Reactive astrocytes and Wnt/ß-catenin signaling link nigrostriatal injury to repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

Emerging evidence points to reactive glia as a pivotal factor in Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of basal ganglia injury, but whether astrocytes and microglia activation may exacerbate dopaminergic (DAergic) neuron demise and/or contribute to DAergic repair is presently the subject of much debate. Here, we have correlated the loss and recovery of the nigrostriatal DAergic functionality upon acute MPTP exposure with extensive gene expression analysis at the level of the ventral midbrain (VM) and striata (Str) and found a major upregulation of pro-inflammatory chemokines and wingless-type MMTV integration site1 (Wnt1), a key transcript involved in midbrain DAergic neurodevelopment. Wnt signaling components (including Frizzled-1 [Fzd-1] and ß-catenin) were dynamically regulated during MPTP-induced DAergic degeneration and reactive glial activation. Activated astrocytes of the ventral midbrain were identified as candidate source of Wnt1 by in situ hybridization and real-time PCR in vitro. Blocking Wnt/Fzd signaling with Dickkopf-1 (Dkk1) counteracted astrocyte-induced neuroprotection against MPP(+) toxicity in primary mesencephalic astrocyte-neuron cultures, in vitro. Moreover, astroglial-derived factors, including Wnt1, promoted neurogenesis and DAergic neurogenesis from adult midbrain stem/neuroprogenitor cells, in vitro. Conversely, lack of Wnt1 transcription in response to MPTP in middle-aged mice and failure of DAergic neurons to recover were reversed by pharmacological activation of Wnt/ß-catenin signaling, in vivo, thus suggesting MPTP-reactive astrocytes in situ and Wnt1 as candidate components of neuroprotective/neurorescue pathways in MPTP-induced nigrostriatal DAergic plasticity.

Experimental demonstration of novel beam characterization using a polarizable X-band transverse deflection structure.

The PolariX TDS (Polarizable X-Band Transverse Deflection Structure) is an innovative TDS-design operating in the X-band frequency-range. The design gives full control of the streaking plane, which can be tuned in order to characterize the projections of the beam distribution onto arbitrary transverse axes. This novel feature opens up new opportunities for detailed characterization of the electron beam. In this paper we present first measurements of the Polarix TDS at the FLASHForward beamline at DESY, including three-dimensional reconstruction of the charge-density distribution of the bunch and slice emittance measurements in both transverse directions. The experimental results open the path toward novel and more extensive beam characterization in the direction of multi-dimensional-beam-phase-space reconstruction.

Experimental demonstration of emittance compensation with velocity bunching.

In this Letter we report the first experiments aimed at the simultaneous demonstration of the emittance compensation process and velocity bunching in a high brightness electron source, the SPARC photoinjector in INFN-LNF. While a maximum compression ratio up to a factor 14 has been observed, in a particular case of interest a compression factor of 3, yielding a slice current of 120 A with less than 2 microm slice emittance, has been measured. This technique may be crucial in achieving high brightness beams in photoinjectors aiming at optimized performance of short wavelength single-pass free electron lasers or other advanced applications in laser-plasma accelerators.

Transcriptional changes induced by bovine papillomavirus type 1 in equine fibroblasts.

Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.

Endoscope drying/storage cabinet: interest and efficacy.

Inadequate drying of endoscope channels is a possible cause of microbial proliferation during storage. This risk could be reduced by any procedure or process used to dry endoscope channels and control storage conditions. The efficacy of a drying and storage cabinet (Hysis Medical) was tested on three different endoscopes: a colonoscope (Olympus); duodenoscope (Fujinon) and an enteroscope (Pentax), all of which had been artificially contaminated with a suspension of Pseudomonas aeruginosa CIP 103467. Changes to the residual internal contamination level of these endoscopes when stored inside or outside the drying cabinet for 12, 24, 48 or 72 h were compared. When stored in the drying and storage cabinet, microbial contamination levels on endoscopes were lower than the number of bacteria initially introduced and could decrease considerably thereafter. For endoscopes stored outside the drying storage cabinet, microbial numbers were stable or even increased. These data demonstrate the advantages of such endoscope drying/storage cabinets that limit the risk of bacterial proliferation in the internal channels of endoscopes during storage, and which ensure that the disinfection level reached at the end of the reprocessing procedure is maintained.

