Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens.

The concentration of cytoplasmic estrogen receptors in the brain of ovariectomized female rats varies during the light-dark cycle. There is no variation in the affinity of the receptors for estradiol, and the rhythm is not due to estrogens from nonovarian sources. Pentobarbital reverses the reduction of receptors that occurs in the dark, and melatonin injection in the light partially mimics the action of darkness in reducing receptor levels. The factors that cause this rhythm is brain estrogen receptors may be one means by which light affects reproductive function.

Antiestrogen inhibits the induction of progestin receptors by estradiol in the hypothalamus-preoptic area and pituitary.

Estradiol treatment of ovariectomized-adrenalectomized rats produced an increase in cytosol progestin binding in the hypothalamus-preoptic area (HPOA), pituitary, and uterus. In the HPOA and pituitary, this induction of progestin receptors by estradiol was inhibited by the antiestrogen CI-628 under a variety of dose and time conditions. In the uterus, inhibition of the full effect of estradiol on progestin binding was observed after 3 days of injection of antiestrogen and estradiol. In the absence of estradiol, the antiestrogen produced a slight induction of progestin receptors in the HPOA and pituitary and a substantial induction of progestin receptors in the uterus. The results suggest that the induction of progestin receptors could be a key intermediate step in some behavioral and neuroendocrine actions of estradiol.

Characterization of a protein that appears in the nervous system of the moth Manduca sexta coincident with neuronal death.

Two-dimensional gel electrophoresis was used to locate potential neuronal death-related proteins in the moth Manduca sexta. Protein patterns of ganglia of pharate adult moths (taken prior to adult ecdysis) compared with protein patterns of one-day-old adults revealed reproducible changes in protein patterns. An acidic protein of approximately 40,000 Da was present in all samples from adult moths undergoing neuronal death and essentially absent from pharate adult samples.

Oestrogen receptors in cell nuclei of the hypothalamus-preoptic area-amygdala following an injection of oestradiol or the antioestrogen CI-628.

Oestrogen receptors were measured by an exchange assay in cell nuclei of the hypothalamus-preoptic area-amygdala (HPA) of ovariectomized-adrenalectomized (OVX-ADX) rats following an intravenous injection of oestradiol or an antioestrogen, CI-628. Receptors were translocated to the nucleus by both compounds. Receptors translocated by the antioestrogen were specific for oestrogens; testosterone, corticosterone and progesterone were not bound by these receptors. Several properties of antioestrogen- and oestradiol-receptor complexes were compared. The time-course in cell nuclei in vivo showed that receptors were still present in HPA nuclei 24 h after administration of CI-628 but not 24 h after oestradiol. This prolonged increase of nuclear receptors after the antioestrogen treatment was attributed to the continued presence of CI-628 and its metabolites in plasma. The maximum level of receptors produced in the HPA cell nuclei following CI-628 treatment was lower than the peak level of nuclear receptors following oestradiol. The dissociation rate in vitro of nuclear antioestrogen-receptor complexes formed in vivo was more rapid at 0 degrees C than that of nuclear oestradiol-receptor complexes. This property may be related to the lower peak level of binding after CI-628 treatment and to the inhibitory actions of antioestrogens.

Long-term adrenalectomy can decrease or increase hippocampal dentate gyrus volumes.

Male and female Long-Evans adult rats were adrenalectomized and sacrificed 6 weeks later to determine whether dentate gyrus damage would differ in females and males. A subset of adrenalectomized rats of both sexes had significantly reduced dentate gyrus volumes compared to the same sex SHAM operated rats. The remainder of the male and female adrenalectomized rats which did not have clear dentate gyrus damage had significantly larger dentate gyrus volumes compared to the same sex SHAM rats. The dentate gyrus volumes of all adrenalectomized rats were significantly correlated with two indices of residual hormonal levels (Na+/K+ ratios and body weight gain 6 weeks after surgery), indicating that endogenous corticosterone levels may be a determining factor in the response of the dentate gyrus to adrenalectomy. These dentate gyrus volumetric changes could not be attributed to tissue shrinkage as there were no changes in CA3 volumes in any of the groups. These results suggest that long-term adrenalectomy can result in either increased or decreased dentate gyrus volumes and that the adrenal steroid levels of each individual adrenalectomized rat may be the factor determining the direction of the dentate gyrus volumetric response.

