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OBJECTIVE To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. METHODS This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n = 108) underwent in utero administration of 1.106 cells on D2, while patients in the control group (n = 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n = 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated. RESULTS Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0. CONCLUSION Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.

With over 30% of current medications targeting this family of proteins, G-protein–coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

The use of opioids for the relief of pain and headache disorders has been studied for years. Nowadays, particularly because of its ability to produce analgesia in various pain models, delta opioid receptor (DOPr) emerges as a promising target for the development of new pain therapies. Indeed, their potential to avoid the unwanted effects commonly observed with clinically used opioids acting at the mu opioid receptor (MOPr) suggests that DOPr agonists could be a therapeutic option. In this review, we discuss the use of opioids in the management of pain in addition to describing the evidence of the analgesic potency of DOPr agonists in animal models.

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of β-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.

Over the last few years, intrauterine administration of autologous peripheral blood mononuclear cells (PBMC) has been proposed as new immunotherapy for patients with unexplained recurrent implantation failure (RIF). In these patients, administration of activated PBMC before embryo transfer results in a 2-fold increase in live birth rates(Yang et al., 2020). In this study we evaluated the role of T cells to promotes human endometrial receptivity. On the day of ovulation, PBMC were isolated from and activated with T cells mitogen, the phytohemagglutinin (PHA) and hCG for 48-h in a conditioned culture medium. Distributions of CD4+ T cells were characterized in 157 patients by flow cytometry before and after PHA/hCG activation. Cytokine production was analyzed by cytometric beads array. We observed in RIF patients a significant decrease in Th2 and natural Treg cells before activation with PHA/hCG and an increase of Th17 cells after activation compared to intrauterine sperm insemination (IUI) and in vitro fertilization (IVF) groups. Furthermore, the hCG/PHA treatment increases anti-inflammatory T cells (Th2 and Treg cells) compared to non-treated T cells. Principal component analysis (PCA) performed on CD4 T cell subtypes revealed a different cellular profile in the RIF compared to the IUI and IVF groups. This inflammatory state change could explain how endometrium immunomodulation by hCG-activated PBMC helps patients with unexplained RIF to reach implantation.

Nowadays, pain represents one of the most important societal burdens. Current treatments are, however, too often ineffective and/or accompanied by debilitating unwanted effects for patients dealing with chronic pain. Indeed, the prototypical opioid morphine, as many other strong analgesics, shows harmful unwanted effects including respiratory depression and constipation, and also produces tolerance, physical dependence, and addiction. The urgency to develop novel treatments against pain while minimizing adverse effects is therefore crucial. Over the years, the delta-opioid receptor (DOP) has emerged as a promising target for the development of new pain therapies. Indeed, targeting DOP to treat chronic pain represents a timely alternative to existing drugs, given the weak unwanted effects spectrum of DOP agonists. Here, we review the current knowledge supporting a role for DOP and its agonists for the treatment of pain. More specifically, we will focus on the cellular and subcellular localization of DOP in the nervous system. We will also discuss in further detail the molecular and cellular mechanisms involved in controlling the cellular trafficking of DOP, known to differ significantly from most G protein-coupled receptors. This review article will allow a better understanding of how DOP represents a promising target to develop new treatments for pain management as well as where we stand as of our ability to control its cellular trafficking and cell surface expression.

Genetically-modified animal models have significantly increased our understanding of the complex central nervous system circuits. Among these models, inducible transgenic mice whose specific gene expression can be modulated through a Cre recombinase/LoxP system are useful to study the role of specific peptides and proteins in a given population of cells. In the present study, we describe an efficient approach to selectively deliver a Cre-GFP to dorsal root ganglia (DRG) neurons. First, mice of different ages were injected in both hindpaws with a recombinant adeno-associated virus (rAAV2/9-CBA-Cre-GFP). Using this route of injection in mice at 5 days of age, we report that approximately 20% of all DRG neurons express GFP, 6 to 8 weeks after the infection. The level of infection was reduced by 50% when the virus was administered at 2 weeks of age. Additionally, the virus-mediated delivery of the Cre-GFP was also investigated via the intrathecal route. When injected intrathecally, the rAAV2/9-CBA-Cre-GFP virus infected a much higher proportion of DRG neurons than the intraplantar injection, with up to 51.6% of infected lumbar DRG neurons. Noteworthy, both routes of injection predominantly transduced DRG neurons over spinal and brain neurons.

Given the putative selectivity of the antagonist TIPP (Tyr-Tic-Phe-Phe) for δ-opioid receptors (DOP), this compound was selected for the design of a novel 64Cu-radiolabeled potent and selective DOP positron emission tomography (PET) imaging agent. Ex vivo autoradiography of TIPPD-PEG-K(NOTA/64Cu)-NH2 on rat brain sections produced a distribution pattern consistent with the known expression of DOP. Taken together, the in vitro and ex vivo data indicate that this 64Cu-tracer holds promise for studying the DOP by means of PET.

The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC-MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co-immunoprecipitating with Flag-DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle-coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di-lysine and di-arginine motifs within the intracellular segments of DOPr. Using cell-surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the K164AK166 (ICL2) or K250EK252 (ICL3) putative COPI binding sites increased the cell-surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells.

Crystal structures provide first atomic-level insights into δ-opioid receptor activation by two structurally diverse agonists., Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.

International audience; Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotropic hypogonadism. Here, we report an original case of GROS, associated with compound heterozygous follicle-stimulating hormone receptor (FSHR) variants, in a woman who achieved a live birth by in vitro maturation (IVM) of her oocytes. This 31-year-old woman consulted our assisted reproduction center for a second opinion after having been advised, because of pervasive high serum follicle-stimulating hormone (FSH) levels, to pursue in vitro fertilization (IVF) with donor oocytes. She presented with primary infertility and progressively prolonged menstrual cycles. Her serum FSH levels were indeed found to be high, but in discordance with a normal anti-Müllerian hormone (AMH) level and antral follicle count. Genetic investigation found the patient to be compound heterozygous for two FSHR variants: I160T, a known pathologic variant, and N558H, which has never been previously reported. As there was no ovarian response to high daily doses of exogenous gonadotropins, IVM was proposed to the patient with success and she finally delivered at term a healthy boy. Effects of the receptor variants were analyzed in heterologous cells. Whereas the I160T mutation blocked FSHR membrane trafficking and FSH-stimulated cAMP-dependent signaling in transfected CHO cells, the novel variant, N558H, functioned equivalently to wild-type FSHR in the assays employed. In conclusion, IVM should always be offered as a first-line therapy to infertile women presenting with GROS. The N558H variant discovered in FSHR is novel, but its functional significance, if any, is unresolved and merits further investigation as it may be associated with a recessive FSHR-related disorder.

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cel...

Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce life-threatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioid-independent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018 mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8 mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8 mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018 mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2-NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio.

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1–4] (i.e. Dmt- d -Ala-Phe-GlyNH2, Dmt = 2′,6′-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.

Given the putative selectivity of the antagonist TIPP (Tyr-Tic-Phe-Phe) for δ-opioid receptors (DOP), this compound was selected for the design of a novel

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1−/− mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity., NLRX1 is a guardian protein that inhibits the inflammatory response of glial cells within the central nervous system and prevents the onset of a spontaneous multiple sclerosis–like disease in mice. This study uses a novel mouse model to provide mechanistic insights into the neurodegenerative origin of multiple sclerosis.

During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7's IDC for its activation and proteolytic activity.

We have cloned and sequenced a cDNA from the surf clam (Spisula solidissima, a pelecypod mollusc) that encodes an octopamine receptor which we have named Spi-OAR. The sequence of Spi-OAR shares many similarities with two Aplysia and three Drosophila octopamine receptors belonging to a sub-group of beta-adrenergic-like octopamine receptors. Using an expression vector and transient transfections of Spi-OAR into HEK 293 cells, we observed an increase of cAMP upon addition of octopamine and, to a lesser extent, of tyramine, but not after addition of dopamine, serotonin, or histamine. Using a battery of known agonists and antagonists for octopamine receptors, we observed a rather unique pharmacological profile for Spi-OAR through measurements of cAMP. Spi-OAR exhibited some constitutive activity in HEK 293 cells and no Ca(2+) responses could be detected following addition of octopamine to Spi-OAR-transfected cells. RT-PCR analysis revealed ubiquitous expression of Spi-OAR mRNA in all adult tissues, oocytes and early embryos examined. While addition of serotonin to isolated clam oocytes resulted in meiotic activation, similar additions of octopamine had no effect, suggesting that its potential role in clam reproductive physiology differs significantly from that of serotonin. This work identifies Spi-OAR as a novel mollusc octopamine receptor closely related to other invertebrate beta-adrenergic-like octopamine receptors, with possible reproductive and other physiological functions. This initial characterization of Spi-OAR makes possible further investigations and comparisons with more studied and familiar insect or gastropod mollusc octopamine receptors.