Comparison of the fixative properties of five disinfectant solutions.

Following a French circular published in 2001, the use of glutaraldehyde for the disinfection of reusable medical devices was abandoned in favour of non-fixative disinfectants such as peracetic-acid-based solutions. Data published regarding the fixative properties of alternative disinfectants remain contradictory. We compared the effect of repetitive treatments of polytetrafluoroethylene (PTFE) tubes, contaminated by a liquid medium inoculated with Pseudomonas aeruginosa, using five different disinfectant solutions: two peracetic acid solutions (with and without an activator), glutaraldehyde, ortho-phthaldehyde and succine dialdehyde. The results confirmed that repeated treatments of a PTFE tube with a 2% glutaraldehyde solution induce an important accumulation and/or fixation of protein, compared to peracetic-acid-based disinfectants, for which the accumulation and/or fixation of proteins remain low and vary from one formulation to another.

The E5 protein of BPV-4 interacts with the heavy chain of MHC class I and irreversibly retains the MHC complex in the Golgi apparatus.

BPV-4 E5 inhibits transcription of the bovine MHC class I heavy chain (HC) gene, increases degradation of HC and downregulates surface expression of MHC class I by retaining the complex in the Golgi apparatus (GA). Here we report that transcription inhibition can be alleviated by interferon treatment and the degradation of HC can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC class I to the cell surface is irreversible. We show that E5 is capable of physically interacting with HC. Together with the inhibition of the vacuolar ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and HC is likely to be responsible for retention of MHC class I in the GA. C-terminus deletion mutants of E5 are incapable of either downregulating surface MHC class I or interacting with HC, establishing that the C-terminus domain of E5 is important in the inhibition of MHC class I.

Estrogen, neuroinflammation and neuroprotection in Parkinson's disease: glia dictates resistance versus vulnerability to neurodegeneration.

Post-menopausal estrogen deficiency is recognized to play a pivotal role in the pathogenesis of a number of age-related diseases in women, such as osteoporosis, coronary heart disease and Alzheimer's disease. There are also sexual differences in the progression of diseases associated with the nigrostriatal dopaminergic system, such as Parkinson's disease, a chronic progressive degenerative disorder characterized by the selective degeneration of mesencephalic dopaminergic neurons in the substancia nigra pars compacta. The mechanism(s) responsible for dopaminergic neuron degeneration in Parkinson's disease are still unknown, but oxidative stress and neuroinflammation are believed to play a key role in nigrostriatal dopaminergic neuron demise. Estrogen neuroprotective effects have been widely reported in a number of neuronal cell systems including the nigrostriatal dopaminergic neurons, via both genomic and non-genomic effects, however, little is known on estrogen modulation of astrocyte and microglia function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. We here highlight estrogen modulation of glial neuroinflammatory reaction in the protection of mesencephalic dopaminergic neurons and emphasize the cardinal role of glia-neuron crosstalk in directing neuroprotection vs neurodegeneration. In particular, the specific role of astroglia and its pro-/anti-inflammatory mechanisms in estrogen neuroprotection are presented. This study shows that astrocyte and microglia response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injury vary according to the estrogenic status with direct consequences for dopaminergic neuron survival, recovery and repair. These findings provide a new insight into the protective action of estrogen that may possibly contribute to the development of novel therapeutic treatment strategies for Parkinson's disease.

GSK-3ß-induced Tau pathology drives hippocampal neuronal cell death in Huntington's disease: involvement of astrocyte-neuron interactions.

Glycogen synthase kinase-3ß (GSK-3ß) has emerged as a critical factor in several pathways involved in hippocampal neuronal maintenance and function. In Huntington's disease (HD), there are early hippocampal deficits both in patients and transgenic mouse models, which prompted us to investigate whether disease-specific changes in GSK-3ß expression may underlie these abnormalities. Thirty-three postmortem hippocampal samples from HD patients (neuropathological grades 2-4) and age- and sex-matched normal control cases were analyzed using real-time quantitative reverse transcription PCRs (qPCRs) and immunohistochemistry. In vitro and in vivo studies looking at hippocampal pathology and GSK-3ß were also undertaken in transgenic R6/2 and wild-type mice. We identified a disease and stage-dependent upregulation of GSK-3ß mRNA and protein levels in the HD hippocampus, with the active isoform pGSK-3ß-Tyr(216) being strongly expressed in dentate gyrus (DG) neurons and astrocytes at a time when phosphorylation of Tau at the AT8 epitope was also present in these same neurons. This upregulation of pGSK-3ß-Tyr(216) was also found in the R6/2 hippocampus in vivo and linked to the increased vulnerability of primary hippocampal neurons in vitro. In addition, the increased expression of GSK-3ß in the astrocytes of R6/2 mice appeared to be the main driver of Tau phosphorylation and caspase3 activation-induced neuronal death, at least in part via an exacerbated production of major proinflammatory mediators. This stage-dependent overactivation of GSK-3ß in HD-affected hippocampal neurons and astrocytes therefore points to GSK-3ß as being a critical factor in the pathological development of this condition. As such, therapeutic targeting of this pathway may help ameliorate neuronal dysfunction in HD.

The immune response evokes up- and down-modulation of beta 2-adrenergic receptor messenger RNA concentration in the male rat thymus.

Important alterations of noradrenergic activity are known to occur in specific brain regions and in different lymphoid tissues during the course of an immune response. Our recent characterization of the beta 2-adrenergic receptor (beta 2AR)-cAMP system of the rat thymus gland, the identification of a thymic beta 2AR gene expression, and the marked modulation of receptor mRNA concentration after castration and replacement with estrogen prompted us to study the ability of products of immune axis activation to modulate beta 2AR number, distribution, and expression in the male rat thymus. Moreover, the effect of adrenergic stimulation of adenylyl cyclase activity on thymus gland membrane preparations was measured. The beta 2AR present in the rat thymus undergoes marked changes in both number and distribution during the course of an immune response. At 3 days after antigenic challenge (injection of BSA in complete Freund's adjuvant), a sharp decrease of receptor number coupled with a significant loss of the autoradiographic reaction in the medullary compartment of the rat thymus gland were observed. These effects were followed by a significant increase in receptor density and number without changes in receptor affinity at 7 and 15 days post immunization, corresponding to the pick of the immune response. Parallel alterations in adenylyl cyclase activity were measured. Northern blot analysis, using a human beta 2AR cDNA as a probe, revealed dramatic alterations of the beta 2AR mRNA in the thymus, characterized by an approximately 75% decrease of mRNA level 3 days after immunization, and by a progressive increase at 7 and 15 days, with beta 2AR mRNA concentration rising to levels even higher than those found in control animals. These results suggest that the immune response evokes marked alterations of the thymic beta 2AR-cAMP signaling pathway. Moreover, antigenic stimulation triggers a down- and up-modulation of beta 2AR gene expression. Although it is presently unknown whether factor(s) released by immune axis activation act at the level of gene transcription to modulate adrenergic receptor function in the rat thymus, such down- and up-regulation of beta 2AR mRNA may play a role in the dynamic regulation of the immune response.

Novel schemes for the optimization of the SPARC narrow band THz source.

A pulsed, tunable, narrow band radiation source with frequency in the THz region can be obtained collecting the coherent transition radiation produced by a train of ultra-short electron bunches having picosecond scale inter-distance. In this paper, we review the techniques feasible at the SPARC_LAB test facility to produce and manipulate the requested train of electron bunches and we examine the dynamics of their acceleration and compression. In addition, we show how the performances of the train compression and the radiation intensity and bandwidth can be significantly improved through the insertion of a fourth order harmonic cavity, working in the X-band and acting as a longitudinal phase space linearizer.

Hormonal regulation of beta-adrenergic receptors in the rat mammary gland during the estrous cycle and lactation: role of sex steroids and prolactin.

To gain further knowledge on the role of ovarian hormones in the regulation of mammary beta-adrenergic receptors, virgin animals were killed during the various phases of the estrous cycle as well as after ovariectomy and treatment with sex steroid. beta-Adrenergic receptor levels fluctuate in the rat mammary gland during the estrous cycle, with higher receptor numbers during the proestrous and estrous phases of the cycle. Ovariectomy caused an almost 50% loss of beta-adrenergic receptor concentration in the mammary gland of virgin rats. Treatment of ovariectomized animals with 17 beta-estradiol or progesterone alone or in combination for 3 weeks induced a marked increase in beta-adrenergic receptor concentration, while administration of the androgen dihydrotestosterone did not modify mammary beta-adrenergic binding sites. While levels of beta-adrenergic receptors in control lactating animals (10 days of lactation) were elevated, chronic treatment with the dopaminergic-mimetic agent 2 alpha-bromoergocryptine (CB-154; for 7 days) reduced beta-adrenergic receptor concentration. Castration of lactating animals decreased beta-adrenergic receptor number to approximately 30% of the value in intact controls, while combined withdrawal of circulating ovarian hormones and inhibition of plasma PRL levels caused an almost complete inhibition of beta-adrenergic receptor concentration. Scatchard analysis of the binding data revealed that the observed alterations in beta-adrenergic receptors resulted from changes in the number of beta-adrenergic binding sites, with no change in binding affinities. The present findings indicate that the beta-adrenergic receptor population of the rat mammary gland is under the control of ovarian hormones and PRL and suggest that circulating or locally released catecholamines could interact with sex steroids and PRL in the regulation of mammary gland growth, differentiation, and activity.

Ovarian adrenergic nerves directly participate in the control of luteinizing hormone-releasing hormone and beta-adrenergic receptors during puberty: a biochemical and autoradiographic study.

The rat ovary receives sympathetic innervation from the superior ovarian nerve (SON) and the plexus nerve (OP). To examine the possibility of a direct adrenergic mechanism controlling ovarian receptor distribution during the onset of puberty, we have studied the acute (48-h) effect of unilateral nervotomy (combined section of SON and OP nerves) on ovarian LHRH and beta-adrenergic receptor concentrations and distribution using both radioreceptor assays and in vitro autoradiography. Ovarian LHRH receptor concentration increased sharply between 12 and 20 days of age. At this time receptors were mostly associated with follicles and interstitial cells, whereas at 37 days of age, when a measurable loss in the receptor concentration was observed, light and diffuse autoradiographic labeling of receptors was also found in the corpora lutea. Complete removal of adrenergic input to the gland produced a sharp decrease in LHRH-binding activity within the denervated ovary at each time interval studied, with no effect in the innervated contralateral gland. Autoradiographic data also revealed a decrease in both the number of labeled follicles and the intensity of the labeling. beta-Adrenergic receptor concentration increased progressively between days 12 and 27, reaching a peak value at 37 days of age. Labeling was very weak at 12 days of age and increased progressively at 20 and 27 days of age. At this time, receptors were mostly localized by autoradiography in the interstitial cells, while at 37 days of age corpora lutea were strongly labeled. Ovarian beta-adrenergic receptors showed a marked drop when acutely deprived of their neural tone, as illustrated by the 2- to 3-fold decrease in receptor-binding capacity within the denervated gland. The autoradiographic data also showed marked changes in beta-adrenergic receptor distribution, specially at 37 days of age. At this time, the labeling of corpora lutea was markedly decreased in denervated ovaries. The present results clearly demonstrate that complete removal of ovarian adrenergic tone produces a profound decrease in the concentrations of LHRH and beta-adrenergic receptors within the ovary, although it cannot be excluded that peptidergic factors also arriving via the SON and OP could have some influence on the regulation of these receptors. The results support the concept of a direct involvement of the central nervous system in ovarian function. They also suggest that during ovarian development a neural efferent system might be involved in the adjustment of ovarian responsiveness to stimulation by the gonadotropins via changes in receptor content and/or distribution in the different ovarian compartments.

Luteinizing hormone-releasing hormone signaling at the lymphocyte involves stimulation of interleukin-2 receptor expression.

The marked modulation of lymphocyte function exerted by the hypothalamic decapetide LHRH prompted us to study the possible involvement of the neuropeptide in one of the major steps of lymphocyte proliferation, namely the expression of interleukin-2 (IL-2) receptor during in vitro treatment of rat lymphocytes with LHRH agonists (LHRH-A) or antagonists (LHRH-ANTA). The basal proliferative activity of splenocytes and thymocytes from proestrous female rats was significantly stimulated after incubation with LHRH and LHRH-A, but not LHRH fragments [LHRH-(1-3), LHRH-(1-5), and LHRH-(2-6)]. Similarly, in the absence of the mitogenic stimulus, IL-2 receptor expression was significantly stimulated in thymocyte and splenocyte cultures incubated with increasing doses of LHRH or its agonists. The amplification of Concanavalin-A-induced increase in blastogenic transformation of lymphocytes by LHRH was paralleled by a significant stimulation of IL-2 receptor expression. The specificity of such effect was demonstrated by 1) the failure of LHRH fragments [LHRH-(1-6)] to mimick the LHRH stimulatory effect; and 2) the complete reversal produced by simultaneous addition of a potent LHRH-ANTA on IL-2 receptor expression induced by LHRH. Moreover, basal and lectin stimulation of IL-2 receptor-positive cells were significantly inhibited by treatment with the LHRH-ANTA. These data clearly demonstrate that 1) LHRH induction of lymphocyte activation in vitro is accompanied by a specific increase in IL-2 receptor-positive cells; 2) endogenous lymphocyte LHRH may participate in stimulation of IL-2 receptor expression under both basal and stimulated conditions, suggesting that LHRH signaling at the lymphocyte may interact synergistically with intracellular mechanisms responsible for lymphocyte activation.

Disruption of hypothalamic-pituitary-adrenocortical system in transgenic mice expressing type II glucocorticoid receptor antisense ribonucleic acid permanently impairs T cell function: effects on T cell trafficking and T cell responsiveness during postnatal development.

We used transgenic mice with impaired corticosteroid receptor function, caused by expression of type II glucocorticoid receptor (GR) antisense RNA, to study the role of glucocorticoid feedback during the developmental maturation of hypothalamus-pituitary-adrenal-immune functions. These mice have increased plasma concentrations of ACTH and corticosterone as well as reduced GR binding capacity. In control mice, a strong sex dimorphism in the development of GR gene expression is apparent, and in males between postnatal days 9-36, the GR gene transcript concentration is approximately twice that in female mice. Endogenous GR messenger RNA levels were markedly reduced in transgenic mice, and the sex dimorphism was abolished. An abnormal developmental pattern of adrenal secretory activity accompanied the postnatal maturation of the hypothalamic-pituitary-adrenocortical system of the transgenic mice, and high plasma corticosterone levels were measured at early postnatal ages through adulthood. Inefficient glucocorticoid inhibitory action on the immune axis was supported by both the inability of high circulating levels of corticosterone to reduce thymus weight and the failure of dexamethasone to influence in vitro thymocyte and splenocyte cell proliferation. Alterations in thymocyte trafficking/migration in transgenic mice was supported by flow cytometric analysis of the distribution of phenotypically distinct lymphocyte subsets accompanying the postnatal maturation of the thymus. A marked increase in CD4+CD8+ double positive cells and a 2-fold increase in the CD4/CD8 (helper/suppressor) ratio caused by a 40-60% increase in the CD4+CD8- (T helper) subset and a decrease in the CD4-CD8+ (T suppressor) subset, was seen. Moreover, in transgenic mice, an absence of sexual dimorphism and a significantly increased immune reactivity were observed. The present study shows that disruption of the hypothalamic-pituitary-adrenocortical system has both developmental and permanent effects on T cell function characterized by a shifting of the T cell balance toward the CD4+CD8- helper-inducer phenotype coupled with hyperresponsiveness of the T (helper) cell compartment. These findings point to the GR as a major factor in the counterregulatory feedback loop controlling autoaggressive immune responses and underline the potential modulatory role of sex steroids in this feedback regulation and in the pathogenesis of autoimmune diseases.

