A peculiar VNTR in the cystathionine ß-synthase gene is a risk factor for Down Syndrome.

In the present study, we analysed a 31bp variable number of tandem repeats (VNTR) of the cystathionine ß-synthase (CBS) gene in 427 subjects: 127 patients with Down syndrome (DS) and in 60 of their mothers; 172 age-and sex-matched controls and in 68 of their mothers. A significant statistical difference in the distribution of the 21 repeat allele was found comparing mothers of subjects with DS versus mothers of children without DS (χ2= 4.166; P = 0.0413; Table 2). Since CBS 21 repeats allele carriers show a decrease of CBS enzyme activity possibly leading to lower intracellular glutathione concentration, these results could be explained by a higher not disjunction probability of chromosome 21 in oocytes, due to poor antioxidative protection against reactive oxygen species (ROS) toxic activity.

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.

Loss of aromatase cytochrome P450 function as a risk factor for Parkinson's disease?

The final step in the physiological synthesis of 17beta estradiol (E(2)) is aromatization of precursor testosterone by a CYP19 gene product, cytochrome P450 estrogen aromatase in the C19 steroid metabolic pathway. Within the central nervous system (CNS) the presence, distribution, and activity of aromatase have been well characterized. Developmental stage and injury are known modulators of brain enzyme activity, where both neurons and glial cells reportedly have the capability to synthesize this key estrogenic enzyme. The gonadal steroid E(2) is a critical survival, neurotrophic and neuroprotective factor for dopaminergic neurons of the substantia nigra pars compacta (SNpc), the cells that degenerate in Parkinson's disease (PD). In previous studies we underlined a crucial role for the estrogenic status at the time of injury in dictating vulnerability to the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our ongoing studies address the contribution of brain aromatase and extragonadal E(2) as vulnerability factors for PD pathology in female brain, by exposing aromatase knockout (ArKO, -/-) female mice which are unable to synthesize estrogens to MPTP. Our initial results indicate that aromatase deficiency from early embryonic life significantly impairs the functional integrity of SNpc tyrosine hydroxylase-positive neurons and dopamine transporter innervation of the caudate-putamen in adulthood. In addition, ArKO females exhibited a far greater vulnerability to MPTP-induced nigrostriatal damage as compared to their Wt type gonadally intact and gonadectomized counterparts. Characterization of this novel implication of P450 aromatase as determining factor for PD vulnerability may unravel new avenues for the understanding and development of novel therapeutic approaches for Parkinson's disease.

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.

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.

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.

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)

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 (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)

Upregulation of lymphocyte beta-adrenergic receptor in Down's syndrome: a biological marker of a neuroimmune deficit.

To test the hypothesis of an altered central nervous system influence upon the immune system of Down's syndrome (DS) patients and in order to establish a peripheral biological marker of neuroimmune deficit, we have studied the characteristics of the beta 2-adrenergic receptor (B2AR) system in peripheral blood monocytes (PBMC) of 12 pre-pubertal (six boys and six girls) individuals and correlated alterations in binding with changes in distribution of lymphocyte subsets. Using the very potent beta-adrenergic antagonist, iodocyanopindolol ([125I]CYP), as a ligand, the present study shows that a typical BAR population of the beta 2-subtype is present in PBMC from DS children, with binding kinetics and structural specificity similar to those measured in PBMC from patients with other (non-genetic) forms of mental retardation, or in PBMC from age-matched healthy subjects. On the other hand, this study revealed a significant increase in B2AR binding capacity of PBMC from DS subjects (Bmax = 5258 +/- 470 sites/cell) compared to the values measured in the control population of retarded children (Bmax = 1965 +/- 280 sites/cell), characterized by an approximately three-fold increase in the Bmax, without changes in binding affinity (KD = 40.5 +/- 2.0 and 36.6 +/- 2.5 pM in DS and retarded patients, respectively). The flowcytometric analysis of lymphocyte subsets using a panel of monoclonal antibodies against a series of lymphocyte markers revealed a profound alteration in the distribution of lymphocyte subtypes with an almost 50% decrease in B cell and T-helper populations, a three-fold increase in T-cytotoxic suppressor, a seven-fold increase in lymphocyte-activated killer cells (LAK) and 30% increase in natural killer (NK) subpopulations. When fluorescence-labelled lymphocytes were visualized in the cytofluorograph and sorted for their use in the radioreceptor assay, B cells had approximately twice the number of B2AR when compared to T cells; and cytotoxic/suppressor showed a higher binding capacity compared to T-helper cells. On the other hand, labelled lymphocytes from DS patients showed a specific increase in receptor number in B cells, T-cytotoxic suppressor and NK subpopulations. It is concluded that a profound catecholaminergic dysfunction not previously appreciated in DS is reflected by a significant alteration in lymphocyte subset distribution and by a specific up-regulation of lymphocyte B2AR in phenotypically and functionally distinct T and B cells as well NK subpopulations, suggesting a possible denervation supersensitivity phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)

Partial blockade of T-cell differentiation during ontogeny and marked alterations of the thymic microenvironment in transgenic mice with impaired glucocorticoid receptor function.

