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.

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 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.

Stress, the immune system and vulnerability to degenerative disorders of the central nervous system in transgenic mice expressing glucocorticoid receptor antisense RNA.

Current research evidence suggests that interactions between genetic and environmental factors contribute to modulate the susceptibility to degenerative disorders, including inflammatory and autoimmune diseases of the central nervous system (CNS). In this context, bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life, thereby influencing the predisposition to several disease entities. Glucocorticoids (GCs), the end products of the hypothalamic-pituitary-adrenocortical (HPA) axis, gender and signals generated by hypothalamic-pituitary-gonadal (HPG) axis are major players coordinating the development of immune system function and exerting powerful effects in the susceptibility to autoimmune disorders, including experimental autoimmune encephalomyelitis (EAE), the experimental model for multiple sclerosis (MS). In particular, GCs exert their beneficial immunosuppressive and anti-inflammatory effects in inflammatory disorders of the CNS, after binding to their cytoplasmic receptors (GRs). Here we review our work using transgenic (Tg) mice with a dysfunctional GR from early embryonic life on programming vulnerability to EAE. The GR-deficiency of these Tg mice confers resistance to active EAE induction. The interplay between GCs, proinflammatory mediators, gender and EAE is summarized. On the basis of our data, it does appear that exposure to a defective GR through development programs major changes in endogenous neuroendocrine and immune mechanisms controlling the vulnerability to EAE. These studies highlight the plasticity of the HPA-immune axis and its pharmacological manipulation in autoimmune diseases of the CNS.

Stress, glucocorticoids and the susceptibility to develop autoimmune disorders of the central nervous system.

Alterations of the immunoendocrine circuit along the hypothalamic-pituitary-adrenocortical (HPA) axis in various autoimmune diseases have recently been observed, suggesting a modulatory role of this feedback regulation in the pathogenesis of autoimmune diseases. Susceptibility to experimental autoimmune encephalomyelitis (EAE) may be influenced by variations in the production of endogenous glucocorticoid hormones (GC). The adrenocortical response is central to recovery from EAE in the Lewis rat, as reflected by increased severity of the disease in adrenalectomized animals. The key role of GC in modifying the induction and progression of EAE is also emphasized by a reversal of corticoid-mediated effects through treatment with glucocorticoid receptor (GR) antagonists. We studied the relationship between defective GR function and susceptibility to EAE in transgenic (Tg) mice expressing GR antisense RNA. EAE was induced with the encephalitogenic myelin oligodendrocyte peptide (pMOG 36-50) in wild type (Wt) and transgenic (Tg) female mice bearing GR antisense RNA. pMOG 36-50 induced typical EAE in Wt mice but not in Tg mice. Histological examination of brains and spinal cords of Wt mice showed the presence of inflammation and/or demyelination, whereas in Tg mice neither were present. Although the mechanisms underlying the resistance of Tg mice to EAE induction are not yet clarified, compensatory changes at different levels of the HPA-immune axis in response to the potent immunogenic challenge are likely to participate in the observed effects. This work underlies the plasticity of the HPA-immune axis and suggests that pharmacological manipulation of neuroendocrine-immune networks may be a therapy of multiple sclerosis.

Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions to function: Focus on gender and HPA-HPG interactions on early programming of the NEI system.

Bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life. Signals generated by the hypothalamic-pituitary-gonadal axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids), and by the hypothalamic-pituitary-adrenocortical axis (glucocorticoids (GC)), are major players coordinating the development of immune system function. Conversely, products generated by immune system activation exert a powerful and long-lasting regulation on neuroendocrine axes activity. The neuroendocrine-immune system is very sensitive to preperinatal experiences, including hormonal manipulations and immune challenges, which may influence the future predisposition to several disease entities. We review our work on the ongoing mutual regulation of neuroendocrine and immune cell activities, both at a cellular and molecular level. In the central nervous system, one chief compartment is represented by the astroglial cell and its mediators. Hence, neuron-glial signalling cascades dictate major changes in response to hormonal manipulations and pro-inflammatory triggers. The interplay between LHRH, sex steroids, GC and pro-inflammatory mediators in some physiological and pathological states, together with the potential clinical implications of these findings, are summarized. The overall study highlights the plasticity of this intersystem cross-talk for pharmacological targeting with drugs acting at the neuroendocrine-immune interface.

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.

Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture.

Luteinizing hormone-releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression of immortalized hypothalamic LHRH (GT(1-1)) neurons in vitro. Culturing GT(1-1) cells in the presence of young glia for different time intervals caused a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT(1-1) cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of processes/cell and greater surface area of growth cones than GT(1-1) cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT(1-1) neurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT(1-1) neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT(1-1) neurons cocultured with young glia exhibited a 50% lower incorporation of [(3)H]thymidine than GT(1-1) neurons cultured without glia. By contrast, in the presence of old glia [(3)H]thymidine incorporation was significantly higher in cells cocultured with glia than in GT(1-1) neurons cultured alone. Localization of the proliferating cells by dual immunohistochemical staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted to nuclei of GT(1-1) neurons when these were cocultured with young glia, but associated with both neurons and astrocytes in the presence of old glia. At the functional level, coculture of GT(1-1) neurons with young glia increased the spontaneous release of LHRH as compared to GT(1-1) neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium of young glia (ACM-Y) induced significant neurotrophic and functional effects on GT(1-1) cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM-Y totally abolished its neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release-promoting effects. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged bFGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth-supporting state when treated with bFGF and this functional shift involves a diffusible molecule with potent neurotrophic and functional effects on immortalized LHRH neurons. (c) 2000 Wiley-Liss, Inc.

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.

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.

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.

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.

Opioid-immune interactions in autism: behavioural and immunological assessment during a double-blind treatment with naltrexone.

The emerging concept of opioid peptides as a new class of chemical messengers of the neuroimmune axis and the presence of a number of immunological abnormalities in infantile autism prompted us to correlate biological (hormonal and immunological) determinations and behavioural performances during treatment with the potent opiate antagonist, naltrexone (NAL). Twelve autistic patients ranging from 7 to 15 years, diagnosed according to DSM-III-R, entered a double-blind crossover study with NAL at the doses of 0.5, 1.0 and 1.5 mg/kg every 48 hours. The behavioural evaluation was conducted using the specific BSE and CARS rating scales NAL treatment produced a significant reduction of the autistic symptomatology in seven ("responders") out of 12 children. The behavioural improvement was accompanied by alterations in the distribution of the major lymphocyte subsets, with a significant increase of the T-helper-inducers (CD4+CD8-) and a significant reduction of the T-cytotoxic-suppressor (CD4-CD8+) resulting in a normalization of the CD4/CD8 ratio. Changes in natural killer cells and activity were inversely related to plasma beta-endorphin levels. It is suggested that the mechanisms underlying opioid-immune interactions are altered in this population of autistic children and that an immunological screening may have prognostic value for the pharmacological therapy with opiate antagonists.