Preservation of steroid receptors in frozen brain and pituitary tissue: use of the cryoprotective agent, dimethylsulfoxide.

This paper examines the effects of freezing and thawing on steroid receptor concentrations in the brain and pituitary of the rat. Storage at -70 degrees C for 1-2 weeks had no detectable effect on levels of cytoplasmic estrogen receptors. However, freezing and thawing resulted in measurable losses of cytoplasmic androgen, progestin and glucocorticoid receptors. Cell nuclear receptors were measured by exchange assay after in vivo administration of non-radioactive steroids. Nuclear estrogen, androgen and progestin receptor concentrations were all reduced by freezing compared to the levels in fresh tissue. In all cases except that of cytoplasmic glucocorticoid receptors, these losses could be prevented by freezing the tissue in 10% aqueous dimethylsulfoxide.

Social stimuli augment estrogen receptor binding in preoptic area of female prairie voles.

In female prairie voles ovarian estrogen secretion is stimulated by exposure to males. The present study determined that social stimuli can also enhance the neural response to estrogen. Ovariectomized female voles given a fixed amount of estradiol and exposed to males had higher levels of estrogen receptor binding in cell nuclei in the preoptic area than did females given estrogen and not exposed to males.

Endogenous aldosterone and corticosterone in brain cell nuclei of adrenal-intact rats: regional distribution and effects of physiological variations in serum steroids.

In vivo brain uptake of labeled aldosterone (ALD) and corticosterone (CORT) in adrenalectomized (ADX) rats indicates a strong cell-nuclear localization of both hormones, predominantly in the hippocampus. The primarily limbic concentration of these hormones is also supported by in vitro assays of ALD and CORT binding in cytosol from ADX rats. However, assays of binding in tissues from ADX rats often fail to account for the normal competition of assorted corticosteroids for binding sites in the adrenal-intact subject. Because the binding affinity of corticoid receptors for CORT is greater than, or equivalent to that for ALD, and plasma concentrations of CORT exceed ALD levels, it is possible that ALD is not actually concentrated by brain cell-nuclei in the normal, adrenal-intact subject. Moreover, description of the brain's in vivo regional uptake of ALD or CORT in ADX rats may reflect labeling of heterogeneous binding sites by the single corticosteroid ligand ([3H]ALD or [3H]CORT) under investigation. Research using subcellular fractionation and radioimmunoassay (RIA) has demonstrated the presence of endogenously secreted CORT in brain cell nuclei of adrenal-intact rats, and confirmed the principally limbic localization of endogenous CORT in the brain. In the present study, subcellular fractionation and RIA were employed to determine whether endogenously secreted ALD is concentrated by cell nuclei of the brain in adrenal-intact rats, and to assess the regional variation in the brain's cell-nuclear uptake of endogenously secreted ALD. Cell-nuclear CORT levels were also measured in this experiment to assess the possible competition between ALD and CORT for brain cell-nuclear uptake. Circadian rhythms, stress and dietary sodium were utilized in this study to induce physiological variations in serum ALD and CORT. Endogenous ALD was found in the nuclear fraction of all brain tissues tested, indicating that ALD is bound and translocated to brain cell nuclei in the presence of normal corticosteroid competition. However, brain cell-nuclear ALD appeared not to vary as a function of physiological variation in serum ALD, suggesting that the receptor population was saturated under most normal circumstances. Unexpectedly, the highest cell-nuclear concentrations of endogenous ALD were found in the hypothalamus, rather than hippocampus. This finding suggests that the predominantly hippocampal localization of ALD observed in previous in vivo autoradiographic studies may have provided an inaccurate profile of the loci of ALD action in brain by failing to control for competitive binding by other corticosteroids in the adrenal-intact preparation.

