Progesterone modulates extracellular heat-shock proteins and interlukin-1ß in human choriodecidual after Escherichia coli infection.

INTRODUCTION: Chorioamnionitis is an adverse condition in human pregnancy caused by many bacterial pathogens including Escherichia coli (E. coli); which has been associated with higher risk of preterm birth. We recently reported that human maternal decidua (MDec) tissue responds to E. coli infection by secreting extracellular heat-shock proteins (eHsp)-60, -70 and interlukin-1ß (IL-1ß). Previous studies have shown that progesterone (P4) regulates the immune response, but it is unknown whether P4 inhibits the secretion of eHsp. The aim of this investigation was to determine the role of P4 on the secretion of eHsp-27, -60, -70 and IL-1ß in MDec after 3, 6, and 24 h of E. coli infection. METHODS: Nine human feto-maternal interface (HFMi) tissues were included and mounted in the Transwell culture system. Only the maternal decidua (MDec) was stimulated for 3, 6 and 24 h with E. coli alone or in combination with progesterone and RU486. After each treatment, the HFMi tissue was recovered to determine histological changes and the culture medium recovered to evaluate the levels of eHsp-27, -60, -70 and IL-1ß by ELISA and mRNA expression by RT-PCR. RESULTS: No structural changes were observed in the HFMi tissue treated with P4 and RU486. However, stimulation with E. coli produces diffuse inflammation and ischemic necrosis. E. coli induced infection decreases, in time- and dose-dependent manner, eHsp-27 and increases eHsp-60, eHsp-70 and IL-1ß levels. In contrast, incubation of HFMi tissue with E. coli + P4 reversed eHsp and IL-1ß secretion levels relative to E. coli stimulation group but not relative to the control group. The same profile was observed on the expression of eHsp-27 and eHsp-60. DISCUSSION: we found that progesterone modulates the anti-inflammatory (eHsp-27) and pro-inflammatory (eHsp-60 and eHsp-70) levels of eHsp induced by E. coli infection in human choriodecidual tissue. eHsp-60 and eHsp-70 levels were not completely reversed; maintaining the secretion of IL-1ß, which has been associated with adverse events during pregnancy.

Increased Levels of Plasma Extracellular Heat-Shock Proteins 60 and 70 kDa Characterized Early-Onset Neonatal Sepsis.

Background: Extracellular heat-shock proteins (eHsp) are highly conserved molecules that play an important role in inflammatory diseases and have been quantified in plasma from patients with infectious diseases, including sepsis. There is a constant search for dependable biochemical markers that, in combination with conventional methods, could deliver a prompt and reliable diagnosis of early-onset neonatal sepsis. Objective: We sought to assess the level of eHsp-27, eHsp-60, eHsp-70, and tumor necrosis factor-alpha (TNFα) in plasma of healthy neonates at term and infants with early-onset neonatal sepsis. Methods: This study included 34 newborns that were classified as healthy neonates at term (blood samples from the umbilical cord, n = 23) or infants with early-onset neonatal sepsis (blood samples obtained from umbilical artery by standard sterile procedures before starting a systemic antibiotic intervention, n = 11). All blood samples were centrifuged, and the plasma recovered to determine eHsp-27, eHsp-60, eHsp-70, and TNFα levels by ELISA. Results: Our results indicate that the level of eHsp-27 in healthy neonates at term was 0.045 ± 0.024 pg/ml. This value decreased 2.5-fold in infants with early-onset neonate sepsis (0.019 ± 0.006 pg/ml, p = 0.004). In contrast, the levels of eHsp-60 and eHsp-70 in healthy neonates at term were 13.69 ± 5.3 and 4.03 ± 2.6 pg/ml, respectively. These protein levels increased significantly 1.8- and 1.9-fold in the plasma of infants with early-onset neonatal sepsis (p ≤ 0.001). The level of TNFα in healthy neonates at term was 2.94 ± 0.46 pg/ml, with a 3.0-fold increase in infants with early-onset neonatal sepsis (8.96 ± 0.72 pm/ml, p ≤ 0.001). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of eHsp compared with that of C-reactive protein were 73.3, 60.0, 47.8, and 33.3%, respectively. Conclusion: This study demonstrated a consistent increase of eHsp-60 and eHsp-70 in the plasma of infants diagnosed with early-onset neonatal sepsis. These proteins showed higher sensitivity and specificity than C-reactive protein and blood culture test.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1ß, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Early post-treatment with 9-cis retinoic acid reduces neurodegeneration of dopaminergic neurons in a rat model of Parkinson's disease.

BACKGROUND: Retinoic acid (RA) is a biologically active derivative of vitamin A. Previous studies have demonstrated that RA has protective effects against damage caused by H2O2 or oxygen-glucose deprivation in mesangial and PC12 cells. Pretreatment with 9-cis-retinoic acid (9cRA) reduced infarction and TUNEL labeling in cerebral cortex as well as attenuated neurological deficits after distal middle cerebral artery occlusion in rats. The purpose of this study was to examine a protective role of 9cRA in dopaminergic (DA) neurons in a typical rodent model of Parkinson's disease (PD). RESULTS: The protective role of 9cRA was first examined in rat primary ventromesencephalic culture. Treatment with 9cRA significantly reduced 6-hydroxydopamine (6-OHDA)-mediated cell death and TUNEL labeling in cultured dopaminergic neurons. The protective effect was also examined in adult male rats. Animals received unilateral 6-OHDA lesioning at the left medial forebrain bundle on day 0. Methamphetamine -induced rotational behavior was examined on days 6, 20 and 30 after lesioning. Animals were separated into 2 groups to balance rotational behavior and lesion extent on day 6 and were treated with either 9cRA or vehicle (i.c.v. on day 7 + intra-nasal from day 8 to day 14). Post-treatment with 9cRA significantly reduced methamphetamine -mediated ipislateral rotation at 20 and 30 days after lesioning. In vivo voltammetry was used to examine DA overflow in striatum. Treatment with 9cRA significantly increased KCl -evoked DA release in the lesioned striatum. 9cRA also increased tyrosine hydroxylase (+) cell number in the lesioned nigra as determined by unbiased stereology. CONCLUSION: Our data suggests that early post-treatment with 9cRA has a protective effect against neurodegeneration in nigrostriatal DA neurons in an animal model of PD.

PTEN deletion enhances survival, neurite outgrowth and function of dopamine neuron grafts to MitoPark mice.

Clinical trials in Parkinson's disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of ß-catenin in dopamine neurons.

In the present study, we analyzed mice with a targeted deletion of ß-catenin in DA neurons (DA-ßcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-ßcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-ßcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-ßcat KO mice. This study demonstrates that ablation of ß-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that ß-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.

NMDA receptors on non-dopaminergic neurons in the VTA support cocaine sensitization.

BACKGROUND: The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1(DATCre) mice). METHODOLOGY AND PRINCIPAL FINDINGS: Using an additional NR1(DATCre) mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1(DATCre) mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1(DATCre) mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1(DATCre) transgenic model, and that application of an NMDAR antagonist within the VTA of NR1(DATCre) animals still blocks sensitization to cocaine. CONCLUSIONS/SIGNIFICANCE: These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1(DATCre) mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior.

Selective deletion of PTEN in dopamine neurons leads to trophic effects and adaptation of striatal medium spiny projecting neurons.

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders.

Stimulation of adenosine receptors selectively activates gene expression in striatal enkephalinergic neurons.

In the striatum, adenosine A2A and dopamine D2 receptors exert reciprocal antagonistic interactions that modulate the function of GABAergic enkephalinergic neurons. We have previously shown that stimulation of adenosine A1 receptors allows the stimulation of A2A receptors to overcome a tonic inhibitory effect of D2 receptors and induce striatal expression of c-fos. In the present work, by studying co-localization of c-Fos immunoreactivity and preproenkephalin and preprodynorphin transcripts, we show that co-administration of the A1 receptor agonist CPA and the A2A receptor agonist CGS 21680 increases the striatal expression of c-fos in GABAergic enkephalinergic but not in GABAergic dynorphinergic neurons. Co-administration of CPA and CGS 21680 also induced a significant increase in the striatal expression of preproenkephalin. The results underscore the role of adenosine in the activation of gene expression in the GABAergic enkephalinergic neuron.

Cannabinoid CB1 receptor and serotonin 3 receptor subunit A (5-HT3A) are co-expressed in GABA neurons in the rat telencephalon.

Among all described serotonin (5-HT) receptors in mammals, the type three (5-HT3) is the only ligand-gated ion channel receptor for serotonin. By using double in situ hybridization histochemistry, we found co-expression of the functional 5-HT3A subunit of the 5-HT3 receptor and the central CB1 cannabinoid receptor in neurons of the rat telencephalon. Double-labeled 5-HT3A/CB1 neurons were found in the anterior olfactory nucleus, superficial and deep layers of the cortex, hippocampal formation (hippocampus, dentate gyrus, subiculum, and entorhinal cortex) and amygdala. Analysis of the proportion of neurons co-expressing 5-HT3A and CB1 receptors in the cortex and amygdala showed that, depending on the brain region, 37-53% of all neurons expressing the 5-HT3A subunit also expressed CB1 transcripts; 16-72% of the total population of neurons expressing CB1 mRNA co-expressed the 5-HT3A subunit. By using a combination of double in situ hybridization and immunohistochemistry, we demonstrated that 5-HT3A/CB1-expressing neurons contained the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). These results imply that in distinct regions of the telencephalon, GABA neurons that react to cannabinoids may also be responsive to serotonin through 5-HT3 receptors. Cellular coexistence of 5-HT3A and CB1 transcripts in interneurons of the cortex, hippocampal formation, and amygdala suggest possible interactions between the cannabinoid and serotonergic systems at the level of GABA neurotransmission in brain areas involved in cognition, memory, and emotion.

The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and noncompetitive manner.

Aspectos moleculares de la demencia asociada con el SIDA / Molecular aspects of dementia associated with AIDS

Se ha observado una variedad de trastornos neuropsiquiátricos entre 20 y 30 por ciento de los pacientes que padecen del síndrome de inmunodeficiencia adquirida (SIDA). Debido a que las neuronas no se infectan directamente con el virus de la inmunodeficiencia adquirida (HIV), las manifestaciones fisiopatológicas de la demencia asociada con el SIDA (ADC) podrían estar relacionadas con mecanismos indirectos. La glucoproteína 120 de la envoltura viral (gp 120) derivada del VIH parece desempeñar un papel importante en el desarrollo de la ADC. Una cantidad cada vez mayor de experimentos han indicado que las concentraciones nanomolares de la gp120 derivada del VIH produce muerte neuronal in vivo, en tanto que las concentraciones picomolares matan a las neuronas in vitro. Los datos recientes sugieren que para inducir el daño neuronal, los receptores a citocinas y el CD4+ desempeñan un papel muy importante en la activación de eventos intracelulares que conducen a un incremento del Ca++ intracelular con la participación de los canales NMDA, lo que dispara los eventos intracelulares y la apoptosis. Entender el mecanismo del daño neuronal desde un punto de vista molecular y conductual, probablemente nos proporcionará el conocimiento para encontrar el tratamiento y la manera de prevenir esta complicación clínica. En esta revisión se incluye también el desarrollo de modelos animales para el estudio del mecanismo fisiopatológico de la demencia asociada con el SIDA