Blocking Placental Class G Immunoglobulin Transfer Prevents NMDA Receptor Antibody Effects in Newborn Mice

To determine in a mouse model whether neonatal Fc receptor (FcRn) blockade prevents the placental transfer of class G immunoglobulin (IgG) derived from patients with anti-NMDA receptor (NMDAR) encephalitis and their pathogenic effects on the fetuses and offspring.Pregnant C57BL/6J mice were administered via tail vein FcRn antibody (FcRn-ab) or saline solution 6 hours before administration of patients' or controls' IgG on days 14, 15, and 16 of gestation. Three experimental groups were established: mice receiving controls' IgG, patients' IgG, or patients' IgG along with pretreatment with FcRn-ab. Immunohistochemical staining, confocal microscopy, hippocampal long-term potentiation, and standardized developmental and behavioral tasks were used to assess the efficacy of treatment with FcRn-ab.In pregnant mice that received patients' IgG, treatment with FcRn-ab prevented the IgG from reaching the fetal brain, abrogating the decrease of NMDAR clusters and the reduction of cortical plate thickness that were observed in fetuses from untreated pregnant mice. Moreover, among the offspring of mothers that received patients' IgG, those whose mothers were treated with FcRn-ab did not develop the alterations that occurred in offspring of untreated mothers, including impairment in hippocampal plasticity, delay in innate reflexes, and visuospatial memory deficits.FcRn blockade prevents placental transfer of IgG from patients with anti-NMDAR encephalitis and abrogates the synaptic and neurodevelopmental alterations caused by patients' antibodies. This model has potential therapeutic implications for other antibody-mediated diseases of the CNS during pregnancy

Allosteric Modulation of NMDARs Reverses Patients' Autoantibody Effects in Mice

Background and Objectives To demonstrate that an analog (SGE-301) of a brain-derived cholesterol metabolite, 24(S)- hydroxycholesterol, which is a selective positive allosteric modulator (PAM) of NMDA re- ceptors (NMDARs), is able to reverse the memory and synaptic alterations caused by CSF from patients with anti-NMDAR encephalitis in an animal model of passive transfer of antibodies. Methods Four groups of mice received (days 1-14) patients' or controls' CSF via osmotic pumps connected to the cerebroventricular system and from day 11 were treated with daily sub- cutaneous injections of SGE-301 or vehicle (no drug). Visuospatial memory, locomotor activity (LA), synaptic NMDAR cluster density, hippocampal long-term potentiation (LTP), and paired-pulse facilitation (PPF) were assessed on days 10, 13, 18, and 26 using reported techniques. Results On day 10, mice infused with patients' CSF, but not controls' CSF, presented a significant visuospatial memory deficit, reduction of NMDAR clusters, and impairment of LTP, whereas LA and PPF were unaffected. These alterations persisted until day 18, the time of maximal deficits in this model. In contrast, mice that received patients' CSF but from day 11 were treated with SGE-301 showed memory recovery (day 13), and on day 18, all paradigms (memory, NMDAR clusters, and LTP) had reversed to values similar to those of controls. On day 26, no differences were observed among experimental groups. Discussion An oxysterol biology-based PAM of NMDARs is able to reverse the synaptic and memory deficits caused by CSF from patients with anti-NMDAR encephalitis. These findings suggest a novel adjuvant treatment approach that deserves future clinical evaluation

Placental transfer of NMDAR antibodies causes reversible alterations in mice

Objective: To determine whether maternofetal transfer of NMDA receptor (NMDAR) antibodies has pathogenic effects on the fetus and offspring, we developed a model of placental transfer of antibodies. Methods: Pregnant C57BL/6J mice were administered via tail vein patients' or controls' immunoglobulin G (IgG) on days 14-16 of gestation, when the placenta is able to transport IgG and the immature fetal blood-brain barrier is less restrictive to IgG crossing. Immunohistochemical and DiOlistic (gene gun delivery of fluorescent dye) staining, confocal microscopy, standardized developmental and behavioral tasks, and hippocampal long-term potentiation were used to determine the antibody effects. Results: In brains of fetuses, patients' IgG, but not controls' IgG, bound to NMDAR, causing a decrease in NMDAR clusters and cortical plate thickness. No increase in neonatal mortality was observed, but offspring exposed in utero to patients' IgG had reduced levels of cell-surface and synaptic NMDAR, increased dendritic arborization, decreased density of mature (mushroom-shaped) spines, microglial activation, and thinning of brain cortical layers II-IV with cellular compaction. These animals also had a delay in innate reflexes and eye opening and during follow-up showed depressive-like behavior, deficits in nest building, poor motor coordination, and impaired social-spatial memory and hippocampal plasticity. Remarkably, all these paradigms progressively improved (becoming similar to those of controls) during follow-up until adulthood. Conclusions: In this model, placental transfer of patients' NMDAR antibodies caused severe but reversible synaptic and neurodevelopmental alterations. Reversible antibody effects may contribute to the infrequent and limited number of complications described in children of patients who develop anti-NMDAR encephalitis during pregnancy

