The role of P-glycoprotein (P-gp) and inwardly rectifying potassium (Kir) channels in sudden unexpected death in epilepsy (SUDEP).

Sudden unexpected death in epilepsy (SUDEP) is the major cause of death that affects patients with epilepsy. The risk of SUDEP increases according to the frequency and severity of uncontrolled seizures; therefore, SUDEP risk is higher in patients with refractory epilepsy (RE), in whom most antiepileptic drugs (AEDs) are ineffective for both seizure control and SUDEP prevention. Consequently, RE and SUDEP share a multidrug resistance (MDR) phenotype, which is mainly associated with brain overexpression of ABC-transporters such as P-glycoprotein (P-gp). The activity of P-gp can also contribute to membrane depolarization and affect the normal function of neurons and cardiomyocytes. Other molecular regulators of membrane potential are the inwardly rectifying potassium channels (Kir), whose genetic variants have been related to both epilepsy and heart dysfunctions. Although it has been suggested that dysfunctions of the cardiac, respiratory, and brainstem arousal systems are the causes of SUDEP, the molecular basis for explaining its dysfunctions remain unknown. In rats, repetitive seizures or status epilepticus induced high expression of P-gp and loss Kir expression in the brain and heart, and promoted membrane depolarization, malignant bradycardia, and the high rate of mortality. Here we reviewed clinical and experimental evidences suggesting that abnormal expression of depolarizing/repolarizing factors as P-gp and Kir could favor persistent depolarization of membranes without any rapid functional recovery capacity. This condition induced by convulsive stress could be the molecular mechanism leading to acquired severe bradycardia, as an ineffective heart response generating the appropriate scenario for SUDEP development. This article is part of the Special Issue "NEWroscience 2018".

New anticonvulsant candidates prevent P-glycoprotein (P-gp) overexpression in a pharmacoresistant seizure model in mice.

Despite the approval of a considerable number of last generation antiepileptic drugs (AEDs) (only in the last decade, six drugs have gained Food and Drug Administration approval), the global figures of seizure control have seemingly not improved, and available AED can still be regarded as symptomatic treatments. Fresh thinking in AEDs drug discovery, including the development of drugs with novel mechanisms of action, is required to achieve truly innovative antiepileptic medications. The transporter hypothesis proposes that inadequate penetration of AEDs across the blood-brain barrier, caused by increased expression of efflux transporters such as P-glycoprotein (P-gp), contributes to drug-resistant epilepsy. Neuroinflammation due to high levels of glutamate has been identified as one of the causes of P-gp upregulation, and several studies in animal models of epilepsy suggest that antiinflammatory drugs might prevent P-gp overexpression and, thus, avoid the development of refractory epilepsy. We have applied ligand-based in silico screening to select compounds that exert dual anticonvulsant and antiinflammatory effects. Five of the hits were tested in animal models of seizure, with protective effects. Later, two of them (sebacic acid (SA) and gamma-decanolactone) were submitted to the recently described MP23 model of drug-resistant seizures. All in all, SA displayed the best profile, showing activity in the maximal electroshock seizure (MES) and pentylenetetrazol (PTZ) seizure models, and reversing resistance to phenytoin (PHT) and decreasing the P-gp upregulation in the MP23 model. Furthermore, pretreatment with SA in the pilocarpine status epilepticus (SE) model resulted in decreased histamine release in comparison with nontreated animals. This is the first report of the use of the MP23 model to screen for novel anticonvulsant compounds that may avoid the development of P-gp-related drug resistance.

Metastatic Niches and the Modulatory Contribution of Mesenchymal Stem Cells and Its Exosomes.

Mesenchymal stem cells (MSCs) represent an interesting population due to their capacity to release a variety of cytokines, chemokines, and growth factors, and due to their motile nature and homing ability. MSCs can be isolated from different sources, like adipose tissue or bone marrow, and have the capacity to differentiate, both in vivo and in vitro, into adipocytes, chondrocytes, and osteoblasts, making them even more interesting in the regenerative medicine field. Tumor associated stroma has been recognized as a key element in tumor progression, necessary for the biological success of the tumor, and MSCs represent a functionally fundamental part of this associated stroma. Exosomes represent one of the dominant signaling pathways within the tumor microenvironment. Their biology raises high interest, with implications in different biological processes involved in cancer progression, such as the formation of the pre-metastatic niche. This is critical during the metastatic cascade, given that it is the formation of a permissive context that would allow metastatic tumor cells survival within the new environment. In this context, we explored the role of exosomes, particularly MSCs-derived exosomes as direct or indirect modulators. All this points out a possible new tool useful for designing better treatment and detection strategies for metastatic progression, including the management of chemoresistance.

