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Microglia, together with astrocytes and pericytes, cooperate to ensure blood–brain barrier (BBB) stability, modulating endothelial responses to inflammatory insults. Agonists of the sphingosine 1 phosphate (S1P) receptors, such as siponimod (BAF-312), are important pharmacological tools in multiple sclerosis and other inflammatory diseases. Modulation of S1P receptors may result in a reduced inflammatory response and increased BBB stability. An in vitro BBB model was reproduced using human-derived endothelial cells, astrocytes and microglia. Co-cultures were exposed to inflammatory cytokines (TNFα, 10 UI and IFNγ, 5 UI) in the presence of BAF-312 (100 nM), and the BBB properties and microglia role were evaluated. The drug facilitated microglial migration towards endothelial/astrocyte co-cultures, involving the activity of the metalloprotease 2 (MMP2). Microglia actively cooperated with astrocytes in the maintenance of endothelial barrier stability: in the triple co-culture, selective treatment of microglial cells with BAF-312 significantly prevented cytokines’ effects on the endothelial barrier. In conclusion, BAF-312, modulating S1P receptors in microglia, may contribute to the reinforcement of the endothelial barrier at the BBB, suggesting an additional effect of the drug in the treatment of multiple sclerosis.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-&gamma, ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-&gamma, (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism, (2) the amelioration of the macrophage energy state, (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde, (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat), (5) the increase of the transforming growth factor-&beta, 1 (TGF-&beta, 1) and the down-regulation of the expressions of interleukins 1&beta, and 6 (IL-1&beta, and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Background: The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer’s Disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ). Methods: We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). Results: In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low molecular weight ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression. Conclusions: These data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leucocytes migration through the endothelial layer.

Background The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer’s disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ). Methods We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). Results In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression. Conclusion These data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.

Chronic wounds often occur in patients with diabetes mellitus due to the impairment of wound healing. This has negative consequences for both the patient and the medical system and considering the growing prevalence of diabetes, it will be a significant medical, social, and economic burden in the near future. Hence, the need for therapeutic alternatives to the current available treatments that, although various, do not guarantee a rapid and definite reparative process, appears necessary. We here analyzed current treatments for wound healing, but mainly focused the attention on few classes of drugs that are already in the market with different indications, but that have shown in preclinical and few clinical trials the potentiality to be used in the treatment of impaired wound healing. In particular, repurposing of the antiglycemic agents dipeptidylpeptidase 4 (DPP4) inhibitors and metformin, but also, statins and phenyotin have been analyzed. All show encouraging results in the treatment of chronic wounds, but additional, well designed studies are needed to allow these drugs access to the clinics in the therapy of impaired wound healing.

Since publication of the results of the Women Health Initiative Memory Study demonstrating that hormone therapy initiated late after menopause increases the risk of dementia in women, attempts have been made to identify a "critical window of intervention". In the meantime, basic research carried out in the last 10-15 years has reinforced the concept of a strong impact of estrogen in neuroprotection, moving also into novel directions that include characterization of estrogen's effect on non-neuronal cells, mitochondrial function, miRNA production and novel targets for their action in the central nervous system (CNS). All these findings, together with a list of recent animal models of Alzheimer's Disease that appear feasible for the study of estrogen's CNS effect are here summarized and accompanied by the most recent data from clinical trials in which hormone therapy was initiated early after menopause.

Carnosine (&beta, alanyl-L-histidine), a dipeptide, is an endogenous antioxidant widely distributed in excitable tissues like muscles and the brain. Carnosine is involved in cellular defense mechanisms against oxidative stress, including the inhibition of amyloid-beta (A&beta, ) aggregation and the scavenging of reactive species. Microglia play a central role in the pathogenesis of Alzheimer&rsquo, s disease, promoting neuroinflammation through the secretion of inflammatory mediators and free radicals. However, the effects of carnosine on microglial cells and neuroinflammation are not well understood. In the present work, carnosine was tested for its ability to protect BV-2 microglial cells against oligomeric A&beta, 1-42-induced oxidative stress and inflammation. Carnosine prevented cell death in BV-2 cells challenged with A&beta, oligomers through multiple mechanisms. Specifically, carnosine lowered the oxidative stress by decreasing NO and O2&minus, &bull, intracellular levels as well as the expression of iNOS and Nox enzymes. Carnosine also decreased the secretion of pro-inflammatory cytokines such as IL-1&beta, simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-&beta, 1. Carnosine also prevented A&beta, induced neurodegeneration in mixed neuronal cultures challenged with A&beta, oligomers, and these neuroprotective effects were completely abolished by SB431542, a selective inhibitor of the type-1 TGF-&beta, receptor. Our data suggest a multimodal mechanism of action of carnosine underlying its protective effects on microglial cells against A&beta, toxicity with a key role of TGF-&beta, 1 in mediating these protective effects.

Selective reuptake inhibitors (SSRIs), such as fluoxetine and sertraline, increase circulating Transforming-Growth-Factor-β1 (TGF-β1) levels in depressed patients, and are currently studied for their neuroprotective properties in Alzheimer’s disease. TGF-β1 is an anti-inflammatory cytokine that exerts neuroprotective effects against β-amyloid (Aβ)-induced neurodegeneration. In the present work, the SSRI, fluoxetine, was tested for the ability to protect cortical neurons against 1 μM oligomeric Aβ1-42-induced toxicity. At therapeutic concentrations (100 nM–1 μM), fluoxetine significantly prevented Aβ-induced toxicity in mixed glia-neuronal cultures, but not in pure neuronal cultures. Though to a lesser extent, also sertraline was neuroprotective in mixed cultures, whereas serotonin (10 nM–10 μM) did not mimick fluoxetine effects. Glia-conditioned medium collected from astrocytes challenged with fluoxetine protected pure cortical neurons against Aβ toxicity. The effect was lost in the presence of a neutralizing antibody against TGF-β1 in the conditioned medium, or when the specific inhibitor of type-1 TGF-β1 receptor, SB431542, was added to pure neuronal cultures. Accordingly, a 24 h treatment of cortical astrocytes with fluoxetine promoted the release of active TGF-β1 in the culture media through the conversion of latent TGF-β1 to mature TGF-β1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-β1. We conclude that fluoxetine is neuroprotective against Aβ toxicity via a paracrine signaling mediated by TGF-β1, which does not result from a simplistic SERT blockade.

Estrogen is known to affect different aspects of β-amyloid (Aβ) synthesis and degradation. The present work was undertaken to evaluate specifically whether matrix metalloproteinases (MMP) -2 and -9 are involved in Aβ degradation induced by estrogen and whether this is relevant to estrogen-induced neuroprotection. In SH-SY5Y human neuroblastoma cells, 10 nM 17β-estradiol (17β-E2) increases mRNA and intracellular protein expression of MMP-2 and -9, as well as the levels of the active forms of both enzymes released in the medium. Specificity of the effect is proved by prevention with the estrogen receptor (ER) antagonist ICI 182,780 (1 μM) and involvement of the ERα subtype is confirmed by the use of selective ERα or ERβ agonists (PPT, DPN) and antagonists (MPP, PHTPP). 17β-E2 significantly increases the degradation of Aβ, either transferred with the conditioned medium of H4-APPSw human neuroglioma cells, engineered to overproduce Aβ(1-40) and Aβ(1-42), or exogenously added as 2 μM Aβ(1-42). Both these effects are completely prevented by preexposure to the broad spectrum MMP inhibitor GM6001 (5 μM). Importantly, the 17β-E2-induced rescue of neuroblastoma cells challenged with 2 μM Aβ(1-42), an effect prevented by ICI 182,780 (1 μM), is mediated by MMPs, as it appears significantly reduced by GM6001 (5 μM) as well as by both MMP-2 (200 nM) and MMP-9 (200nM) selective inhibitors. In conclusion, the present study shows for the first time that MMP-2 and -9 give a main contribution to estrogen's neuroprotective effect.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic β-amyloid protein (Aβ) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aβ-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aβ toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-β1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aβ neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aβ toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aβ, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aβ-induced toxicity.

Alzheimer's disease is associated to a cerebral amyloid angiopathy with dysregulation of cerebral blood flow (CBF). In vitro studies have shown that short-term application of beta-amyloid (Abeta) peptides to isolated vessels affects vascular tone within 1h, but no studies have examined the effect of long-term incubation with Abeta. Here we evaluate the effect of Abeta((1-40)) and Abeta((25-35)) in rat basilar artery for up to 24h. Basilar artery segments were incubated with 25microeta((1-40)) or Abeta((25-35)), for 6 or 24h. After treatment, arteries were mounted in a wire myograph, in physiological salt solution gassed with O(2)/CO(2), in the absence of Abeta, and challenged with vasoconstrictors and vasodilators. Vasomotor responses were not significantly changed by 6h treatment with Abeta peptides whereas 24h treatment with either Abeta((25-35)) or Abeta((1-40)) increased vasoconstriction to 5-hydroxytryptamine (5-HT) and reduced endothelium-dependent vasodilatation to acetylcholine (ACh). Analysis of endothelial cells did not show apoptotic changes associated to endothelial dysfunction, as assessed by TUNEL immunostaining and examination of nuclear morphology, but basal phosphorylation of endothelial nitric oxide synthase (at serine 1177) appeared reduced. These data suggest that long incubation with Abeta peptides induces an alteration of endothelial function in isolated basilar artery, involving eNOS activity without changing cell morphology. This endothelial dysfunction may play a role in the pathogenesis of CBF dysregulation occurring in cerebral amyloid angiopathy and Alzheimer's disease.

Calixarenes are synthetic macrocyclic compounds that may serve as scaffolds for biologically active molecules and have been proposed as potential anticancer agents. We synthesized a ureido-calix[8]arene carrying N-acetyl-D-glucosamine residue (compound 1) and had previously demonstrated that it inhibits C6 glioma cell migration and proliferation, with divergent mechanisms. In the present work we explored in more detail the antiproliferative effect of compound 1, comparing it to related compounds lacking either the sugar moieties (compound 2), the multiple ureido groups (compound 3) or both (compound 4). The results show that the action of compound 1 is independent of the N-acetyl-D-glucosamine residues, requires the presence of multiple ureido groups and does not seem to involve focal adhesion kinase signaling. Inhibition of proliferation is reduced by preincubation with epidermal growth factor (EGF) and vascular endothelial growth factor (20 ng/ml) with compound 1, and extracellular-related kinase phosphorylation is reduced by treatment with compound 1 in both basal and EGF-stimulated conditions, suggesting that the observed effect depends on a direct interference with growth factor signaling.

Dipeptidyl peptidase 4 (DPP4) is expressed in various tissues, including the skin, and DPP4 inhibitors, that are currently used for the treatment of diabetes, may be effective also for complications of diabetes that affect the skin. To assess the role of DPP4 in keratinocytes, after creating a scratch wound in a monolayer of NTCC 2544 cells, we evaluated DPP4 expression and monitored wound repair over time, after treatment with the DPP4 inhibitor 1(((1-(hydroxymethyl)cyclopentyl)amino)acetyl)2,5-cis-pyrrolidinedicarbonitrile (DPP4-In). Expression of DPP4 increased early and was maintained up to 48 h following the scratch as shown by western blot and immunostaining. Treatment with 10 μM DPP4-In reduced DPP4 expression and significantly accelerated wound repair. This effect did not involve enhanced cell proliferation as shown by MTT proliferation assay, the lack of changes of cell cycle profiles and the slight inhibition of ERK phosphorylation. Enhancement of wound repair by DPP4 inhibition was prevented by the non specific MMPs inhibitor GM6100 (5 μM). Treatment with DPP4-In increased the expression of high mobility group box 1 (HMGB1), a substrate of this enzyme, and exposure of NCTC 2544 cells to DPP4-In and exogenous HMGB1 (10 nM) produced a non additive effect. Finally the healing promoting effect of DPP4-In was prevented by pretreatment with a neutralizing anti-HMGB1 antibody. The present results suggest that DPP4 inhibition contributes to enhanced wound healing by inducing keratinocytes to migrate into a scratched area. This effect seems to be independent of cell proliferation and involves enhanced production of HMGB1.

Alzheimer's disease pathology involves β-amyloid and tau. Various potential pharmacological targets are discussed that may be able to alleviate the accumulation of β-amyloid and tau. Possible causes of Alzheimer's disease are discussed involving impaired glucose and lipid metabolism and obesity. Adipokines may be involved in the etiology of Alzheimer's disease. An extensive discussion of the evidence concerning the adipokines leptin, adiponectin, resistin, visfatin, plasminogen activator inhibitor, interleukin-6 and transforming growth factor β1 as causes of Alzheimer's disease is presented.

Alzheimer's disease pathology involves β-amyloid and tau. Various potential pharmacological targets are discussed that may be able to alleviate the accumulation of β-amyloid and tau. Possible causes of Alzheimer's disease are discussed involving impaired glucose and lipid metabolism and obesity. Adipokines may be involved in the etiology of Alzheimer's disease. An extensive discussion of the evidence concerning the adipokines leptin, adiponectin, resistin, visfatin, plasminogen activator inhibitor, interleukin-6 and transforming growth factor β1 as causes of Alzheimer's disease is presented.

Alzheimer's disease (AD) is the most common form of dementia and is of rapidly increasing health, social and economic impact. Recent evidence suggests a strict link between metabolic disorders and AD. In the last decade much attention has focused specifically on the connection between dysfunction of lipid metabolism and AD. Here we discuss aspects of lipid regulation, including changes in cholesterol levels, function of apolipoproteins and leptin, and how these relate to AD pathogenesis. Despite the vast literature available, many aspects still need clarification. Nevertheless, the route is already delineated to directly connect aspects of lipid regulation to AD. This could represent a starting point to identify novel potential targets for a preventive and/or treatment strategy of the disease.

Estrogen affects proliferation and migration of different skin components, thus influencing wound healing processes. The human keratinocyte cell line NCTC 2544 has been used to examine the effects of estrogen, dissect its mechanism of action and characterize receptor subtypes involved. Western blot and immunocytochemical analyses confirmed the expression of estrogen receptors (ERs) α and β, with prevalence in the nuclear and extranuclear compartment, for ERα and ERβ respectively. Treatment with 10 nM 17β-estradiol (17β-E2) and the ERα and ERβ selective agonists, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; 100 nM), and diarylpropionitrile (DPN; 1 nM) produced a slight but significant increase in cell proliferation, as by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays, only after a long-term treatment (96 h). Analysis of cell migration by a scratch wound assay showed that 17β-E2 (10 nM) accelerated migration between 5 and 24 h after scratching, an effect confirmed by the transwell migration assay. PPT and DPN elicited similar effects. Pre-treatment with the mitogen-activated protein kinase inhibitor, U0126 (1 μM), abolished the ability of 17β-E2 and DPN, but not of PPT, to accelerate wound closure. TGF-β1 (10 ng/ml) produced a similar positive effect on wound closure and the TGF-β1 receptor antagonist, SB431542 (10 μM), reduced the ability of 17β-E2 and PPT to accelerate cell migration, but did not modify DPN effect. It is suggested that estrogen positively affects in vitro wound healing by stimulating cell proliferation after long-term exposure but mainly by accelerating cell migration within a few hours from treatment. Selective activation of ERβ may result in favorable stimulation of wound healing without any increase of transforming growth factor-β1 production.

Neurospheres from the subventricular zone of adult mice were used as an experimental model to analyse the early differential effects of 17beta-estradiol (17beta-E2). Both floating and differentiating neurospheres expressed estrogen receptors (ERs) alpha and beta. The initial phases of differentiation coincided with a peak of ERalpha expression as by Western blot analysis. Treatment with 10 nM 17beta-E2 induced a significant increase in the glial fibrillary acidic protein (GFAP)-positive population and a greater expression of GFAP, an effect sensitive to the estrogen receptor antagonist ICI 182,780. The GFAP-positive cell population induced by 17beta-E2 was characterized by a highly differentiated phenotype and intense immunostaining as by immunocytochemistry and flow cytometry. These cells co-expressed ERalpha and were positive to BrdU. 17beta-E2 also affected neuronal differentiation by rapidly and transiently increasing the percentage of polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive progenitors, and by accelerating the appearance of a mature neuronal phenotype, as evaluated by microtubule-associated protein 2 (MAP2) staining. Our results point to a key role for ERalpha during initial phases of differentiation of brain cells and to an effect of 17beta-E2 that sequentially involves both glia and neurons.

17beta-Estradiol (17beta-E(2)) is known to exert neuroprotective activity against beta-amyloid, but its exact target and mechanism of action in this effect have not been elucidated. The involvement of astroglia in neuroprotection of 17beta-E(2) against the beta-amyloid fragment [betaAP((25-35))] has been evaluated using an experimental paradigm in which medium conditioned from rat astroglia pretreated with 17beta-E2 was transferred to pure rat cortical neurons challenged with 25 microm betaAP((25-35)) for 24 h. The toxicity of betaAP((25-35)) was assessed by flow cytometry, evaluating the ability of the peptide to induce an aberrant mitotic cell cycle in neurons. The results obtained indicate that conditioned medium from astrocytes preexposed to 17beta-E(2) for 4 h increased the viability of cortical neurons treated with betaAP((25-35)). This effect was not modified by treatment with the estrogen receptor antagonist ICI 182,780, added directly to neurons, nor was it mimicked by direct addition of 17beta-E(2) to neuronal cultures during exposure to betaAP((25-35)). A soluble factor stimulated by 17beta-E(2) seemed to be involved, and accordingly, the intracellular and released levels of TGF-beta1 were increased by 17beta-E(2) treatment, as established by Western blot analysis. In addition, the intracellular content of TGF-beta1 in immunopositive cells, as detected by flow cytometry, was reduced, suggesting that 17beta-E(2) stimulated mainly the release of the cytokine. In support of a role for TGF-beta1 in astrocyte-mediated 17beta-E(2) neuroprotective activity, incubation with a neutralizing anti-TGF-beta1 antibody significantly modified the reduction of neuronal death induced by 17beta-E(2)-treated astrocyte-conditioned medium.