Henry Uhlman Bryant

Immunosuppressive Effects of Chronic Morphine Treatment in Mice

In this report we describe the immunomodulatory effects of subcutaneous morphine pellets in mice, a model commonly used in the study of opiate tolerance and dependence. Mice given a single 75 mg morphine pellet displayed marked atrophy and reduced cellularity of the spleen and thymus, and an attenuated lymphocyte proliferative response to T- and B-cell mitogens (concanavalin A and bacterial lipopolysaccharide, respectively). These immunosuppressive effects were observed 72 hr following implantation of the pellet, a time point by which the mice also had developed tolerance to the antinociceptive effect of the pellet. Splenic and thymic atrophy with reduced mitogen-induced lymphocyte proliferative responses and opiate tolerance were also apparent in mice subjected to a multiple pellet implantation schedule. However, implantation of a pellet containing 37.5 mg morphine did not suppress mitogen-stimulated lymphocyte proliferation, which was slightly elevated in this group. These findings concur with other observations suggesting immunosuppression with morphine tolerance. Furthermore, we suggest that chronic morphine treatment acts as a pharmacologic stressor that mimics behavioral stress.

An estrogen receptor basis for raloxifene action in boneProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

Although controversy remains regarding direct effects of estrogen on bone, in vivo data clearly show that estrogens suppress bone turnover, resulting in decreased bone resorption and formation activity. Selective estrogen receptor modulators (SERMs), such as raloxifene, produce effects on bone which are very similar to those of estrogen. In vitro, both raloxifene and estrogen inhibit mammalian osteoclast differentiation and bone resorption activity, but only in the presence of IL-6. Data from a number of ovariectomized rat model manipulations (i.e. hypophysectomy, low calcium diet and drug combinations) demonstrate a strong parallel between the antiosteopenic effects of raloxifene and estrogen. A characteristic action of estrogens on the skeleton is inhibition of longitudinal bone growth, an effect which is not observed with other resorption inhibitors, including calcitonin and bisphosphonates. Consistent with an estrogen-like mechanism on bone, raloxifene inhibits longitudinal bone growth in growing rats. In addition to the overall similarity of the bone activity profile in animals, estrogen and raloxifene also produce similar effects on various signaling pathways relative to the antiosteopenic effect of these two agents. For example, IL-6, a cytokine involved in high turnover bone resorption following estrogen deficiency in rats, is suppressed by both raloxifene and estrogen. Raloxifene and estrogen also produce a similar activation of TGF-beta3 (a cytokine associated with inhibition of osteoclast differentiation and activity) in ovariectomized rats. Like 17beta-estradiol, raloxifene binds with high affinity to both estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta). Crystal structure analyses have shown that 17beta-estradiol and raloxifene bind to ER alpha with small, but important, differences in three dimensional structure. These subtle differences in the conformation of the ligand:receptor complex are likely the basis for the key pharmacological differences between estrogens and the various SERMs (i.e. raloxifene vs tamoxifen). Raloxifene also produces estrogen-like effects on serum cholesterol metabolism and the vasculature. Thus, while raloxifene exhibits a complete estrogen antagonist in mammary tissue and the uterus, it produces beneficial effects on the cardiovascular system and prevents bone loss via an estrogen receptor mediated mechanism.

Morphine-induced immunomodulation is not related to serum morphine concentrations☆

Morphine pellets produced atrophy of the spleen and thymus and affected mitogen-induced lymphocyte proliferation in mice as characterized by marked suppression of concanavalin A-induced blastogenesis at 48 h, and mild stimulation at 120 h. Morphine blood levels in these animals indicated that changes in the immunomodulatory effects of morphine over time were not related to dramatic shifts in circulating morphine. Enclosure of the pellet in nylon mesh did not alter blood levels or morphine-induced immunomodulation.

Mechanism of action of estrogens and selective estrogen receptor modulators

Estrogen, one of several sex steroid hormones, mediates its actions through the estrogen receptor. The estrogen receptor (ER) has two subtypes, ER alpha and ER beta, each of which predominates in specific tissues and organs. Cofactor proteins interact with the ER to maximize ligand-dependent transactivation of target-gene promoters. The estrogen response element is the final step in estrogen-mediated gene regulation, and current research is focused on alternate response elements. The resulting biologic action can vary according to the specific type of ER, cofactor milieu, response element, and ligand. Selective estrogen receptor modulators (SERMs) exhibit tissue-specific estrogen agonist or antagonist activity. The SERM raloxifene, which binds to ER and targets a distinct DNA element, may distinguish agonist vs antagonist activity by ER subtype and has unique activity among other SERMs because of its molecular conformation. Phytoestrogens, a potential alternative to hormone replacement therapy and for cancer prevention, do not consistently mimic estrogens activity. Different types of phytoestrogens have different potencies, and taking high-dose supplements after menopause may not emulate the apparent benefits of lifelong consumption of phytoestrogen-rich diets. In conclusion, the complexity of estrogen action--through different ER subtypes, with various cofactors, on alternate response element--is further enhanced by ligands with selective estrogen activity. Additional research is needed to elucidate these pathways and the resulting biological effects.

Long‐Term Dosing of Arzoxifene Lowers Cholesterol, Reduces Bone Turnover, and Preserves Bone Quality in Ovariectomized Rats

Long‐term effects of a new selective estrogen receptor modulator (SERM) arzoxifene were examined in ovariectomized (OVX) rats. Arzoxifene was administered postoperatively (po) at 0.1 mg/kg per day or 0.5 mg/kg per day to 4‐month‐old rats, starting 1 week after OVX for 12 months. At study termination, body weights for arzoxifene groups were 16–17% lower than OVX control, which was caused by mainly reduced gain of fat mass. Longitudinal analysis of the proximal tibial metaphysis (PTM) by computed tomography (CT) at 0, 2, 4, 6, 9, and 12 months showed that OVX induced a 22% reduction in bone mineral density (BMD) at 2 months, which narrowed to a 12% difference between sham‐operated (sham) and OVX rats by 12 months. Both doses of arzoxifene prevented the OVX‐induced decline in BMD. Histomorphometry of the PTM showed that arzoxifene prevented bone loss by reducing osteoclast number in OVX rats. Arzoxifene maintained bone formation indices at sham levels and preserved trabecular number above OVX controls. Micro‐CT analysis of lumbar vertebrae showed similar preservation of BMD compared with OVX, which were not different from sham. Compression testing of the vertebra and three‐point bending testing of femoral shaft showed that strength and toughness were higher for arzoxifene‐treated animals compared with OVX animals. Arzoxifene reduced serum cholesterol by 44–59% compared with OVX. Uteri wet weight from arzoxifene animals was 38–40% of sham compared with OVX rats, which were 29% of sham. Histology of the uterine endometrium showed that cell heights from both doses of arzoxifene were not significantly different from OVX controls. In summary, treatment of OVX rats with arzoxifene for nearly one‐half of a lifetime maintained beneficial effects on cholesterol and the skeleton. These data suggest that arzoxifene may be a useful therapeutic agent for osteoporosis in postmenopausal women.

Prolactin and Immune Function

This chapter discusses the role of prolactin in immune function. The release of prolactin from pituitary lactotrophes in vivo varies according to time of day and is further modulated by behavioral and environmental stimuli, the reproductive cycle, steroid hormones, and possibly by immune cytokines. Among the primary neuroendocrine processes involved in regulating circulating levels of prolactin, tonic inhibition of the hormone results from the release of dopamine from impinging on dopamine-2 (DA-2) receptors located on lactotrophe cells of the anterior pituitary. At the cellular level, DA-2 receptor-mediated inhibition of adenylate cyclase results in decreased cyclic AMP and ultimately decreased prolactin release. Secretion of pituitary prolactin seems to be necessary for normal immune function in rodents, particularly when stress or infection results in increased secretion of immunosuppressive glucocorticoids. A prolactin-like protein also appears to be produced in an autocrine fashion by lymphocytes and may endow these cells with competence to progress through the cell cycle. Drugs that modify prolactin secretion, many of which are in common use in medicine, may have the ability to modify the immune function.