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND: Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS: We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS: Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS: The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

Twenty-One Novel EGFR Kinase Domain variants in Patients with Nonsmall Cell Lung Cancer.

Somatic sequence variants in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with nonsmall cell lung cancer (NSCLC). Patients exhibiting sequence variants in this domain that produce kinase activity enhancement, are more likely to benefit from TKIs than patients with EGFR wild-type disease. Although most NSCLC EGFR-related alleles are concentrated in a few positions, established protocols recommend sequencing EGFR exons 18-21. In this study, 21 novel somatic variants belonging to such exons in adult Argentinean patients affected with NSCLC are reported. Of these, 18 were single amino acid substitutions (SASs), occurring alone or in combination with another genetic alteration (complex cases), one was a short deletion, one was a short deletion-short insertion combination, and one was a duplication. New variants and different combinations of previously reported variants were also found. Moreover, two of the reported SASs occurred in previously unreported positions of the EGFR kinase domain. In order to characterize the new sequence variants, physicochemical, sequence and conformational analyses were also performed. A better understanding of sequence variants in NSCLC may facilitate the most appropriate treatment choice for this complex disease.

In vitro Effect of Cannabidiol on Red Blood Cells: Implication in Long-Lasting Pathology Treatment.

BACKGROUND: Cannabidiol (CBD) is the principal non-hallucinogenic compound of Cannabis plants with high clinical interest because CBD has been described as having anti-inflammatory, analgesic and anticonvulsant properties. CBD is considered a multitarget compound as it can interact with a wide range of targets, explaining their multiplicity of effects. Some clinical studies have indicated certain side effects of CBD, including somnolence, anemia and diarrhea, while the elevation of transaminases is considered as an exclusion criterion from the trial. Since the red blood cells (RBCs) are a source of transaminase, we assayed in vitro effect on RBCs stability. METHODS: We performed in vitro experiments with RBCs obtained from human peripheral blood with normal hematological parameters exposed to CBD in the range of therapeutic uses. We evaluated RBCs morphological changes, membrane fragility and hemoglobin release as a reflection of hemolysis. RESULTS: CBD induced an increase in the hemoglobin release (3.27 µg/106 RBC), without altered RBC osmotic fragility. When RBCs suspensions were incubated with CBD the initial number of elements (RBCs + vesicles) was increased up to 65% after 20 min and returned to basal level after 40 min of incubation. In the first 20 min, the accounts of elements were enriched in the smaller vesicles that disappeared after the remaining 20 minutes. CONCLUSION: These results suggest that CBD affects the indemnity of erythrocytes in vitro, inducing the formation of hemolytic vesicles that can provide the basis for the development of anemia, transaminase elevation and underlying tissular iron overload in patients chronically treated with CBD.

COVID-19 and Alzheimer's Disease: Neuroinflammation, Oxidative Stress, Ferroptosis, and Mechanisms Involved.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by marked cognitive decline, memory loss, and spatio-temporal troubles and, in severe cases, lack of recognition of family members. Neurological symptoms, cognitive disturbances, and the inflammatory frame due to COVID-19, together with long-term effects, have fueled renewed interest in AD based on similar damage. COVID-19 also caused the acceleration of AD symptom onset. In this regard, the morbidity and mortality of COVID-19 were reported to be increased in patients with AD due to multiple pathological changes such as excessive expression of the viral receptor angiotensin-converting enzyme 2 (ACE2), comorbidities such as diabetes, hypertension, or drug-drug interactions in patients receiving polypharmacy and the high presence of proinflammatory molecules. Furthermore, the release of cytokines, neuroinflammation, oxidative stress, and ferroptosis in both diseases showed common underlying mechanisms, which together worsen the clinical picture and prognosis of these patients.

Enlightening the mechanism of ferroptosis in epileptic heart.

Epilepsy is a chronic neurological degenerative disease with a high incidence, affecting all age groups. Refractory Epilepsy (RE) occurs in approximately 30-40% of cases with a higher risk of sudden unexpected death in epilepsy (SUDEP). Recent studies have shown that spontaneous seizures developed in epilepsy can be related to an increase in oxidative stress and reactive oxygen derivatives (ROS) production. Increasing ROS concentration causes lipid peroxidation, protein oxidation, destruction of nuclear genetic material, enzyme inhibition, and cell death by a mechanism known as "ferroptosis" (Fts). Inactivation of glutathione peroxidase 4 (GPX4) induces Fts, while oxidative stress is linked with increased intracellular free iron (Fe+2) concentration. Fts is also a non-apoptotic programmed cell death mechanism, where a hypoxia-inducible factor 1 alpha (HIF-141) dependent hypoxic stress-like condition appears to occur with accumulation of iron and cytotoxic ROS in affected cells. Assuming convulsive crises as hypoxic stress, repetitive convulsive/hypoxic stress can be an effective inducer of the "epileptic heart" (EH), which is characterized by altered autonomic function and a high risk of malignant or fatal bradycardia. We previously reported that experimental recurrent seizures induce cardiomyocyte Fts associated with SUDEP. Furthermore, several genes related to Fts and hypoxia have recently been identified in acute myocardial infarction. An emerging theme from recent studies indicates that inhibition of GPX4 through modulating expression or activities of the xCT antiporter system (SLC7A11) governs cell sensitivity to oxidative stress from ferroptosis. Furthermore, during hypoxia, an increased expression of stress transcriptional factor ATF3 can promote Fts induced by erastin in a HIF-141-dependent manner. We propose that inhibition of Fts with ROS scavengers, iron chelators, antioxidants, and transaminase inhibitors could provide a therapeutic effect in epilepsy and improve the prognosis of SUDEP risk by protecting the heart from ferroptosis.

Uridine Diphosphate Glucose (UDP-G) Activates Oxidative Stress and Respiratory Burst in Isolated Neutrophils.

The extracellular purinergic agonist uridine diphosphate glucose (UDP-G) activates chemotaxis of human neutrophils (PMN) and the recruitment of PMN at the lung level, via P2Y14 purinergic receptor signaling. This effect is similar to the activation of PMN with N-formyl-methionyl-leucyl-phenylalanine (fMLP), a mechanism that also triggers the production of superoxide anion and hydrogen peroxide via the NADPH oxidase system. However, the effects of UDP-G on this system have not been studied. Defects in the intracellular phagocyte respiratory burst (RB) cause recurrent infections, immunodeficiency, and chronic and severe diseases in affected patients, often with sepsis and hypoxia. The extracellular activation of PMN by UDP-G could affect the RB and oxidative stress (OS) in situations of inflammation, infection and/or sepsis. The association of PMNs activation by UDP-G with OS and RB was studied. OS was evaluated by measuring spontaneous chemiluminescence (CL) of PMNs with a scintillation photon counter, and RB by measuring oxygen consumption with an oxygen Clark electrode at 37 °C, in non-stimulated cells and after activation (15 min) with lipopolysaccharides (LPS, 2 µg/mL), phorbol myristate acetate (PMA, 20 ng/mL), or UDP-G (100 µM). The stimulation index (SI) was calculated in order to establish the activation effect of the three agonists. After stimulation with LPS or PMA, the activated PMNs (0.1 × 106 cells/mL) showed an increase in CL (35%, p < 0.05 and 56%, p < 0.01, SI of 1.56 and 2.20, respectively). Contrariwise, the stimulation with UDP-G led to a decreased CL in a dose-dependent manner (60%, 25 µM, p < 0.05; 90%, 50-150 µM, p < 0.001). Nonetheless, despite the lack of oxidative damage, UDP-G triggered RB (SI 1.8) in a dose-dependent manner (38-50%, 100-200 µM, p < 0.0001). UDP-G is able to trigger NADPH oxidase activation in PMNs. Therefore, the prevention of OS and oxidative damage observed upon PMN stimulation with UDP-G indicates an antioxidant property of this molecule which is likely due to the activation of antioxidant defenses. Altogether, LPS and UDP-G have a synergistic effect, suggesting a key role in infection and/or sepsis.