Binding of [3-H]estradiol by brain cell nuclei and female rat sexual behavior: inhibition by antiestrogens.

The antiestrogens MER-25, CI-628, and nafoxidine inhibit the uptake of [3H]-estradiol in whole homogenates and isolated cell nuclei tissues and the pituitary, and inhibit estradiol-induced female sexual behavior. The antiestrogens were injected intraperitoneally 2 h prior to an intravenous injection of [3H]estradiol, and the animals were killed 2 h after the estradiol. CI-628 reduces radioactivity in whole homogenates and isolated cell nuclei of cerebral cortex, hypothalamus, preoptic area -septum and pituitary. Nafoxidine reduces uptake in cell nuclei of the hypothalamus, preoptic area-septum and pituitary. In this paradigm, MER-25 inhibited uptake only in the pituitary. In the analogous behavioral experiments, with antiestrogens injected 2 h prior to an intravenous injection of unesterified estradiol, CI-628 and nafoxidine totally inhibited lordosis responding. MER-25 shows no inhibition of behavior in this paradigm. However, when MER-25 is injected 12 h prior to the estradiol, it inhibits retention of [3H]estradiol at 2 h in brain and pituitary cell nuclei, and lordosis responding is also inhibited. Additionally, the antiestrogens can apparently displace previously bound [3H]estrdiol. When the antiestrogens are injected 2 h prior to an injection of [3H]estradiol, MER-25, CI-628 and nafoxidine all show greater inhibition of nuclear estradiol retention at 12 h after the [3H]estradiol injection than 2 h. Analogously, when CI-628 is injected 2 h after an intravenous injection of [3H]estradiol, it displaces most of the radioactivity present in hypothalamic-preoptic area nuclei at 12 h after the estradiol injection. These results indicate that antiestrogens can prevent or reverse the nuclear concentration of estradiol in brain cells and are consistent with a role of the cell nucleus in the induction of estrous behavior by estradiol.

PIP: In order to clarify the relationship between estradiol binding in the brain and its behavior actions, the effects of 3 antiestrogens (MER-25, CI-628, and nafoxidine) on in vivo tritiated estradiol uptake and retention in cell nuclear fractions or regions of the brain and pituitary were investigated. The ability of antiestrogens to block estradiol-induced female sexual behavior was also studied. Antiestroge were injected 2 hours prior to injection of tritiated estradiol and animals killed 2 hours later. Radioactivity in whole homogenates and isolated cell nuclei of cerebral cortex, hypothalamus, preoptic area-suptum, and pituitary was reduced by CI-628. Nafoxidine reduced uptake in cell nuclei of the hypothalamus, preoptic area-septum, and pituitary. MER-25 only inhibited uptake in the pituitary. CI-628 and nafoxidine totally inhibited lordosis while MER-25 was ineffective when injected 2 hours prior to testing. However, when MER-25 was injected 12 hours prior to testing it inhibited retention of tritiated estradiol in brain and pituitary cell nuclei and lordosis response. When antiestrogens were injected prior to estradiol injection all had greater inhibition of nuclear estradiol retention at 12 hours after the estradiol injection than at 2 hours. When CI-628 was injected 2 hours following injection of tritiated estradiol, most of the radioactivity present in the hypothalamic preoptic area nuclei at 12 hours after the estradiol injection was displaced. Thus antiestrogens can prevent or reverse the nuclear concentration of estradiol in the brain. Female sexual behavior seems to be induced by the presence of estradiol in the cell nucleus.

Inhibition of sexual receptivity by anesthesia during estrogen priming.