Blockade of central and peripheral luteinizing hormone-releasing hormone (LHRH) receptors in neonatal rats with a potent LHRH-antagonist inhibits the morphofunctional development of the thymus and maturation of the cell-mediated and humoral immune responses.

The development of the thymus and the hypothalamic-pituitary-gonadal axis are linked by bidirectional hormonally mediated relationships. In the present study, the direct involvement of the neuropeptide LHRH in the maturation of the thymus and development of the cell-mediated and humoral immune responses were assessed after treatment of neonatal (from post-natal day 1-day 5) female rats with a potent LHRH-antagonist (LHRH-anta, p-Glu-D-Phe 2.6,Pro3-LHRH, 50 micrograms/rat), and the effects compared to those resulting from neonatal castration. Whereas in control animals the maturation of mitogenic potential in thymocyte cultures showed a progressive and age-dependent increase, reaching a maximal activity at 30 days of age and then decreasing after puberty onset, in LHRH-anta-treated rats, the thymocyte's proliferative response was completely blocked at 7 days of age and remained very low at each time interval studied, until 3 months of age. A similar effect of the LHRH-anta treatment on splenocyte cultures was measured. Moreover, a reduced percentage of the T-helper lymphocyte subpopulation followed LHRH-anta administration. By contrast, in neonatally castrated rats, blastogenic activity was significantly higher, compared to control cultures, at each stage studied. Treatment with LHRH-anta produced a significant decrease in thymus wt, an alteration of the maturational pattern characterized by a cellular monomorphism, reduced thymocyte volume, reduction of the cortical area, and depauperation of the epithelial microenvironment. Moreover, a morphometric analysis revealed a selective decrease in the large lymphoid cell population of the subcapsular cortex at 7 and 15 days. On the other hand, neonatal castration produced an opposite effect, leading to a marked hypertrophy of the cortical area, and counteracted the post-puberal thymus atrophy. When LHRH-anta-treated adult (3-month-old) rats were challenged with an antigenic stimulus (multiple sc injections of complete Freund adjuvant and BSA) and antibody (anti-BSA antibodies of the immunoglobulin G class) production measured in the serum after 15 days, a marked and significant decrease in immunoglobulin G levels was observed, compared to the values measured in untreated control. The described immune deficiencies in LHRH-anta-treated rats were associated with a clear inhibition of sexual maturation. This study clearly indicates that the blockade of central and peripheral LHRH receptors during a critical period for maturation of both hypothalamus-hypophyseal-gonadal axis and brain-thymus-lymphoid axis dramatically impairs immune system development, suggesting a potential role of the neuropeptide LHRH in the bidirectional programming of both neuroendocrine and immune functions.

Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function.