An updated examination of the perception of barriers for pharmacogenomics implementation and the usefulness of drug/gene pairs in Latin America and the Caribbean.

Pharmacogenomics (PGx) is considered an emergent field in developing countries. Research on PGx in the Latin American and the Caribbean (LAC) region remains scarce, with limited information in some populations. Thus, extrapolations are complicated, especially in mixed populations. In this paper, we reviewed and analyzed pharmacogenomic knowledge among the LAC scientific and clinical community and examined barriers to clinical application. We performed a search for publications and clinical trials in the field worldwide and evaluated the contribution of LAC. Next, we conducted a regional structured survey that evaluated a list of 14 potential barriers to the clinical implementation of biomarkers based on their importance. In addition, a paired list of 54 genes/drugs was analyzed to determine an association between biomarkers and response to genomic medicine. This survey was compared to a previous survey performed in 2014 to assess progress in the region. The search results indicated that Latin American and Caribbean countries have contributed 3.44% of the total publications and 2.45% of the PGx-related clinical trials worldwide thus far. A total of 106 professionals from 17 countries answered the survey. Six major groups of barriers were identified. Despite the region's continuous efforts in the last decade, the primary barrier to PGx implementation in LAC remains the same, the "need for guidelines, processes, and protocols for the clinical application of pharmacogenetics/pharmacogenomics". Cost-effectiveness issues are considered critical factors in the region. Items related to the reluctance of clinicians are currently less relevant. Based on the survey results, the highest ranked (96%-99%) gene/drug pairs perceived as important were CYP2D6/tamoxifen, CYP3A5/tacrolimus, CYP2D6/opioids, DPYD/fluoropyrimidines, TMPT/thiopurines, CYP2D6/tricyclic antidepressants, CYP2C19/tricyclic antidepressants, NUDT15/thiopurines, CYP2B6/efavirenz, and CYP2C19/clopidogrel. In conclusion, although the global contribution of LAC countries remains low in the PGx field, a relevant improvement has been observed in the region. The perception of the usefulness of PGx tests in biomedical community has drastically changed, raising awareness among physicians, which suggests a promising future in the clinical applications of PGx in LAC.

Neuroproteomics Chip-Based Mass Spectrometry and Other Techniques for Alzheimer's Disease Biomarkers - Update.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease of growing interest given that there is cognitive damage and symptom onset acceleration. Therefore, it is important to find AD biomarkers for early diagnosis, disease progression, and discrimination of AD and other diseases. OBJECTIVE: The objective of this study is to update the relevance of mass spectrometry for the identification of peptides and proteins involved in AD useful as discriminating biomarkers. METHODS: Proteomics and peptidomics technologies that show the highest possible specificity and selectivity for AD biomarkers are analyzed, together with the biological fluids used. In addition to positron emission tomography and magnetic resonance imaging, MALDI-TOF mass spectrometry is widely used to identify proteins and peptides involved in AD. The use of protein chips in SELDI technology and electroblotting chips for peptides makes feasible small amounts (µL) of samples for analysis. RESULTS: Suitable biomarkers are related to AD pathology, such as intracellular neurofibrillary tangles; extraneuronal senile plaques; neuronal and axonal degeneration; inflammation and oxidative stress. Recently, peptides were added to the candidate list, which are not amyloid-ß or tau fragments, but are related to coagulation, brain plasticity, and complement/neuroinflammation systems involving the neurovascular unit. CONCLUSION: The progress made in the application of mass spectrometry and recent chip techniques is promising for discriminating between AD, mild cognitive impairment, and matched healthy controls. The application of this technique to blood samples from patients with AD has shown to be less invasive and fast enough to determine the diagnosis, stage of the disease, prognosis, and follow-up of the therapeutic response.

Uncommon Noninvasive Biomarkers for the Evaluation and Monitoring of the Etiopathogenesis of Alzheimer's Disease.