Allosteric modulation of NMDA receptors prevents the antibody effects of patients with anti-NMDAR encephalitis

Anti-N-Methyl-D-Aspartate Receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a complex neuropsychiatric syndrome in association with an antibody-mediated decrease of NMDAR. About 85% of patients respond to immunotherapy (and removal of an associated tumor if it applies), but it often takes several months or more than 1 year for patients to recover. There are no complementary treatments, beyond immunotherapy, to accelerate this recovery. Previous studies showed that SGE-301, a synthetic analog of 24(S)-hydroxycholesterol, which is a potent, and selective positive allosteric modulator of NMDAR, reverted the memory deficit caused by phencyclidine (a non-competitive antagonist of NMDAR), and prevented the NMDAR dysfunction caused by patients' NMDAR antibodies in cultured neurons. An advantage of SGE-301 is that it is optimized for systemic delivery such that plasma and brain exposures are sufficient to modulate NMDAR activity. Here, we used SGE-301 to confirm that in cultured neurons it prevented the antibody-mediated reduction of receptors, and then we applied it to a previously reported mouse model of passive cerebroventricular transfer of patients' CSF antibodies. Four groups were established: mice receiving continuous (14-day) infusion of patients' or controls' CSF, treated with daily subcutaneous administration of SGE-301 or vehicle (no drug). The effects on memory were examined with the novel object location (NOL) test at different time points, and the effects on synaptic levels of NMDAR (assessed with confocal microscopy) and plasticity (long-term potentiation [LTP]) were examined in the hippocampus on day 18, which in this model corresponds to the last day of maximal clinical and synaptic alterations. As expected, mice infused with patients' CSF antibodies, but not those infused with controls' CSF, and treated with vehicle developed severe memory deficit without locomotor alteration, accompanied by a decrease of NMDAR clusters and impairment of LTP. All antibody-mediated pathogenic effects (memory, synaptic NMDAR, LTP) were prevented in the animals that were treated with SGE-301, despite that this compound did not antagonize antibody binding. Additional investigations on the potential mechanisms related to these SGE-301 effects showed that (1) in cultured neurons SGE-301 prolonged the decay time of NMDAR-dependent spontaneous excitatory postsynaptic currents suggesting a prolonged open time of the channel, and (2) it significantly decreased the internalization of antibody-bound receptors suggesting that additional, yet unclear mechanisms, contribute in keeping unchanged the surface NMDAR density. Overall, these findings suggest that SGE-301, or similar modulators of NMDAR, could potentially serve as complementary treatment for anti-NMDAR encephalitis and deserve future investigations

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures

Impacts of Use and Abuse of Nature in Catalonia with Proposals for Sustainable Management

This paper provides an overview of the last 40 years of use, and in many cases abuse, of the natural resources in Catalonia, a country that is representative of European countries in general, and especially those in the Mediterranean region. It analyses the use of natural resources made by mining, agriculture, livestock, logging, fishing, nature tourism, and energy production and consumption. This use results in an ecological footprint, i.e., the productive land and sea surface required to generate the consumed resources and absorb the resulting waste, which is about seven times the amount available, a very high number but very similar to other European countries. This overexploitation of natural resources has a huge impact on land and its different forms of cover, air, and water. For the last 25 years, forests and urban areas have each gained almost 3% more of the territory at the expense of agricultural land; those municipalities bordering the sea have increased their number of inhabitants and activity, and although they only occupy 6.7% of the total surface area, they account for 43.3% of the population; air quality has stabilized since the turn of the century, and there has been some improvement in the state of aquatic ecosystems, but still only 36% are in good condition, while the remainder have suffered morphological changes and different forms of nonpoint source pollution; meanwhile the biodiversity of flora and fauna remains still under threat. Environmental policies do not go far enough so there is a need for revision of the legislation related to environmental impact and the protection of natural areas, flora, and fauna. The promotion of environmental research must be accompanied by environmental education to foster a society which is Land 2021, 10, 144 3 of 53 more knowledgeable, has more control and influence over the decisions that deeply affect it. Indeed, nature conservation goes hand in hand with other social and economic challenges that require a more sustainable vision. Today’s problems with nature derive from the current economic model, which is environmentally unsustainable in that it does not take into account environmental impacts. Lastly, we propose a series of reasonable and feasible priority measures and actions related to each use made of the country’s natural resources, to the impacts they have had, and to their management, in the hope that these can contribute to improving the conservation and management of the environment and biodiversity and move towards sustainability.info:eu-repo/semantics/publishedVersio

