18 months, and 7 days after orchidectomy there was still no evidence of a thymus. By 30 days after the operation, however, there was a well-defined and well developed bilobular thymus overlying the heart, although it was smaller than those observed in 10-week-old rats. Histologically, the tissue appeared normal, was well vascularized, filled with lymphocytes and several mitotic figures were also seen. When compared with sham-operated animals, blood from these animals had a significantly higher lymphocyte count. These results have important implications for the possible enhancement of the immune system with associated improvement of health during ageing.
    Journal of Endocrinology 10/1985; 106(3):R17-9. · 4.06 Impact Factor