Prolactin and prolactin secretagogues reverse immunosuppression in mice treated with cysteamine, glucocorticoids, or cyclosporin-A

Suppression of prolactin (PRL) secretion with the dopamine agonist, bromocriptine, has been shown in rodents to diminish a variety of immunologic responses, including delayed type hypersensitivity, primary antibody response, T-cell dependent macrophage activation, and ex vivo T- and B-lymphocyte proliferation in response to mitogens. These same responses can be suppressed by endogenous or exogenous glucocorticosteroids and, in large measure, the immunosuppressant peptide cyclosporin A. The sulfhydryl reducing agent cysteamine (2-aminoethanethiol) is known to reduce pituitary and plasma prolactin levels. Treatment of mice with cysteamine at doses which suppressed circulating PRL levels resulted in suppression of ex vivo blastogenic responses of lymphocytes from treated mice. The T-cell-dependent primary IgM response to immunization with sheep red blood cells was also suppressed by cysteamine treatment. Treatment of mice with drugs stimulating the release of endogenous PRL, or with exogenous ovine PRL, was found to antagonize the suppression of lymphocyte proliferative responses to mitogens induced in mice by glucocorticoid or cyclosporin treatment. These data suggest that many drugs in common clinical use could have potential immunomodulatory actions due to suppression or stimulation of pituitary PRL secretion. Furthermore, lactogenic hormones appear to exert counterregulatory actions which may modify glucocorticosteroid actions on immune and other target issues.

DNA microarray data integration by ortholog gene analysis reveals potential molecular mechanisms of estrogen-dependent growth of human uterine fibroids

BackgroundUterine fibroids or leiomyoma are a common benign smooth muscle tumor. The tumor growth is well known to be estrogen-dependent. However, the molecular mechanisms of its estrogen-dependency is not well understood.MethodsDifferentially expressed genes in human uterine fibroids were either retrieved from published papers or from our own statistical analysis of downloaded array data. Probes for the same genes on different Affymetrix chips were mapped based on probe comparison information provided by Affymetrix. Genes identified by two or three array studies were submitted for ortholog analysis. Human and rat ortholog genes were identified by using ortholog gene databases, HomoloGene and TOGA and were confirmed by synteny analysis with MultiContigView tool in the Ensembl genome browser.ResultsBy integrated analysis of three recently published DNA microarray studies with human tissue, thirty-eight genes were found to be differentially expressed in the same direction in fibroid compared to adjacent uterine myometrium by at least two research groups. Among these genes, twelve with rat orthologs were identified as estrogen-regulated from our array study investigating uterine expression in ovariectomized rats treated with estrogen. Functional and pathway analyses of the twelve genes suggested multiple molecular mechanisms for estrogen-dependent cell survival and tumor growth. Firstly, estrogen increased expression of the anti-apoptotic PCP4 gene and suppressed the expression of growth inhibitory receptors PTGER3 and TGFBR2. Secondly, estrogen may antagonize PPARγ signaling, thought to inhibit fibroid growth and survival, at two points in the PPAR pathway: 1) through increased ANXA1 gene expression which can inhibit phospholipase A2 activity and in turn decrease arachidonic acid synthesis, and 2) by decreasing L-PGDS expression which would reduce synthesis of PGJ2, an endogenous ligand for PPARγ. Lastly, estrogen affects retinoic acid (RA) synthesis and mobilization by regulating expression of CRABP2 and ALDH1A1. RA has been shown to play a significant role in the development of uterine fibroids in an animal model.ConclusionIntegrated analysis of multiple array datasets revealed twelve human and rat ortholog genes that were differentially expressed in human uterine fibroids and transcriptionally responsive to estrogen in the rat uterus. Functional and pathway analysis of these genes suggest multiple potential molecular mechanisms for the poorly understood estrogen-dependent growth of uterine fibroids. Fully understanding the exact molecular interactions among these gene products requires further study to validate their roles in uterine fibroids. This work provides new avenues of study which could influence the future direction of therapeutic intervention for the disease.