Efectes dels inhibidors de la ciclooxigenasa en cèl·lules hepàtiques i el seu paper en la inflamació i fibrosi hepàtica experimental

[cat] INTRODUCCIÓ:La inflamació és una reacció defensiva de l'organisme contra tot tipus de lesió. Si la inflamació aguda persisteix s'arriba a un estat d'inflamació crònica generant-se una neoformació de teixit connectiu que pot arribar a derivar a teixit fibrós. Els eicosanoids constitueixen els mediadors d'inflamació més importants ja que modulen la iniciació, progressió i resolució de la resposta inflamatòria. L'acid araquidonic (AA) és el precursor més important dels eicosanoids. L'oxidació de l'AA es pot donar de manera enzimàtica a través de l'acció la ciclooxigenasa (COX) que catalitza la síntesi de prostaglandines (PG). L'aspirina (ASA) inhibeix la producció de PG al bloquejar l'activitat de la COX. Interessantment, l'ASA desencadena la biosíntesi de compostos antiinflamatoris endògens (anomenats 15-epi-lipoxines, 15-epi-LXs), que actuen com a senyals de reclutament de leucòcits i juguen un paper important en la resolució de la inflamació. Així doncs, en el primer estudi vam examinar els efectes de l'ASA en les vies de la COX i la 5-lipooxigenasa i el seu impacte en els nivells del receptor activat per proliferadors peroxisomals (PPAR) i CINC-1 (citoquina pro-inflamatoria homòloga de la IL-8 humana) en cèl·lules hepàtiques de rata. La inflamació és un dels factors determinants de la patogènesi de la fibrosi hepàtica ja que precedeix o coexisteix amb el desenvolupament d'alteracions de la matriu extracel·lular. La COX-2 és l'enzim clau en el procés inflamatori i la seva inhibició selectiva constitueix una diana farmacològica interessant en el tractament de la inflamació i la fibrosi hepàtica. En el moment actual no es coneix en precisió la via de la COX en el fetge i les conseqüències de la seva inhibició a nivell de reducció de la inflamació i fibrosi hepàtica. Ja que les cèl·lules de Kupffer (KC) són el tipus cel·lular majoritàriament responsable de l'increment de la COX-2 i de la síntesi de PGs en el fetge i perquè la població d'aquestes cèl·lules incrementa durant la progressió de la inflamació hepàtica, el segon estudi es va basar en l'efecte d'un inhibidor selectiu de COX-2 sobre aquest tipus cel·lular.OBJECTIUS: 1. Avaluar els efectes de l'ASA sobre la síntesi d'eicosanoids i de productes proinflamatoris en cèl·lules hepàtiques en cultiu. 2. Estudiar els efectes d'un inhibidor selectiu de la COX-2, l'SC-236, i el seu possible paper en la resolució de la fibrosi hepàtica.RESULTATS I CONCLUSIONS:En el primer estudi observàrem que l'ASA produïa en KC un redireccionament del metabolisme dels eicosanoids, de la producció de PGE2 cap a la formació de leucotriè B4 i 15-epi-LXA4. Al mateix temps l'ASA i LXA4 i 15-epi-LXA4, a mes d'inhibir l'activitat de la 5-LO, reduiren els nivells de PPAR i CINC-1 en hepatòcilts. En conjunt, i degut a que els productes derivats de l'AA, els nivells de PPAR, i la secreció de CINC-1 estan involucrats en la duració de la resposta inflamatòria, aquests resultats proporcionen mecanismes moleculars addicionals a les propietats farmacològiques de l'ASA.Els resultats del segon estudi ens indicaren que l'expressió de COX-2 es trobava augmentada en fetges de rates tractades amb CCl4. L'administració d'SC-236 disminuïa els nivells incrementats de 15d-PGJ2, el grau de fibrosi hepàtica, el contingut d'hidroxiprolina, l'activitat de les metaloproteinases 2 i 9, i l'expressió de l'alfa-SMA en els fetges de les rates tractades amb CCl4. Addicionalment, l'SC-236 inhibia el creixement i induia apoptosi en cèl·lules hepàtiques estrellades (HSCs) i KC, augmentava l'expressió del PPAR-gamma en HSCs i actuava com a agonista d'aquest receptor nuclear. Així doncs, tots aquests resultats ens indiquen que l'inhibidor selectiu de COX-2, SC-236, mitjançant mecanismes que involucren l'apoptosi de cèl·lules no parenquimals i l'activació del PPAR-gamma, pot tenir un potencial terapèutic en la prevenció de la fibrosi hepàtica.[eng] The mechanism of action of aspirin (ASA) is related to cyclooxygenase (COX) inhibition, but additional actions cannot be excluded for their antiinflammatory and antithrombotic properties. We examined the effects of ASA on COX and 5-lipoxygenase (5-LO) pathways and its impact on peroxisome proliferator-activated receptor alpha (PPAR) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels in rat liver cells. In Kupffer cells (KC), the liver resident macrophages, ASA switched eicosanoid biosynthesis from prostaglandin (PG) E2 to leukotriene B4 and 15-epi-lipoxin A4 (15-epi-LXA4) formation. In hepatocytes, ASA significantly inhibited PPAR-alpha protein expression and CINC-1 secretion. Interestingly, the endogenous antiinflammatory eicosanoids LXA4 and 15-epi-LXA4, in addition to inhibiting macrophage 5-LO activity, reduced PPAR levels and CINC-1 secretion in hepatocytes. Taken together and because arachidonic acid-derived products, PPAR expression and CINC-1 levels are involved in the extent and duration of an inflammatory response, these findings provide additional molecular mechanisms for the pharmacological properties of ASA.The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. For this reason, we tested the effects of SC-236, a selective COX-2 inhibitor, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Livers from CCl4-treated rats showed increased COX-2 expression and 15d-PGJ2 formation. In these animals, SC-236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase-2 activity, and alpha-smooth muscle actin expression. Interestingly, SC-236 normalized 15d-PGJ2 levels and restored PPAR-alpha expression in the liver of CCl4-treated rats. In isolated hepatic stellate cells (HSC)--the major player in liver fibrogenesis--and KC--the cell type primarily responsible for increased hepatic COX-2-- SC-236 exhibited remarkable pro-apoptotic and growth inhibitory properties. Of interest, SC-236 decreased HSC viability to a similar extent than the PPAR-gamma ligand rosiglitazone. Moreover, SC-236 significantly induced PPAR-gamma expression in HSCs and acted as a potent PPAR-gamma agonist in a luciferase-reporter trans-activation assay. These data indicate that, by mechanisms involving non-parenchymal cell apoptosis and PPAR-gamma activation, the selective COX-2 inhibitor SC-236 might have therapeutic potential for prevention of liver fibrosis