BACKGROUND: Alzheimer´s disease (AD) is the most widespread dementia in the world, followed by vascular dementia. Since AD is a heterogeneous disease that shows several varied phenotypes, it is not easy to make an accurate diagnosis, so it arises when the symptoms are clear and the disease is already at an advanced stage. Therefore, it is important to find out biomarkers for early AD diagnosis that facilitate treatment or slow down the disease. Classic biomarkers are obtained from cerebrospinal fluid and plasma, along with brain imaging by positron emission tomography. Attempts have been made to discover uncommon biomarkers from other body fluids, which are addressed in this update. OBJECTIVE: This update aims to describe recent biomarkers from minimally invasive body fluids for the patients, such as saliva, urine, eye fluid or tears. METHODS: Biomarkers were determined in patients versus controls by single tandem mass spectrometry and immunoassays. Metabolites were identified by nuclear magnetic resonance and microRNAs with genome-wide high-throughput real-time polymerase chain reaction-based platforms. RESULTS: Biomarkers from urine, saliva, and eye fluid were described, including peptides/proteins, metabolites, and some microRNAs. The association with AD neuroinflammation and neurodegeneration was analyzed, highlighting the contribution of matrix metalloproteinases, the immune system and microglia, as well as the vascular system. CONCLUSION: Unusual biomarkers have been developed, which distinguish each stage and progression of the disease, and are suitable for the early AD diagnosis. An outstanding relationship of biomarkers with neuroinflammation and neurodegeneration was assessed, clearing up concerns about the etiopathogenesis of AD.

Transporter hypothesis in pharmacoresistant epilepsies. Is it at the central or peripheral level?

The multidrug-resistance (MDR) phenotype is typically observed in patients with refractory epilepsy (RE) whose seizures are not controlled despite receiving several combinations of more than two antiseizure medications (ASMs) directed against different ion channels or neurotransmitter receptors. Since the use of bromide in 1860, more than 20 ASMs have been developed; however, historically ~30% of cases of RE with MDR phenotype remains unchanged. Irrespective of metabolic biotransformation, the biodistribution of ASMs and their metabolites depends on the functional expression of some ATP-binding cassette transporters (ABC-t) in different organs, such as the blood-brain barrier (BBB), bowel, liver, and kidney, among others. ABC-t, such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP-1), and breast cancer-resistance protein (BCRP), are mainly expressed in excretory organs and play a critical role in the pharmacokinetics (PK) of all drugs. The transporter hypothesis can explain pharmacoresistance to a broad spectrum of ASMs, even when administered simultaneously. Since ABC-t expression can be induced by hypoxia, inflammation, or seizures, a high frequency of uncontrolled seizures increases the risk of RE. These stimuli can induce ABC-t expression in excretory organs and in previously non-expressing (electrically responsive) cells, such as neurons or cardiomyocytes. In this regard, an alternative mechanism to the classical pumping function of P-gp indicates that P-gp activity can also produce a significant reduction in resting membrane potential (ΔΨ0 = -60 to -10 mV). P-gp expression in neurons and cardiomyocytes can produce membrane depolarization and participate in epileptogenesis, heart failure, and sudden unexpected death in epilepsy. On this basis, ABC-t play a peripheral role in controlling the PK of ASMs and their access to the brain and act at a central level, favoring neuronal depolarization by mechanisms independent of ion channels or neurotransmitters that current ASMs cannot control.

Potential role of multidrug resistant proteins in refractory epilepsy and antiepileptic drugs interactions.

Epilepsy is a common neurological disorder. About one-third of epilepsy patients have a multidrug resistance (MDR) phenotype and develop refractory epilepsy (RE). Changes in the properties of the antiepileptic drugs (AEDs) targets resulting in reduced drug sensitivity, can't explain the MDR phenotype. This particular refractoriness is now attributed to overexpression of multidrug transporters in brain, leading to impaired access of AEDs to CNS targets, and it was documented in both human as well as in experimental models of RE. Single nucleotide polymorphism (SNP) identified in the MDR1-ABCB1 gene (C3435T/CC-genotype) is associated with increased intestinal expression of P-glycoprotein (P-gp) that affects levels of AEDs in plasma. The functional studies of P-gp using P-gp inhibitors could show the still unclear clinical impact of ABCB1 polymorphisms on AEDs resistance. Some drug-drug interactions previously believed to be cytochrome P450 (CYP) mediated are now also considered to be due to the modulation of multidrug-transporters. Because in certain cases pharmacoresistance can be overcome by add-on therapy, co-administered P-gp inhibitors could contribute to the effectiveness of AEDs treatment in RE. And in this regard, perhaps we can postulate to P-gp as a new clinical therapeutic target in multidrug-refractory epilepsy.

Experimental evidence of the potential use of erythropoietin by intranasal administration as a neuroprotective agent in cerebral hypoxia.

Stroke is a major human health problem without efficient available therapeutics. Ischemic brain injury can induce cell death as well as upregulation of endogenous adaptive mechanisms depending on the severity and duration of hypoxia, and the activity of transcription factors, such as hypoxia inducible factor 1-α (HIF-1α). HIF-1α induces gene expression as multidrug resistance (MDR-1) gene associated with drug-refractory phenotype, as well as erythropoietin (Epo) and erythropoietin receptor (Epo-R) associated with O(2) supply. The spontaneous stimulation of the Epo/Epo-R system is not enough for brain protection. Therefore, administration of exogenous recombinant human Epo (rHu-Epo) was suggested as an alternative therapy in stroke. In several experimental models of brain hypoxia, Epo and Epo variants, including rHu-Epo, showed neuroprotective effects. Intranasal administration of these Epo-compounds can reach the central nervous system and protect the brain against ischemia, avoiding hematopoietic effects. However, it has been reported that high expression of Epo-R in neurons must be available to be activated by Epo. According to these considerations, intranasal delivery of rHu-Epo could be an interesting approach in the treatment of cerebral hypoxias avoiding both (i) adverse peripheral effects of treatment with Epo in stroke, and (ii) the pharmacoresistant phenotype depending on MDR-1 expression.

Hypoxia, Oxidative Stress, and Inflammation: Three Faces of Neurodegenerative Diseases.

The cerebral hypoxia-ischemia can induce a wide spectrum of biologic responses that include depolarization, excitotoxicity, oxidative stress, inflammation, and apoptosis, and result in neurodegeneration. Several adaptive and survival endogenous mechanisms can also be activated giving an opportunity for the affected cells to remain alive, waiting for helper signals that avoid apoptosis. These signals appear to help cells, depending on intensity, chronicity, and proximity to the central hypoxic area of the affected tissue. These mechanisms are present not only in a large list of brain pathologies affecting commonly older individuals, but also in other pathologies such as refractory epilepsies, encephalopathies, or brain trauma, where neurodegenerative features such as cognitive and/or motor deficits sequelae can be developed. The hypoxia inducible factor 1α (HIF-1α) is a master transcription factor driving a wide spectrum cellular response. HIF-1α may induce erythropoietin (EPO) receptor overexpression, which provides the therapeutic opportunity to administer pharmacological doses of EPO to rescue and/or repair affected brain tissue. Intranasal administration of EPO combined with other antioxidant and anti-inflammatory compounds could become an effective therapeutic alternative, to avoid and/or slow down neurodegenerative deterioration without producing adverse peripheral effects.

Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment.

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti-epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1-CB2) or multiple non-canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P-glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration-dependent manner, while low CBD concentration decreased PMN viability in a time-dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti-inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.

Myocardial Iron Overload in an Experimental Model of Sudden Unexpected Death in Epilepsy.