Summary The deltaic aquifer system of the Valcartier sector in Quebec, Canada, is part of a quaternary valley fill contaminated by dissolved trichloroethene (TCE). The objective of our study is to define the aquifer system heterogeneity that should influence TCE transport. Heterogeneity is defined by the distribution of both hydrofacies and hydraulic conductivity ( K ). Hydrofacies are defined as lithologic facies with distinctive hydraulic conductivity ranges. Our approach was developed to take advantage of the abundant stratigraphic and lithologic data provided by borehole logs (7000 m logged from 430 locations). Four site-specific deltaic hydrofacies were defined on the basis of lithologic descriptions, supported by data from grain size analyses, slug tests and cone penetration tests. Each hydrofacies includes a group of geologic facies found in borehole log descriptors to which an initial mean horizontal conductivity K H is associated based on slug tests. Borehole logs were converted to hydrofacies proportions over 5-m intervals to provide 1350 data points. The spatial distribution of hydrofacies was interpolated by three successive interconnected 3D kriging steps using the new technique of “imbricated kriging”. Global dual kriging is directly carried out on the 3D grid of a numerical model. Finally, the proportions of hydrofacies were used to estimate horizontal ( K H ) and vertical ( K V ) hydraulic conductivity fields using generalized means for layered media. Final K H values assigned to the hydrofacies are calibrated by comparison with 2D trends in K H shown by slug tests. This approach also provides an estimated vertical K V field with a spatially varying proportion to K H , rather than a fixed anisotropy ratio. Imbricated kriging does not preserve the statistical variability of fine scale hydrofacies distribution representative of geological variability. However, the approach provides K H and K V estimates over a 3D numerical grid that are coherent with hydrofacies distribution, which in this case control K H and K V .

Non-steroidal anti-inflammatory drugs (NSAIDs) and inhibitors of the cyclooxygenase (COX) pathways are currently recommended for the prevention and treatment of several inflammatory diseases, including neurodegenerative disorders. However non-selective blockade of COX was found to have pro-inflammatory properties, because they have the ability to alter the plasma glucocorticoid levels that play a critical role in the control of the innate immune response. The present study investigated the role of non-selective (ketorolac or indomethacin) or specific inhibitors of COX-1 (SC-560) and COX-2 (NS-398) in these effects. Mice challenged systemically with the endotoxin lipopolysaccharide (LPS) exhibited a robust hybridization signal for numerous inflammatory genes in vascular-associated cells of the brain and microglia across the cerebral tissue. Ketorolac, indomethacin and NS-398 significantly increased the ability of LPS to trigger such an innate immune response at time 3 h post challenge, whereas SC-560 failed to change gene expression in the brain of animals treated with the endotoxin. These data together with the crucial role of COX-2-derived prostaglandin E2 (PGE2) in the increase of glucocorticoids during systemic immune stimuli provide evidence that inhibition of this pathway results in an exacerbated early innate immune reaction. This may have a major impact on the use of these drugs in diseases where inflammation is believed to be a contributing and detrimental factor.

The present study investigated the interaction between highly reactive gaseous-free radical NO and cytokines that are produced by activated Th-1 cells on the cerebral immune response and neuronal integrity. CD-1 mice received an intrastriatal infusion of different solutions containing the NO synthase inhibitor N(G)-nitro-l-arginine methylester, NO-releasing substance sodium nitroprusside (SNP), IFN-γ, and/or TNF-α. The solution containing both cytokines caused a profound and transient transcriptional activation of numerous genes encoding proinflammatory proteins in microglial/monocytic cells ipsilateral to infusion site. This increase in gene expression peaked 1 day after the cerebral bolus of cytokines and returned to basal levels from 3 to 7 days post administration. N(G)-nitro-l-arginine methylester further stimulated this immune reaction to IFN-γ and TNF-α, but the brain of these mice failed to exhibit signs of neurodegeneration and demyelination. In contrast, a single bolus of SNP in the striatal region caused neuronal death and demyelination as early as 1 to 3 days following the infusion with the NO donor. This phenomenon was greatly exacerbated by the coadministration of both cytokines, although TNF-α remained the most critical cytokine to enhance the damage of cerebral elements. These data provide evidence that NO has the ability to modulate the immune response, which is not by itself detrimental for the brain. However, SNP-induced NO production together with TNF-α in the cerebral environment are critical events leading to intense neurodegeneration and demyelination in vivo.