Glucocorticoids (GCs) are widely known to be potent modulators of the immune system. The role of GCs in thymopoiesis as well as the integration of the thymus with the neuroendocrine system is, however, poorly understood. In the present work, we have studied, in transgenic mice with an impaired GC function, the alterations which occur in both T-cell differentiation and thymic stroma maturation, throughout ontogeny as well as in adult condition, analyzing their possible rebounding on the status of adult splenic T lymphocyte populations. These transgenic mice have been described to present a significant decrease (60-70%) of thymic and splenic GC receptor binding capacity but maintain normal their basal plasma ACTH and corticosterone levels. The animals showed a partial blockade of T-cell differentiation and decreased percentages of apoptotic cells during fetal development but not in adult life, when thymic cellularity was significantly increased although thymocyte apoptosis response was not affected. In contrast, thymic stroma was profoundly altered from early fetal stages and large epithelium-free areas appeared in adult thymus. On the other hand, our study revealed a reduction of the splenic TcRalphabeta population accompanied by an increase in the CD4/CD8 ratio. The analysis of different adhesion molecules as well as activation markers demonstrated that most of them (CD5, CD11a, CD11b, CD69 and MHC Class II) were normally expressed in transgenic lymphocytes, whereas CD44 and CD62L expression was altered indicating the existence of an increased proportion of primed T-cells in these animals. In view of the mutual interdependence of thymic stroma and thymocyte maturation, the partial blockade of T-cell differentiation during ontogeny and the profound alterations of the stromal cell compartment in transgenic mice with impaired GR function suggest a key role for GCs in coordinating the physiological dialogue between the developing thymocytes and their microenvironment.

Circadian melatonin and young-to-old pineal grafting postpone aging and maintain juvenile conditions of reproductive functions in mice and rats.

Chronic, night administration of melatonin to aging mice and transplantation of a young pineal gland into the thymic rudiment of older mice and rats have been studied with the aim of evaluating their effects on aging of gonadal, sexual, and reproductive functions. Both melatonin administration and young-to-old pineal grafting positively affect size and function of testes and maintenance of juvenile hippocampal and testicular LHRH-receptors and beta-adrenergic receptors in the tests of old rats and mice. These results demonstrate that a pineal-directed circadian function and cyclicity is fundamental for the regulation of sexual, reproductive physiology, and that proper intervention with melatonin may potentially postpone aging of both neural and gonadal sexual function.

Basic fibroblast growth factor priming increases the responsiveness of immortalized hypothalamic luteinizing hormone releasing hormone neurones to neurotrophic factors.

The participation of growth factors (GFs) in the regulation of luteinizing hormone releasing hormone (LHRH) neuronal function has recently been proposed, but little is known about the role played by GFs during early LHRH neurone differentiation. In the present study, we have used combined biochemical and morphological approaches to study the ability of a number of GFs normally expressed during brain development, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I) to induce survival, differentiation, proliferation, and phenotypic expression of immortalized (GT1-1) LHRH neurones in vitro, at early (3-days in vitro, 3-DIV) and late (8-DIV) stages of neuronal differentiation. Comparison of GF-treated vs untreated neurones grown in serum-deprived (SD) medium demonstrated bFGF to be the most potent, and insulin the least active in promoting neuronal differentiation. Thus, at both 3-DIV and 8-DIV, but especially at 8-DIV, bFGF induced the greatest increase in the total length and number of LHRH processes/cell and in growth cone surface area. bFGF was also the most active at 3-DIV, and IGF-I at 8-DIV, in counteracting SD-induced cell death, whereas EGF was the most potent in increasing [3H]thymidine incorporation. All GFs studied decreased the spontaneous release of LHRH from GT1-1 cells when applied at 3-DIV or 8-DIV, except for insulin which was inactive at both time-points and bFGF which was inactive at 8-DIV. Pre-treatment of GT1-1 cells with a suboptimal ('priming') dose of bFGF for 12 h followed by application of the different GFs induced a sharp potentiation of the neurotrophic and proliferative effects of the latter and particularly of those of IGF-I. Moreover, bFGF priming counteracted EGF-induced decrease in LHRH release and significantly stimulated LHRH secretion following IGF-I or insulin application, suggesting that bFGF may sensitize LHRH neurones to differentiating effects of specific GFs during development.

Luteinizing hormone-releasing hormone (LHRH) receptors in the neuroendocrine-immune network. Biochemical bases and implications for reproductive physiopathology.

It seems apparent that the brain-pituitary-reproductive axis and the brain-thymus-lymphoid axis are linked by an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets. Moreover, such communication networks form the basis and control of each step and every level of reproductive physiology. This work has focused on the LHRH system, a primary central and peripheral clock of both neuroendocrine and immune functions. From the initiation of a sexually organized response, the detection of sexual odors, and the induction of mating behavior, extrahypothalamic and hypothalamic LHRH orchestrates the neuroendocrine modulation of gonadotropin secretion, while its expression within the ovary directly controls specific events such as follicular atresia. The presence of LHRH receptors in oocytes clearly anticipates a potential action of the decapeptide during the process of fertilization and/or implantation. Within the thymus and other peripheral immune organs, LHRH plays a unique role of immunomodulator, contributing to the sex-dependent changes in immune responsiveness during the estrous-menstrual cycle as well as pregnancy. The reciprocity of the neuroendocrine-immune signaling systems is further supported by the ability of sex steroids to modulate thymus-dependent immune functions via direct effects on specific target genes involved in the development of sex dimorphism and sex-dimorphic immune responses, including the downregulation of immune response observed during pregnancy. Such cyclic changes in immune responsiveness could have a physiological implication, such as the decrease or suppression in cell-mediated immunity observed in the postovulatory phase of the cycle and in pregnancy, respectively, and might play a role during the implantation process and the establishment of pregnancy. In this context, the ability of corticosterone to directly inhibit both GR transcript levels as well as a cell-mediated immune response within the thymus, and the modulation of such an inhibitory effect by the sex steroid hormone milieu, may offer an explanation and a molecular mechanism whereby stress may be deleterious for reproduction, also via immunomodulation. On the other hand, hormonally mediated alterations in immunity might also have a pathological implication in sexually related immune diseases. For example, in mouse and humans, lupus erythematosus is more prevalent in females and estrogen accelerates the disease process, while menstruation is known to exacerbate idiopathic thrombocytopenia purpura. Sex steroid hormone milieu might also have a role in controlling the stress response through immunomodulation. Within the placenta, an intricate network of signaling systems controls a delicate interplay between the neuroendocrine hormones, growth factors, and cytokines that are susceptible to play a major local role in the processes of implantation and the establishment and completion of pregnancy. The neuroendocrine and immunomodulatory role of LHRH continues well after parturition because the presence of LHRH-like material within the mammary gland and milk participates in the physiological modulation of hypophyseal, gonadal, and immune functions of the pups. Such a significant role played by the hypothalamic peptide in the modulation of immune responsiveness would indicate LHRH as the signal conveying information to both neuroendocrine and immune cells, with the role of informing and then transducing the messages into appropriate biological responses.(ABSTRACT TRUNCATED)

Gender, neuroendocrine-immune interactions and neuron-glial plasticity. Role of luteinizing hormone-releasing hormone (LHRH).

Signals generated by the hypothalamic-pitutary-gonadal (HPG) axis powerfully modulate immune system function. This article summarizes some aspects of the impact of gender in neuroendocrine immunomodulation. Emphasis is given to the astroglial cell compartment, defined as a key actor in neuroendocrine immune communications. In the brain, the principal hormones of the HPG axis directly interact with astroglial cells. Thus, luteinizing hormone releasing hormone, LHRH, influences hypothalamic astrocyte development and growth, and hypothalamic astrocytes direct LHRH neuron differentiation. Hormonally induced changes in neuron-glial plasticity may dictate major changes in CNS output, and thus actively participate in sex dimorphic immune responses. The impact of gender in neuroimmunomodulation is further underlined by the sex dimorphism in the expression of genes encoding for neuroendocrine hormones and their receptors within the thymus, and by the potent modulation exerted by circulating sex steroids during development and immunization. The central role of glucocorticoids in the interactive communication between neuroendocrine and immune systems, and the impact of gender on hypothalamic-pituitary-adrenocortical (HPA) axis modulation is underscored in transgenic mice expressing a glucocorticoid receptor antisense RNA.