Uncontrolled repetitive generalized tonic-clonic seizures (GTCS) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). GTCS can be observed in models such as Pentylenetetrazole kindling (PTZ-K) or pilocarpine-induced Status Epilepticus (SE-P), which share similar alterations in cardiac function, with a high risk of SUDEP. Terminal cardiac arrhythmia in SUDEP can develop as a result of a high rate of hypoxic stress-induced by convulsions with excessive sympathetic overstimulation that triggers a neurocardiogenic injury, recently defined as "Epileptic Heart" and characterized by heart rhythm disturbances, such as bradycardia and lengthening of the QT interval. Recently, an iron overload-dependent form of non-apoptotic cell death called ferroptosis was described at the brain level in both the PTZ-K and SE-P experimental models. However, seizure-related cardiac ferroptosis has not yet been reported. Iron overload cardiomyopathy (IOC) results from the accumulation of iron in the myocardium, with high production of reactive oxygen species (ROS), lipid peroxidation, and accumulation of hemosiderin as the final biomarker related to cardiomyocyte ferroptosis. Iron overload cardiomyopathy is the leading cause of death in patients with iron overload secondary to chronic blood transfusion therapy; it is also described in hereditary hemochromatosis. GTCS, through repeated hypoxic stress, can increase ROS production in the heart and cause cardiomyocyte ferroptosis. We hypothesized that iron accumulation in the "Epileptic Heart" could be associated with a terminal cardiac arrhythmia described in the IOC and the development of state-potentially in the development of SUDEP. Using the aforementioned PTZ-K and SE-P experimental models, after SUDEP-related repetitive GTCS, we observed an increase in the cardiac expression of hypoxic inducible factor 1α, indicating hypoxic-ischemic damage, and both necrotic cells and hemorrhagic areas were related to the possible hemosiderin production in the PTZ-K model. Furthermore, we demonstrated for the first time an accumulation of hemosiderin in the heart in the SE-P model. These results suggest that uncontrolled recurrent seizures, as described in refractory epilepsy, can give rise to high hypoxic stress in the heart, thus inducing hemosiderin accumulation as in IOC, and can act as an underlying hidden mechanism contributing to the development of a terminal cardiac arrhythmia in SUDEP. Because iron accumulation in tissues can be detected by non-invasive imaging methods, cardiac iron overload in refractory epilepsy patients could be treated with chelation therapy to reduce the risk of SUDEP.

From Genome to Drugs: New Approaches in Antimicrobial Discovery.

Decades of successful use of antibiotics is currently challenged by the emergence of increasingly resistant bacterial strains. Novel drugs are urgently required but, in a scenario where private investment in the development of new antimicrobials is declining, efforts to combat drug-resistant infections become a worldwide public health problem. Reasons behind unsuccessful new antimicrobial development projects range from inadequate selection of the molecular targets to a lack of innovation. In this context, increasingly available omics data for multiple pathogens has created new drug discovery and development opportunities to fight infectious diseases. Identification of an appropriate molecular target is currently accepted as a critical step of the drug discovery process. Here, we review how diverse layers of multi-omics data in conjunction with structural/functional analysis and systems biology can be used to prioritize the best candidate proteins. Once the target is selected, virtual screening can be used as a robust methodology to explore molecular scaffolds that could act as inhibitors, guiding the development of new drug lead compounds. This review focuses on how the advent of omics and the development and application of bioinformatics strategies conduct a "big-data era" that improves target selection and lead compound identification in a cost-effective and shortened timeline.

Intermittent hypoxia during sleep induces reactive gliosis and limited neuronal death in rats: implications for sleep apnea.

Sleep apnea (SA) can be effectively managed in humans but it is recognized that when left untreated, SA causes long-lasting changes in neuronal circuitry in the brain. Recent neuroimaging studies gave suggested that these neuronal changes are also present even in patients successfully treated for the acute effects of SA. The cellular mechanisms that account for these changes are not certain but animal models of intermittent hypoxia (IH) during sleep have shown neuronal death and impairment in learning and memory. Reactive gliosis has a drastic effect on neuronal survival and circuitry and in this study we examined the neuro-glial response in brain areas affected by SA. Glial and neuronal alterations were analyzed after 1, 3, 5 and 10 days of exposure to IH (8 h/day during the sleep phase, cycles of 6 min each, 10-21% O2) and observed significant astroglial hyperplasia and hypertrophy in parietal brain cortex and hippocampus by studying gliofibrillary acidic protein, Vimentin, S100B and proliferating cell nuclear antigen expression. In addition, altered morphology, reduced dendrite branching and caspase activation were observed in the CA-1 hippocampal and cortical (layers IV-V) pyramidal neurons at short exposure times (1-3 days). Surprisingly, longer exposure to IH reduced the neuronal death rate and increased neuronal branching in the presence of persistent reactive gliosis. Up-regulation of hypoxia inducible factor 1 alpha (HIF-1alpha) and mdr-1, a HIF-1alpha target gene, were observed and increased expression of receptor for advanced end glycated products and its binding partner S100B were also noted. Our results show that a low number of hypoxic cycles induce reactive gliosis and neuronal death whereas continuous exposure to IH cycles reduced the rate of neuronal death and induced neuronal branching on surviving neurons. We hypothesize that HIF-1alpha and S100B glial factor may improve neuronal survival under hypoxic conditions and propose that the death/survival/re-growth process observed here may underlie brain circuitry changes in humans with SA.