A nonsecosteroidal vitamin D receptor ligand with improved therapeutic window of bone efficacy over hypercalcemia

Vitamin D3 analogues were shown to be beneficial for osteoporosis and other indications, but their narrow therapeutic window between efficacy and hypercalcemia has limited their clinical utility. A nonsecosteroidal, tissue‐selective, orally bioavailable, vitamin D receptor (VDR) ligand was ascertained to be efficacious in bone while having modest calcemic effects in vivo. This compound (VDRM2) potently induced Retinoid X Receptor alpha (RXR)‐VDR heterodimerization (EC50 = 7.1 ± 1.6 nM) and induced osteocalcin promoter activity (EC50 = 1.9 ± 1.6 nM). VDRM2 was less potent in inducing Ca2+ channel transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression (EC50 = 37 ± 12 nM). VDRM2 then was evaluated in osteopenic ovariectomized (OVX) rats and shown to dose‐dependently restore vertebral bone mineral density (BMD) from OVX to sham levels at 0.08 µg/kg per day. Hypercalcemia was observed at a dose of 4.6 µg/kg per day of VDRM2, suggesting a safety margin of 57 [90% confidence interval (CI) 35–91]. 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D], ED71, and alfacalcidol restored BMD at 0.030, 0.0055, and 0.046 µg/kg per day, respectively, whereas hypercalcemia was observed at 0.22, 0.027, and 0.23 µg/kg per day, indicating a safety margin of 7.3, 4.9, and 5.0, respectively (90% CIs 4.1–13, 3.2–7.7, and 3.5–6.7, respectively). Histomorphometry showed that VDRM2 increased cortical bone area and stimulated the periosteal bone‐formation rate relative to OVX at doses below the hypercalcemic dose. By contrast, ED71 increased the periosteal bone‐formation rate only above the hypercalcemic dose. VDRM2 suppressed eroded surface on trabecular bone surfaces at normal serum calcium dosage levels, suggesting dual anabolic and antiresorptive activity. In summary, vitamin D analogues were more potent than VDRM2, but VDRM2 had a greater safety margin, suggesting possible therapeutic potential.

A novel class of 5-HT2a receptor antagonists: Aryl aminoguanidines

Local delivery of serotonin (5-HT) produces a rapid edematous response in soft tissues via increased fluid extravasation which is prevented by 5-HT2 antagonists such as ketanserin or mianserin. Here we report the effects of a new class of aminoguanidine 5-HT2 antagonists, with relative selectivity for 5-HT2A receptors which are potent inhibitors of 5-HT-induced paw edema in the rat. Radioligand binding studies with 125I DOI on human 5-HT2A and 5-HT2C receptors and with 3H-5-HT on human 5-HT2B receptors demonstrated that, LY314228, and LY320954 displayed some selectivity for the 5-HT2A receptor. When compared to binding at other 5-HT2 receptor subtypes, LY314228 had an 18.6-fold greater affinity for the 5-HT2A site over the 5-HT2B site, and 2.6 fold greater at the 5-HT2C site. LY320954 displayed similar preference for 5-HT2A sites. Both compounds also inhibited 5-HT-induced paw swelling in rats, with ED50s of 6.4 and 4.8 mg/kg (for LY314228 and LY320954, respectively). These studies offer evidence for a novel class of pharmacophores for the 5-HT2 receptor family which show greater relative affinities for the 5-HT2A receptor subclass.