Circulating tumor necrosis factor alpha (TNF-alpha) has a profound stimulatory influence on mitogen-activated protein kinases that lead to nuclear factor kappa B (NF-kappaB) activity and transcription of the cyclooxygenase 2 (COX-2) gene in cells associated with the blood-brain barrier (BBB). This study investigated the hypothesis that nitric oxide (NO) acts as an endogenous modulator of TNF-induced NF-kappaB signaling and COX-2 transcription in the endothelium of the cerebral capillaries. To this end, rats were pretreated with the nonselective inhibitor of NO synthase (NOS) N(G)-nitro-L-arginine methyl ester (L-NAME) and killed 15, 45, and 90 minutes (min) after an i.v. injection of recombinant rat TNF-alpha. De novo expression of the inhibitory factor kappa B alpha (IkappaB alpha) was used as an index of NF-kappaB activity, whereas COX-2 mRNA induction was evaluated throughout the brain by in situ hybridization combined with immunohistochemistry. A single i.v. bolus of TNF caused a rapid expression of IkappaB alpha transcript first along large arterioles and small capillaries and thereafter within microglia across the brain parenchyma. The proinflammatory cytokine also provoked a strong transcriptional activation of the COX-2 gene that was quite specific to the cerebral endothelium as revealed by dual labeling using an antisera directed against the von Willebrand factor. Inhibition of NO synthesis did not by itself activate these proinflammatory molecules, but it enhanced the effects of circulating TNF-alpha in the BBB; the IkappaB alpha and COX-2 signal was significantly higher in microvascular-associated cells of animals that received both L-NAME and TNF-alpha treatments than those challenged with the proinflammatory cytokine alone. Rats treated with specific NOS inhibitors provided the evidence that these effects were mediated via the constitutive endothelial NOS (eNOS) and not the inducible form. These results indicate that eNOS-derived NO acts as an endogenous inhibitor of TNF-alpha-induced NF-kappaB activity and COX-2 transcription in the endothelium of the cerebral capillaries. This autoregulatory feedback of NO on these proinflammatory signal transduction events may be an essential element to prevent an exaggerated response that takes place in cells of the BBB during systemic immune challenges.

Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.

There is an innate immune system in the brain. It is inducible in a transient manner from the structures that are devoid of blood brain barrier and thereafter within parenchymal microglia during systemic infection. Transcriptional activation of genes encoding proteins of the innate immunity also takes place in diseases of the central nervous system. This recent discovery raised the hypothesis that inflammation and innate immunity may be involved in the etiology of neurodegenerative disorders. Nevertheless, this system is able to trigger the release of neurotrophic factors and to protect neuronal elements during brain infection and trauma. The innate immune response may play a critical role in protecting neurons and be a possible cause of neurodegeneration. The fate of this newly identified cascade of events is therefore likely to have a determinant impact on the central nervous system during infection and injury.

Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for their antiinflammatory, antipyretic, and analgesic properties. The molecular basis for the therapeutic action of NSAIDs is believed to be in their ability to inhibit cyclooxygenase (COX) activity and thereby blocking the production of prostaglandins. Emerging evidence now suggests that NSAIDs can exert their pharmacological effects through other mechanisms. This study investigated the influence of a nonselective COX-inhibitor ketorolac on IL-1beta- and TNFalpha-induced expression of proinflammatory genes in the brain. Systemic injection of both cytokines caused a rapid and transient transcriptional activation of COX-2 gene within the cerebral microvasculature, which was significantly enhanced by ketorolac. Expression of genes encoding the index of nuclear factor kappaB activity and the chemokine monocyte chemoattractant protein-1 was also increased by the NSAID. We speculated here that such effect was indirectly mediated via an altered secretion of plasma glucocorticoids because ketorolac is a potent inhibitor of the hypothalamic-pituitary-adrenal axis during systemic inflammation. As expected, pretreatment with the glucocorticoid receptor antagonist RU-486 exacerbated the influence of systemic immune stimuli on proinflammatory signaling. In contrast, exogenous corticosterone abolished the effects of ketorolac on IL-1beta-induced COX-2 and monocyte chemoattractant protein-1 gene expression in the cerebral endothelium. This drug plays therefore a paradoxical role in its ability to inhibit the circulating levels of glucocorticoids that are essential inhibitory feedback on the proinflammatory signal transduction pathways and gene transcription. In altering the production of key prostaglandins that are involved in the control of hypothalamic-pituitary-adrenal axis, ketorolac may have proinflammatory properties in the central nervous system during systemic immune stimuli.