Role of Filamin A in endocytic trafficking of GPCRs

Póster presentado en el XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrada del 3 al 6 de septiembre de 2013 en Madrid (España)Chemokines and their receptors (GPCR) are known to play a crucial role in directing the movement of circulating leukocytes to sites of inflammation or injury. Monocyte chemoattractant protein 1 (CCL2), and its receptor CCR2 are implicated in a wide variety of pathophysiological inflammatory processes. We have previously demonstrated that the Filamin A protein binds to the carboxyl terminal tail of the CCR2B and its binding is important for the early steps of receptor-vesicle internalization [1,2]. We have now observed using a melanoma cell line M2, devoid of filamin A, that both the ligand-activated CCR2B and Transferrin did not reach the Rab11 recycling compartment and the internalized CCR2B-vesicles remained closer to the plasma membrane in M2 cells. In order to deep into the mechanism of action of filamin A, we have investigated its role in the entry of the receptor to the different recycling compartments, Rab4-short loop and Rab11-long loop recycling. We used immunofluorescence antibody labeling, as well as, fluorescence recovery after photobleaching (FRAP) with shFLNA Hela cells to study the influence of FLNA in trafficking of CCR2B. Transferrin and CCR2B receptors recycling is affected by the lack of FLNA in shFLNA cells. In addition, we also report here that most Rab11 recovered the bleached area more rapidly in scramble control cells than in shFLNA. Rab4 recovered equally in both types of cells. Thus, our data demonstrate that FLNA has an important role in the internalization and recycling of the CCR2B and affects the localization and trafficking of Rab11-endosomes.Peer Reviewe

Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time

Background: Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B. Methodology and Principal Findings: In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers. Significance: Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles