Combining imaging mass spectrometry and immunohistochemistry to analyse the lipidome of spinal cord inflammation.

Inflammation is a complex process that accompanies many pathologies. Actually, dysregulation of the inflammatory process is behind many autoimmune diseases. Thus, treatment of such pathologies may benefit from in-depth knowledge of the metabolic changes associated with inflammation. Here, we developed a strategy to characterize the lipid fingerprint of inflammation in a mouse model of spinal cord injury. Using lipid imaging mass spectrometry (LIMS), we scanned spinal cord sections from nine animals injected with lysophosphatidylcholine, a chemical model of demyelination. The lesions were demonstrated to be highly heterogeneous, and therefore, comparison with immunofluorescence experiments carried out in the same section scanned by LIMS was required to accurately identify the morphology of the lesion. Following this protocol, three main areas were defined: the lesion core, the peri-lesion, which is the front of the lesion and is rich in infiltrating cells, and the uninvolved tissue. Segmentation of the LIMS experiments allowed us to isolate the lipid fingerprint of each area in a precise way, as demonstrated by the analysis using classification models. A clear difference in lipid signature was observed between the lesion front and the epicentre, where the damage was maximized. This study is a first step to unravel the changes in the lipidome associated with inflammation in the context of diverse pathologies, such as multiple sclerosis.

Cloning, expression, solubilization, and purification of a functionally active recombinant cAMP-dependent protein kinase catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum.

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni2+-chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP ¼ GTP â‰… ITP and Mg2+ â‰… Mn2+ â‰… Fe2+ ¼ Ca2+ â‰… Zn2, respectively.

Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age.

Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor's health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors. Two different PRP pools were prepared from donors 65‒85 and 20‒25 years old. The cellular and molecular composition of both PRPs were analyzed. Subsequently, the cellular response was evaluated in CNS in vitro models, studying proliferation, neurogenesis, synaptogenesis, and inflammation. While no differences in the cellular composition of PRPs were found, the molecular composition of the Young PRP showed lower levels of inflammatory molecules such as CCL-11, as well as the presence of other factors not found in Aged PRP (GDF-11). Although both PRPs had effects in terms of reducing neural progenitor cell apoptosis, stabilizing neuronal synapses, and decreasing inflammation in the microglia, the effect of the Young PRP was more pronounced. In conclusion, the molecular composition of the PRP, conditioned by the age of the donors, affects the magnitude of the biological response.

Contribution of P2X4 Receptors to CNS Function and Pathophysiology.

The release and extracellular action of ATP are a widespread mechanism for cell-to-cell communication in living organisms through activation of P2X and P2Y receptors expressed at the cell surface of most tissues, including the nervous system. Among ionototropic receptors, P2X4 receptors have emerged in the last decade as a potential target for CNS disorders such as epilepsy, ischemia, chronic pain, anxiety, multiple sclerosis and neurodegenerative diseases. However, the role of P2X4 receptor in each pathology ranges from beneficial to detrimental, although the mechanisms are still mostly unknown. P2X4 is expressed at low levels in CNS cells including neurons and glial cells. In normal conditions, P2X4 activation contributes to synaptic transmission and synaptic plasticity. Importantly, one of the genes present in the transcriptional program of myeloid cell activation is P2X4. Microglial P2X4 upregulation, the P2X4+ state of microglia, seems to be common in most acute and chronic neurodegenerative diseases associated with inflammation. In this review, we summarize knowledge about the role of P2X4 receptors in the CNS physiology and discuss potential pitfalls and open questions about the therapeutic potential of blocking or potentiation of P2X4 for different pathologies.

IL-6 trans-signaling in the brain influences the behavioral and physio-pathological phenotype of the Tg2576 and 3xTgAD mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is the most commonly diagnosed dementia but its underlying pathological mechanisms still unclear. Neuroinflammation and secretion of cytokines such as interleukin-6 (IL-6) accompany the main hallmarks of the disease: amyloid plaques and neurofibrillary tangles. In this study, we analyzed the role of IL-6 trans-signaling in two mouse models of AD, Tg2576 and 3xTg-AD mice. The inhibition of IL-6 trans-signaling partially rescued the AD-induced mortality in females of both models. Before amyloid plaques deposition, it reversed AD-induced changes in exploration and anxiety (but did not affect locomotion) in Tg2576 female mice. However, after plaque deposition the only behavioral trait affected by the inhibition of IL-6 trans-signaling was locomotion. Results in the Morris water maze suggest that cognitive flexibility was reduced by the blocking of the IL-6 trans-signaling in young and old Tg2576 female mice. The inhibition of IL-6 trans-signaling also decreased amyloid plaque burden in cortex and hippocampus, and Aß40 and Aß42 levels in the cortex, of Tg2576 female mice. The aforementioned changes might be correlated with changes in blood vessels and matrix structure and organization rather than changes in neuroinflammation. 3xTgAD mice showed a very mild phenotype regarding amyloid cascade, but results were in accordance with those of Tg2576 mice. These results strongly suggest that the inhibition of the IL-6 trans-signaling could represent a powerful therapeutic target in AD.

Glucose deprivation activates a cAMP-independent protein kinase from Trypanosoma equiperdum.

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07-51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22-1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, ß,γ-imidoadenosine 5'-triphosphate and 5'-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.

Microglia and meningeal macrophages depletion delays the onset of experimental autoimmune encephalomyelitis.