Studies of mouse models of endotoxemia and sepsis with gram-negative bacteria have shown that the host response is genetically controlled. Mice infected with the gram-negative bacteriumSalmonella entericaserovar Typhimurium exhibit marked genetic differences in disease manifestation, and the wild-derived strainMus musculus molossinusMOLF/Ei is extremely susceptible toS. entericaserovar Typhimurium. The kinetics of bacterial proliferation within the liver and the spleen and histological examination of tissue sections have suggested that MOLF/Ei mice do not succumb to infection because of overwhelming bacterial growth in the reticuloendothelial organs or massive tissue necrosis, as observed in otherSalmonella-susceptible strains. MOLF/Ei mice respond normally to lipopolysaccharide (LPS) in vivo and in vitro, as determined by the production of tumor necrosis factor alpha and spleen cell mitogenesis. However, they have a unique cytokine profile in response to infection compared to that observed for otherSalmonella-susceptible mice. There was increased expression of mRNA of the interleukin-1α (IL-1α) and IL-1β genes as the infection in the spleens and livers of MOLF/Ei mice progressed. Despite the fact that MOLF/Ei mice have the ability to respond to LPS and the fact that there are significant increases in IL-1α and IL-1β mRNA,Nos2in the spleen is not upregulated and nitrite production by spleen cells is reduced. At the central level, the inflammatory response is characterized by strong upregulation of the inhibitory factor kappa B alpha and Toll-like receptor 2 genes, two genes known to be regulated by LPS and IL-1 in the brain. The high levels of IL-1 expression in the spleens and livers of MOLF/Ei mice may have important implications for the activation of peripheral and central innate immune mechanisms.

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin-1 (proET-1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET-1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET-1. These data reveal that furin and PC7 have similar specificities towards proET-1 and suggest that both enzymes may participate in the maturation of proET-1 in ECs.

Nous savons depuis peu qu’il existe une réponse immunitaire innée dans le système nerveux central. Cette réponse s’amorce à partir des structures non protégées par la barrière hémato-encéphalique et du réseau microvasculaire cérébral et s’étend progressivement à l’ensemble du cerveau au cours d’une infection systémique. L’activité transcriptionnelle des gènes codant pour les protéines de la réponse immunitaire innée est aussi fortement stimulée lors de différentes perturbations neurologiques. Cette découverte récente a soulevé l’hypothèse selon laquelle l’inflammation pourrait contribuer à la neurodégénérescence et à la démyélinisation. Les molécules immunitaires peuvent également stimuler la libération des facteurs neurotrophiques et, par conséquent, promouvoir la réparation et la remyélinisation des neurones. Ainsi, la réponse innée inflammatoire cérébrale peut, d’une part, protéger les neurones mais, d’autre part, être une cause directe de certaines maladies neurodégénératives., There is an innate immune system in the brain. It is inducible in a transient manner from the structures that are devoid of blood brain barrier and thereafter within parenchymal microglia during systemic infection. Transcriptional activation of genes encoding proteins of the innate immunity also takes place in diseases of the central nervous system. This recent discovery raised the hypothesis that inflammation and innate immunity may be involved in the etiology of neurodegenerative disorders. Nevertheless, this system is able to trigger the release of neurotrophic factors and to protect neuronal elements during brain infection and trauma. The innate immune response may play a critical role in protecting neurons and be a possible cause of neurodegeneration. The fate of this newly identified cascade of events is therefore likely to have a determinant impact on the central nervous system during infection and injury.