The present study was designed to explore the effects of LHRH and its agonists on immune system function. As a first step, to identify a putative site of action, the very potent and stable LHRH agonist (LHRH-A), [D-Ser(TBU6)] des-Gly10-LHRH ethylamide (buserelin), was used as an iodinated ligand to characterize LHRH receptors in a membrane preparation of rat thymus, a key organ of the immune system. The effects of LHRH and LHRH-A were then investigated on the proliferative capacity of rat thymocytes exposed in vitro to a mitogen and on ornithine decarboxylase specific activity. In addition, to determine whether LHRH-A treatment in vivo might directly influence thymic function, we treated hypophysectomized (hypox) rats with a moderately high dose of LHRH-A for a period of 2 weeks, and thymocyte mitogenic capacity, thymus weight, and the histological and functional appearance of the thymus were then assessed. Specific binding of LHRH-A to rat thymic membrane preparations is a saturable process, depending on both time and temperature of incubation, but differs markedly from binding to the rat pituitary or ovarian LHRH receptor in its low binding affinity. Binding is optimal in the absence of chelating agents (EDTA) or divalent metal ions, and increases linearly with increasing protein concentration. Binding is specific for LHRH, LHRH-A, and antagonists. Both the C-terminal amide and N-terminal regions of the LHRH molecule were required for binding, and amino acid substitutions at position 6 markedly enhanced and at position 8 markedly reduced binding potencies in rat thymic tissue. A number of peptides, proteins, and other agents had no effect on the specific binding of LHRH-A to thymic membrane preparations. The binding affinity (Ka) of the membrane receptor of the rat thymus for the LHRH superagonist buserelin was 8.4 x 10(8) M-1, while a higher binding affinity (Ka = 2.8 x 10(9) M-1) was calculated for the ovarian LHRH-binding site. Preincubation of rat thymocytes with LHRH-A for 20 h induced a significant dose-dependent increase in the proliferative response to the mitogen Concanavalin-A, monitored by [3H]thymidine incorporation. Using native LHRH, it was also possible to elicit stimulatory effects on the same parameter, although much higher concentrations were required than with LHRH-A. Furthermore, simultaneous addition of a LHRH antagonist, abolished the LHRH effect on thymocytes. Ornithine decarboxylase specific activity under lectin stimulation was also significantly increased by LHRH-A in cultures of rat thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta-adrenergic receptors in the rat mammary gland during pregnancy and lactation: characterization, distribution, and coupling to adenylate cyclase.

To investigate a possible role of catecholamines in mammary gland growth and differentiation, we have studied the characteristics of a specific beta-adrenergic receptor population during the different reproductive phases of the rat mammary gland, namely pregnancy and lactation. The functional response to mammary beta-adrenergic receptor stimulation was assessed by measurement of adenylate cyclase activity during the same physiological states of the gland [125I]Cyanopindolol (CYP) binds specifically to membranes prepared from lactating mammary glands. Scatchard analysis of the binding data shows the presence of a single class of high affinity sites, with an apparent Kd value of 25.0 +/- 0.4 pM and a binding capacity of 32.5 +/- 1.2 fmol/mg protein in lactating mammary glands at random stages of lactation. The order of potency of a series of agonists to compete for [125I]CYP binding is consistent with the interactions with a beta 2-subtype receptor. The binding of [125I]CYP to mammary glands also shows a marked stereoselectivity; the (-)isomers of isoproterenol and propranolol are more potent than their respective enantiomers. The radioautographic localization of [125I]CYP reveals the presence of specific beta-adrenergic receptors in the epithelial cells, alveoles, ducts, as well as adipocytes. [125I]CYP binding shows a 2- to 3-fold increase during pregnancy. Such a result correlates with parallel increases in stimulation of adenylate cyclase activity, the cytosolic progesterone receptor concentration, as well as plasma 17 beta-estradiol and progesterone levels. At parturition, a sharp decline in beta-adrenergic receptor concentration is observed, a finding concomitant with a drop in progesterone receptor levels as well as plasma estradiol and progesterone concentrations. During midlactation, beta-adrenergic receptors reach their maximal levels. The presence of specific beta-adrenergic receptors functionally coupled to the adenylate cyclase system and the marked changes in receptor capacity and distribution measured during the different physiological states of the mammary gland suggest that the mammary beta-adrenergic receptors are highly sensitive to changes in the hormonal milieu and provide a mechanism for a direct catecholaminergic influence on mammary gland growth and differentiation.