In multiple sclerosis and the experimental autoimmune encephalomyelitis (EAE) model, both resident microglia and infiltrating macrophages contribute to demyelination as well as spontaneous remyelination. Nevertheless, the specific roles of microglia versus macrophages are unknown. We investigated the influence of microglia in EAE using the colony stimulating factor 1 receptor (CSF-1R) inhibitor, PLX5622, to deplete microglial population and Ccr2RFP/+ fmsEGFP/+ mice, to distinguish blood-derived macrophages from microglia. PLX5622 treatment depleted microglia and meningeal macrophages, and provoked a massive infiltration of CCR2+ macrophages into demyelinating lesions and spinal cord parenchyma, albeit it did not alter EAE chronic phase. In contrast, microglia and meningeal macrophages depletion reduced the expression of major histocompatibility complex II and CD80 co-stimulatory molecule in dendritic cells, macrophages and microglia. In addition, it diminished T cell reactivation and proliferation in the spinal cord parenchyma, inducing a significant delay in EAE onset. Altogether, these data point to a specific role of CNS microglia and meningeal macrophages in antigen presentation and T cell reactivation at initial stages of EAE.

Clemastine Induces an Impairment in Developmental Myelination.

Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c+ microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.

Role of Mitochondrial Dynamics in Microglial Activation and Metabolic Switch.

Microglia act as sensors of injury in the brain, favoring its homeostasis. Their activation and polarization toward a proinflammatory phenotype are associated with injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolysis and suppressed oxidative phosphorylation. This metabolic switch can be reproduced in vitro by microglial stimulation with LPS plus IFN-γ. To understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during this switch using rat primary microglia. Cells did not show any change in mitochondrial membrane potential, suggesting a limited impact in the mitochondrial viability. We provide evidence that reverse operation of F0F1-ATP synthase contributes to mitochondrial membrane potential. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. Mdivi-1 significantly reduced the expression of proinflammatory markers in LPS plus IFN-γ-treated microglia. However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS plus IFN-γ or to a microglia repolarization. Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia.

In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke.

Adenosine A1 receptors (A1ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A1ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A1ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [18F]CPFPX and immunohistochemistry (IHC). In addition, the role of A1ARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [18F]DPA-714 (TSPO) and [18F]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [18F]CPFPX signal and IHC showed the overexpression of A1ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the A1AR agonist ENBA showed a significant decrease in both [18F]DPA-714 and [18F]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A1ARs promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A1ARs expression after cerebral ischemia in rats and the application of [18F]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A1ARs playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.

Functional and Metabolic Characterization of Microglia Culture in a Defined Medium.

Microglia are the endogenous immune cells of the brain and act as sensor of infection and pathologic injury to the brain, leading to a rapid plastic process of activation that culminates in the endocytosis and phagocytosis of damaged tissue. Microglia cells are the most plastic cells in the brain. Microglia isolation from their environment as well as culturing them in the presence of serum alter their function and lead to a rapid loss of their signature gene expression. Previous studies have identified pivotal factors allowing microglia culture in the absence of serum. Here, we have further characterized the function, expression of markers, metabolic status and response to pro and anti-inflammatory stimulus of microglia isolated by magnetic-activated cell sorting and cultured in a chemically defined medium. We have compared this new method with previous traditional protocols of culturing microglia that use high concentrations of serum.

Glomerulopatía colapsante con insuficiencia renal terminal de rápida progresión / Collapsing glomerulopathy with rapid progression to terminal renal failure

La glomerulopatía colapsante (GC) constituye una variedad de la glomeruloesclerosis focal y segmentaria. Afecta tanto a la población adulta (40%) como a la infantil (20%); presentándose con mayor frecuencia en hombres jóvenes y afrodescendientes. Clínicamente se presenta como un síndrome nefrótico, con niveles elevados de úrea y creatinina. Se presenta el caso de paciente femenino de 22 años, quien acude por presentar fiebre, edema matutino en miembros inferiores, e intolerancia oral de 9 días de evolución. Al examen físico: Hipertensión arterial y ascitis. La GC es una entidad poco diagnosticada, que progresa rápidamente a insuficiencia renal terminal a pesar de recibir cualquier tratamiento sistémico descrito hasta la actualidad, por lo que amerita mayor investigación en el ámbito terapéutico(AU)

Collapsing glomerulopathy (GC) is a variety of focal segmental glomerulosclerosis. It affects both adult population (40%) and children (20%); it occurs most often in young people, male and of African descent. Clinically it is presented as a nephrotic syndrome, with high levels of urea and creatinine serum. There is insufficient evidence regarding the treatment of this entity, so that steroids and immunosuppressants are used at high doses. We present the case of a 22-year old female, who presented fever, edema in the lower limbs and oral intolerance of 9 days of evolution. Physical examination showed: high blood pressure and ascitis. This nephropathy is an underdiagnosed entity rapidly progressing to kidney failure despite receiving any systemic treatment described until now, so it merits further research in the therapeutic field(AU)

Primeros embarazos con el uso del antagonista de la hormona liberadora de gonadotrofinas ganirelix: reporte de dos casos / First pregnancies with the use of the gonadotropin release hormone antagonist ganirelix: report of two cases

Se reportan los dos primeros embarazos obtenidos con el uso del antagonista de la hormona liberadora de gonadotrofinas, Ganirelix. El primer embarazo se obtuvo en una paciente con diagnóstico de falla ovárica precoz a quien se le realizó donación de óvulos e inyección intracitoplasmática de espermatozoides. Actualmente tiene embarazo de 10 semanas de gestación con evolución normal. A la segunda paciente se realizó ciclo de inyección intracitoplasmática de espermatozoides por infertilidad primaria debida a factor masculino quien cursa en la actualidad con embarazo gemelar de 9 semanas