Anesthetics such as pentobarbital have been used to infer a role of neural activity in several neuroendocrine responses. The present experiments tested the hypothesis that neural activity during estrogen priming is necessary for the induction of sexual receptivity. Sexual behavior was induced in ovariectomized female rats by two pulses of unesterified estradiol 6 h apart, followed by progesterone at 19 h and testing at 24 h. Anesthetizing animals for approximately 1 h during each exposure to estradiol inhibited the induction of receptivity. Anesthesia immediately before each exposure to estradiol did not interfere with the induction of sexual receptivity. The induction of neural progestin receptors by estradiol was not prevented by anesthesia. Anesthesia had no effect on the uterine weight response to estradiol. These results suggest that neural activity during estrogen priming is required for the induction of sexual receptivity, and point to differences in the mechanism of action of estrogens in the brain and the uterus.

Alpha 1-noradrenergic receptor blockade decreases nuclear estrogen receptor binding in guinea pig hypothalamus and preoptic area.

Prazosin, a putative alpha 1-noradrenergic antagonist, reduced nuclear estrogen receptor levels in the hypothalamus and preoptic area of the estrogen-primed guinea pig. Noradrenergic transmission may alter steroid-dependent processes and neuroendocrine function through actions on steroid-concentrating cells.

Age alters the observed response of imipramine binding sites to chronic antidepressant treatment in female rats.

Age alters hypothalamic imipramine binding sites in female rats. Older females have increased levels of imipramine binding with decreased affinity. The response of hypothalamic imipramine binding sites to chronic antidepressant treatment varies depending on the age of the animals treated. In juvenile female rats imipramine binding sites decrease after antidepressant treatment, consistent with previous reports. However, in young females chronic imipramine treatment produces no change in levels of imipramine binding with a slight decrease in binding affinity. In middle-aged female rats, the same treatment causes an apparent increase in levels of imipramine binding sites along with a decrease in affinity. These decreases in the affinity of imipramine binding sites in older female rats, and the concomitant changes in the levels of imipramine binding are the result of imipramine metabolites remaining in the tissue after antidepressant treatment and interfering with the binding assay, despite extensive washing of the membrane preparation. Thus, the observed changes in the response of imipramine binding sites to chronic imipramine treatment with age are probably due to age-related alterations in the metabolism of imipramine in female rats.

Age alters dopaminergic responses to estradiol.

Young (3-5 months) and middle-aged (17-19 months) ovariectomized rats were treated with estradiol benzoate for three days and striatal dopaminergic function was assessed behaviorally (circling in response to apomorphine) and neurochemically (binding of [3H]spiroperidol by striatal membranes). The results suggest that striatal dopaminergic function may respond differently to estrogen in older animals compared to younger animals.

Targeting tumor cells with bispecific antibodies and T cells.

It has been known for some time that mammalian immune systems are capable of eliminating large tumor burdens. Redirecting the immune response of a patient to an established tumor has now become the focus of various therapeutic strategies. In this report, two projects toward this goal are described. The first project involves the development of a transgenic mouse model for T cell directed therapeutics. These mice express specific T cell receptor alpha and beta transgenes on a background in which the recombinational-activating-gene-1 (RAG) has been knocked out. The mice express cytotoxic T cells but not either T helper cells or B cells. Despite these deficiencies, the animals are capable of eliminating tumors that express the appropriate peptide/major histocompatibility complex ligand that is recognized by the alphabeta transgenic T cell receptor. Human tumors grow as transplants in these mice, thereby allowing various agents that redirect the endogenous T cells against human tumors to be tested. The second project involves a description of such agents: bispecific antibodies that simultaneously bind to an immune effector cell and a tumor cell. The bispecific antibody described here consists of folate attached to anti-T cell receptor antibodies, or their fragments. A single-chain Fv coupled with folate can redirect the lysis of human tumor cells that bear the high affinity folate receptor. Preliminary in vivo data showed that the folate/antibody conjugates were also capable of mediating rejection of the human tumor. This transgenic mouse model should now allow the evaluation and optimization of bispecific agents that can redirect a patient's own T cell response.

Direct effects of ovarian hormones on antidepressant binding sites.

Tricyclic antidepressants such as imipramine interact with a membrane binding site associated with the uptake of serotonin. Ovarian hormones estradiol and progesterone were found to affect the concentration and affinity of imipramine binding sites in the hypothalamus but not the cortex of female rats. In vivo and in vitro, estradiol increased the amount of imipramine binding at physiological concentrations; at high concentrations estradiol decreased imipramine binding. Ovariectomy (OVX) had no effect on imipramine binding, consonant with the biphasic dose-response relationship for estradiol. The effects of progesterone were dependent upon the concentration of estradiol. Effects of physiological concentrations of both estradiol and progesterone on imipramine binding in an isolated membrane preparation suggest that the hormones are affecting this aspect of serotonergic neuronal function by nongenomic mechanisms.

Estradiol in the striatum: effects on behavior and dopamine receptors but no evidence for membrane steroid receptors.

Estradiol was applied directly to the striatum of ovariectomized female rats by a unilateral intracerebral cannula for three hr or four days. Following four days of estradiol treatment, rats increased the number of rotations in the direction away from the side of the hormone treatment. Cholesterol-treated animals did not change their rotational behavior. Dopamine receptors were assayed in the same animals by autoradiography; D2 receptors increased on the hormone-treated side relative to the untreated side after four days of treatment, only in the lateral striatum. D1 dopamine receptors did not change. The D2:D1 receptor ratio was related to the direction of rotation. Measurements of membrane fluidity with a fluorescent probe revealed no effect of estradiol on striatal membrane fluidity. Membrane proteins were labeled with estrogen agonist and antagonist affinity labels and analyzed by gel electrophoresis, but no saturable membrane binding sites were detected. The results indicate that estradiol acts directly in the striatum to affect behavior and dopamine receptors, but the neurochemical mechanisms remain to be determined.

Pharmacokinetics of imipramine are affected by age and sex in rats.

Chronic treatment with the antidepressant imipramine (IMI) leads to accumulation of imipramine's major metabolite desmethylimipramine (DMI) in the brain. Juvenile, young and middle-aged female rats, as well as juvenile and young male rats were treated chronically with imipramine (14 days) and analyzed 24 hours later for levels of IMI and DMI in the hypothalamus-preoptic area (HPA) and serum. Older animals of both sexes showed higher levels of DMI than juvenile animals, in both the HPA and serum. Females also had higher DMI levels than males at comparable ages. Analysis of IMI and DMI levels at intervals after a single imipramine injection suggested that the initial metabolism of imipramine is slower in older animals and in females (compared to males). The results indicate that age and gender alter the initial metabolism of imipramine, leading to enhanced accumulation of metabolites during chronic treatment in older animals and in female rats, compared to younger rats and males, respectively.

An exchange assay for estrogen receptors in cell nuclei of the adult rat brain.

A nuclear exchange assay was developed for brain estrogen receptors. The assay employs an extraction procedure which solubilizes essentially all of the estradiol from brain cell nuclei: purified cell nuclei are evenly dispersed in a hypotonic buffer prior to the addition of an equal volume of 0.8M KC1. Experiments with an in vivo injection of 3H-estradiol established that this procedure extracts even normally salt-resistant binding. For exchange, aliquots of the extract are incubated with 3H-estradiol or 3h-estradiol plus 100-fold excess unlabeled estradiol. Bound 3H-estradiol is separated from free 3H-estradiol on Sephdex LH-20 columns. Loss of estradiol binding activity can occur with brain nuclear extracts under conditions required for exchange. This loss of binding is inhibited by the addition of bacitracin to the incubation buffer. The exchange is complete within 5 hrs at 25 degrees C and specific binding activity is stable for at least 16 hrs. The assay was validated by comparing levels of macromolecular-bound radioactivity after an in vivo injection of 3H-estradiol and levels determined by exchange after an injection of unlabeled estradiol. Scatchard analysis confirmed the high affinity nature of the binding measured by exchange.