Dissociated changes of pituitary luteinizing hormone-releasing hormone (LHRH) receptors and responsiveness to the neurohormone induced by 17 beta-estradiol and LHRH in vivo in the rat.

A single injection of 17 beta-estradiol into castrated male or female rats results in an initial decrease in plasma concentrations of LH and pituitary responsiveness to LHRH, followed by a rapid return to normal or slightly elevated values. Under such experimental conditions, no acute change of binding of [125I-labeled D-Ser(TBU)6]LHRH ethylamide to anterior pituitary homogenate could be observed. Moreover, the self-priming effect of LHRH, as illustrated by a 10-fold increase in the LH response to a second injection of LHRH in the afternoon of proestrus, is accompanied by a 40% loss of pituitary LHRH receptors. During the estrous cycle, a 100% increase in pituitary LHRH receptors is already found on diestrus II, while the maximal LH responsiveness to LHRH occurs later, namely on the afternoon of proestrus. The present findings of a dissociation between changes in LHRH receptor levels and LH responsiveness to the neurohormone suggest that postreceptor events play a predominant role in the control of gonadotropin secretion by sex steroids and LHRH itself. Moreover, LHRH can cause an acute down-regulation of its own receptor in the anterior pituitary gland.

Luteinizing hormone-releasing hormone (LHRH) agonist restoration of age-associated decline of thymus weight, thymic LHRH receptors, and thymocyte proliferative capacity.

The presence of specific LHRH-binding sites within the rat thymus gland and the ability of LHRH and its agonistic and antagonistic analogs to directly modulate thymus function prompted us to study the possible changes in the number of thymic LHRH-binding sites during aging-induced physiological immunosenescence. Moreover, the effects of chronic treatment of aging rats with a potent LHRH agonist (LHRH-A) on thymic LHRH receptors, thymus weight and histology, as well as thymocyte proliferative capacity were assessed. For comparison, the effects of castration on the same parameters was also investigated. The process of aging is accompanied by a sharp reduction in LHRH-A-binding sites within the thymus gland of both female and male rats. Starting at 7 months of age, a 50% decrease in thymic LHRH-A binding was followed, at 11-13 months of age, by a nearly 65% inhibition of receptor numbers. In 16- to 19-month-old rats, LHRH-A binding was almost completely lost. Thymus weight was 30% reduced in 7-month-old animals, while a 50% reduction in thymic size was reached at 11 months of age in males and 13 months in female rats. A further decrease in thymic mass was observed at 16 and 19 months. Chronic (45-day) treatment of aging (15-16 months old) female and male rates with the potent LHRH-A, [D-Trp6,Des-Gly10]LHRH-N-ethylamide, reversed the age-related decreases in both thymus weight and thymic LHRH-binding sites. Similarly, surgical removal of testicular hormones by castration restored thymus weight and increased LHRH-A binding in the thymus of aged rats. While thymus histology in 3-month-old rats was characterized by a clear demarcation of cortical and medullary regions, only thymic remnants were present in 16- to 17-month-old animals. Castration of old rats resulted in a partial restoration of thymic structure, while chronic treatment of aging rats with the LHRH-A produced a homogeneous organization of both cortical and medullary compartments accompanied by a marked increase in the width of the cortical layer, densely packed with lymphocytes. While the process of aging was accompanied by an almost complete loss of the proliferative response of thymocytes to optimal concentrations of the mitogen Concanavalin-A, thymocyte cultures from old rats treated with LHRH-A or from castrated animals, displayed significantly greater proliferative responses. Furthermore, the combination of both manipulations resulted in a further significant increase in thymocyte proliferative capacity.(ABSTRACT TRUNCATED AT 400 WORDS)