Luteinizing hormone-releasing hormone is a primary signaling molecule in the neuroimmune network.

The brain-pituitary-reproductive axis and the brain thymus-lymphoid axis are linked by an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets. Moreover, such communication networks form the basis and control each step and every level of reproductive physiology. This presentation highlights the extent to which endocrine, neural, glial, or immunologically competent cells may achieve their specific functions using common mechanisms, but employing them to different degrees. In particular, this work will focus on LHRH, the chief hormone orchestrating reproductive events. Within the thymus LHRH plays a unique role of immunomodulator, contributing to the sex-dependent changes in immune responsiveness during the estrous-menstrual cycle as well as pregnancy. From the recent cloning and sequencing of lymphocyte LHRH, the expression of LHRH receptor mRNA in lymphocyte, the transduction mechanisms involved, and the steroidogenic sensitivity of the intralymphocyte LHRH system. It would appear that this peptide may act as an immunological response modifier in the brain-pituitary-lymphoid-gonadal axis. The interplay between neuronal, endocrine, and immune compartments is further emphasized in the study of LHRH-astroglial interactions. Astrocytes are able to manufacture a wide variety of signaling agents and can secrete immunoregulatory molecules that influence immune cells, as well as the glial cells themselves. Astroglia and the immortalized hypothalamic LHRH (GT1-1) neurons communicate with an array of mechanisms, via soluble mediators as well as cell-to-cell contacts. Manipulation of astroglial-derived cytokines and nitric oxide (NO) in GT1-1 neuron-astroglia cocultures, underscores a potential cross-talk between different intra/inter-cellular mediators in the dynamic control of LHRH release. Further studies aimed to disclose at a biochemical and a molecular level such bidirectional, informative network will give us new insights into more general issues concerned with the malfunction of the neuroendocrine-immune axis.

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor.

Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 microg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.

The reproductive system at the neuroendocrine-immune interface: focus on LHRH, estrogens and growth factors in LHRH neuron-glial interactions.

Bidirectional communication between the neuroendocrine and immune systems plays a pivotal role in health and disease. Signals generated by the hypothalamic-pituitary-gonadal (HPG) axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids) are major players coordinating the development immune system function. Conversely, products generated by immune system activation exert powerful and longlasting effects on HPG axis activity. In the central nervous system (CNS), one chief neuroendocrine-immune (NEI) compartment is represented by the astroglial cell population and its mediators. Of special interest, the major supporting cells of the brain and the thymus, astrocytes and thymic epithelial cells, share a similar origin and a similar set of peptides, transmitters, hormones and cytokines functioning as paracrine/autocrine regulators. This may explain some fundamental analogies in LHRH regulation of both cell types during ontogeny and in adult life. Hence, the neuropeptide LHRH significantly modulates astrocyte and thymic cell development and function. Here we focus this work on LHRH neuron-glial signaling cascades which dictate major changes during LHRH neuronal differentiation and growth as well as in response to hormonal manipulations and pro-inflammatory challenges. The interplay between LHRH, growth factors, estrogens and pro-inflammatory mediators will be discussed, and the potential physiopathological implications of these findings summarized. The overall study highlights the plasticity of this intersystem cross-talk and emphasize neuron-glial interactions as a key regulatory level of neuroendocrine axes activity.

Stimulation of cell division in mouse fibroblast line 3T3 by an extract derived from Triticum vulgare.

The ability of an extract derived from Triticum vulgare, the common wheat plant, to stimulate cellular proliferation of mouse 3T3 fibroblast cells was investigated. Cellular response to Triticum extract (TE) was most evident in sparse cultures made quiescent by growing cells on low concentrations (0.6%) of calf serum. The growth-promoting activity in the extract was lost after dialysis but was resistant to heat treatment and digestion with trypsin or chymotrypsin, suggesting a low-molecular-weight non-protein substance(s). Growth-curve experiments showed that TE was capable of supporting continuous cell division. Cellular proliferation showed a dose-dependent response in the range of 2%-10% TE, and addition of 10% TE to cell culture medium caused a level of cell-growth stimulation approximately 72% that of 20 ng/ml fibroblast growth factor (FGF). Measurement of ornithine decarboxylase (ODC) activity of 3T3 cells after addition of 10% TE showed a significant rise in the specific activity of the enzyme.