Prophylactic zinc and therapeutic selenium administration in adult rats prevents long-term cognitive and behavioral sequelae by a transient ischemic attack.

The transient hypoxic-ischemic attack, also known as a minor stroke, can result in long-term neurological issues such as memory loss, depression, and anxiety due to an increase in nitrosative stress. The individual or combined administration of chronic prophylactic zinc and therapeutic selenium is known to reduce nitrosative stress in the first seven days post-reperfusion and, due to an antioxidant effect, prevent cell death. Besides, zinc or selenium, individually administered, also causes antidepressant and anxiolytic effects. Therefore, this work evaluated whether combining zinc and selenium could prevent stroke-elicited cognition and behavior deficits after 30 days post-reperfusion. Accordingly, we assessed the expression of growth factors at 7 days post-reperfusion, a four-time course of memory (from 7 to 28 days post-learning test), and cell proliferation, depression, and anxiety-like behavior at 30 days post-reperfusion. Male Wistar rats with a weight between 190 and 240 g) were treated with chronic prophylactic zinc administration with a concentration of 0.2 mg/kg for 15 days before common carotid artery occlusion (10 min) and then with therapeutic selenium (6 µg/kg) for 7 days post-reperfusion. Compared with individual administrations, the administration combined of prophylactic zinc and therapeutic selenium decreased astrogliosis, increased growth factor expression, and improved cell proliferation and survival in two regions, the hippocampus, and cerebral cortex. These effects prevented memory loss, depression, and anxiety-like behaviors. In conclusion, these results demonstrate that the prophylactic zinc administration combined with therapeutic selenium can reduce the long-term sequelae caused by the transient ischemic attack. Significance statement. A minor stroke caused by a transient ischemic attack can result in psychomotor sequelae that affect not only the living conditions of patients and their families but also the economy. The incidence of these micro-events among young people has increased in the world. Nonetheless, there is no deep understanding of how this population group responds to regular treatments (Ekker and et al., 2018) [1]. On the basis that zinc and selenium have antioxidant, anti-inflammatory, and regenerative properties in stroke animal models, our work explored whether the chronic combined administration of prophylactic zinc and therapeutic selenium could prevent neurological sequelae in the long term in a stroke rat model of unilateral common carotid artery occlusion (CCAO) by 10-min. Our results showed that this combined treatment provided a long-term neuroprotective effect by decreasing astrogliosis, memory loss, anxiety, and depression-like behavior.

Prophylactic Zinc Administration Combined with Swimming Exercise Prevents Cognitive-Emotional Disturbances and Tissue Injury following a Transient Hypoxic-Ischemic Insult in the Rat.

Exercise performance and zinc administration individually yield a protective effect on various neurodegenerative models, including ischemic brain injury. Therefore, this work was aimed at evaluating the combined effect of subacute prophylactic zinc administration and swimming exercise in a transient cerebral ischemia model. The prophylactic zinc administration (2.5 mg/kg of body weight) was provided every 24 h for four days before a 30 min common carotid artery occlusion (CCAO), and 24 h after reperfusion, the rats were subjected to swimming exercise in the Morris Water Maze (MWM). Learning was evaluated daily for five days, and memory on day 12 postreperfusion; anxiety or depression-like behavior was measured by the elevated plus maze and the motor activity by open-field test. Nitrites, lipid peroxidation, and the activity of superoxide dismutase (SOD) and catalase (CAT) were assessed in the temporoparietal cortex and hippocampus. The three nitric oxide (NO) synthase isoforms, chemokines, and their receptor levels were measured by ELISA. Nissl staining evaluated hippocampus cytoarchitecture and Iba-1 immunohistochemistry activated the microglia. Swimming exercise alone could not prevent ischemic damage but, combined with prophylactic zinc administration, reversed the cognitive deficit, decreased NOS and chemokine levels, prevented tissue damage, and increased Iba-1 (+) cell number. These results suggest that the subacute prophylactic zinc administration combined with swimming exercise, but not the individual treatment, prevents the ischemic damage on day 12 postreperfusion in the transient ischemia model.

OX40 agonists for cancer treatment: a patent review.

INTRODUCTION: OX40 is an immune checkpoint in cancer and its presence in cancer is a good prognosis, making it a highly relevant target for the development of new immunotherapies. AREAS COVERED: The patent literature reveals vital information on new trends in cancer therapies. The authors used the patent databases of the six major patent offices in the world: United States Patent and Trademark Office, European Patent Office, World Intellectual Property Organization, Japan Patent Office, State Office of Intellectual Property of China and Korean Intellectual Property Office, to generate a panorama of patents related to OX40 agonists. Specific patents have been grouped into innovative patents and adoption patents. EXPERT OPINION: An increasing trend in the development of OX40 agonists in cancer, particularly in the years 2018 and 2019. United States was the leader in generating patents, followed by China and England. Major pharmaceutical companies have at least one anti-OX40 agonist, MEDI6469 and MEDI-0562 (AstraZeneca), PF-04518600 (Pfizer), GSK3174998 (GlaxoSmithKline), BMS-986,178 (Bristol-Myers Squibb) and MOXR0916 (Roche), which represent 68% of clinical trials conducted with OX40 agonists.

Bispecific anti-PD-1/LAG-3 antibodies for treatment of advanced or metastatic solid tumors: a patent evaluation of US2018326054.

INTRODUCTION: Due to the primary role of PD-1 and LAG-3 in regulating the immune response in tumors, there is a need to develop therapies focused on the inhibition of PD-1 and LAG-3 in order to improve the immune response in patients with cancer. The authors of US2018326054 patent propose a method to eradicate cancer by using bispecific anti-PD-1/LAG-3 antibodies. AREAS COVERED: The US2018326054 patent describes anti-PD-1/LAG3 antibodies, pharmaceutical composition that contains it, and their application for cancer treatment, particularly pancreatic carcinoma. Proof concept and preclinical results show anti-PD-1/LAG-3 bispecific antibodies bind and are internalized by CD4 + T cells thereby increasing their effector functions (release of Granzyme B and INF-γ) in the presence of tumor cells, and completely suppress tumors in a murine model. EXPERT OPINION: Anti-PD-1/LAG-3 bispecific antibodies of the US2018326054 patent are new in a general concept, but treatment data is only shown for pancreatic carcinoma. The results to be obtained in future clinical trials of safety and efficacy could conclude whether these bispecific anti-PD-1/LAG-3 antibodies will be useful in a cancer treatment scheme.

LAG-3 antagonists by cancer treatment: a patent review.

Introduction: LAG-3 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of LAG-3 and consequently improving the immune response in the various types of cancer. Areas covered: The patent literature reveals novel therapies, which provide information on cancer therapies. The authors used the patent databases of the six main patent offices of the world: United States Patent and Trademark Office, European Patent Office, World Intellectual Property Organization, Japan Patent Office, State Intellectual Property Office of China and Korean Intellectual Property Office, to generate a detailed landscape of patents and patent applications of active companies related to LAG-3 inhibitors. Specific patents have been grouped into innovative patents and adopting patents. Expert opinion: There is a continuing development of LAG-3 inhibitors, and these inhibitors are being used in combination with other cancer treatment schemes, for example, antibodies against PD-1, PD-L1, and CTLA-4. Immutep and IO Therapeutics were the leaders in generating innovator patents, followed by Gustave Roussy Institute, and Applied Research Systems ARS. Dana-Farber Cancer Institute was the leader in the generation of adopter patents, followed by Novartis .

Prophylactic Zinc and Therapeutic Selenium Administration Increases the Antioxidant Enzyme Activity in the Rat Temporoparietal Cortex and Improves Memory after a Transient Hypoxia-Ischemia.

In the cerebral hypoxia-ischemia rat model, the prophylactic administration of zinc can cause either cytotoxicity or preconditioning effect, whereas the therapeutic administration of selenium decreases the ischemic damage. Herein, we aimed to explore whether supplementation of low doses of prophylactic zinc and therapeutic selenium could protect from a transient hypoxic-ischemic event. We administrated zinc (0.2 mg/kg of body weight; ip) daily for 14 days before a 10 min common carotid artery occlusion (CCAO). After CCAO, we administrated sodium selenite (6 µg/kg of body weight; ip) daily for 7 days. In the temporoparietal cerebral cortex, we determined nitrites by the Griess method and lipid peroxidation by the Gerard-Monnier assay. qPCR was used to measure mRNA of nitric oxide synthases, antioxidant enzymes, chemokines, and their receptors. We measured the enzymatic activity of SOD and GPx and protein levels of chemokines and their receptors by ELISA. We evaluated long-term memory using the Morris-Water maze test. Our results showed that prophylactic administration of zinc caused a preconditioning effect, decreasing nitrosative/oxidative stress and increasing GPx and SOD expression and activity, as well as eNOS expression. The therapeutic administration of selenium maintained this preconditioning effect up to the late phase of hypoxia-ischemia. Ccl2, Ccr2, Cxcl12, and Cxcr4 were upregulated, and long-term memory was improved. Pyknotic cells were decreased suggesting prevention of neuronal cell death. Our results show that the prophylactic zinc and therapeutic selenium administration induces effective neuroprotection in the early and late phases after CCAO.

Prophylactic Chronic Zinc Administration Increases Neuroinflammation in a Hypoxia-Ischemia Model.

Acute and subacute administration of zinc exert neuroprotective effects in hypoxia-ischemia animal models; yet the effect of chronic administration of zinc still remains unknown. We addressed this issue by injecting zinc at a tolerable dose (0.5 mg/kg weight, i.p.) for 14 days before common carotid artery occlusion (CCAO) in a rat. After CCAO, the level of zinc was measured by atomic absorption spectrophotometry, nitrites were determined by Griess method, lipoperoxidation was measured by Gerard-Monnier assay, and mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors was measured by qRT-PCR, whereas nitrotyrosine, chemokines, and their receptors were assessed by ELISA and histopathological changes in the temporoparietal cortex-hippocampus at different time points. Long-term memory was evaluated using Morris water maze. Following CCAO, a significant increase in nitrosative stress, inflammatory chemokines/receptors, and cell death was observed after 8 h, and a 2.5-fold increase in zinc levels was detected after 7 days. Although CXCL12 and FGF2 protein levels were significantly increased, the long-term memory was impaired 12 days after reperfusion in the Zn+CCAO group. Our data suggest that the chronic administration of zinc at tolerable doses causes nitrosative stress, toxic zinc accumulation, and neuroinflammation, which might account for the neuronal death and cerebral dysfunction after CCAO.

Firing properties of auditory primary afferents from the basilar papilla in the chick.

We performed intracellular and single-unit extracellular recordings of neurons from different regions of the basilar papilla in the isolated chicken inner ear. We compared the spontaneous activity and the response properties of these neurons in embryos at E15 versus posthatching animals at P1. The recordings were carried out from the apical (position 0) to the basal extension at three positions of the basilar papilla, at 5%, 10% and 40% of the entire length of the cochlea. We found that the neurons at E15 recorded from these three regions exhibited a significant higher coefficient of variation compared with those neurons at P1 recorded in the same positions. This shows that in the posthatching age P1 the neurons from the whole basilar papilla become less irregular. We found that the intracellular action potential waveforms generated at E15 had small amplitudes and small depolarization slopes in comparison to those recorded at P1, respectively (53 ± 1 mV vs. 62 ± 2 mV; 66 ± 12 mV/msec vs. 166 ± 23 mV/msec). Furthermore, we also found that the response patterns to injection of current steps were phasic, tonic, or in the form of a not yet reported "burst" pattern. Our study shows that the low irregular discharge, the immature action potential waveforms, and the differences in the response patterns to current injection, highlights the important differences between neurons at E15 and P1, consistent with the incapacity of auditory neurons at embryonic age E16, to respond at sound levels <100 decibels.

Subacute zinc administration and L-NAME caused an increase of NO, zinc, lipoperoxidation, and caspase-3 during a cerebral hypoxia-ischemia process in the rat.

Zinc or L-NAME administration has been shown to be protector agents, decreasing oxidative stress and cell death. However, the treatment with zinc and L-NAME by intraperitoneal injection has not been studied. The aim of our work was to study the effect of zinc and L-NAME administration on nitrosative stress and cell death. Male Wistar rats were treated with ZnCl2 (2.5 mg/kg each 24 h, for 4 days) and N-ω-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg) on the day 5 (1 hour before a common carotid-artery occlusion (CCAO)). The temporoparietal cortex and hippocampus were dissected, and zinc, nitrites, and lipoperoxidation were assayed at different times. Cell death was assayed by histopathology using hematoxylin-eosin staining and caspase-3 active by immunostaining. The subacute administration of zinc before CCAO decreases the levels of zinc, nitrites, lipoperoxidation, and cell death in the late phase of the ischemia. L-NAME administration in the rats treated with zinc showed an increase of zinc levels in the early phase and increase of zinc, nitrites, and lipoperoxidation levels, cell death by necrosis, and the apoptosis in the late phase. These results suggest that the use of these two therapeutic strategies increased the injury caused by the CCAO, unlike the alone administration of zinc.

Functional expression of P2 receptors in the inner ear of chicken embryo.

The purpose of the present study was to investigate the modulation of spontaneous afferent activity by ATP during embryonic development in a preparation isolated chicken inner ear. This work was performed using multiunit and single-unit extracellular recordings from the posterior semicircular canal nerve and the basilar papilla nerve. α,ß-meATP, a P2X receptor agonist, notably increased the discharge frequency of the vestibular afferents between E15 and E18, but not in the basilar papilla. In contrast, the P2Y receptor agonist UTP produced a slight increase in the discharge frequency of basilar papilla afferents, without apparent changes in the vestibular afferent activity. 2-MeSATP, a P2Y agonist, increased the basal discharge of the primary afferents in a dose-age dependent way, but when we applied the antagonist of P2Y receptor, Reactive Blue 2 (10(-4)M), the effect of 2-MeSATP decreased significantly. This was observed both in vestibule and basilar papilla. Using RT-PCR the presence of P2X3, P2Y1, P2Y2 and P2Y6 mRNA was documented in the vestibular system with more important presence during the early stage (E15) than the later stage (E21), however in the basilar papilla we found only the P2Y1, P2Y2 and P2Y6 mRNA with the same temporal course as in the vestibule. These results confirm our pharmacological findings. Together this data suggests a role for P2X receptors-mediated purinergic signaling in vestibular synaptic organization. Temporal changes in P2Y receptors during development might be involved in the establishment of the endolymphatic ion composition needed for normal vestibular and auditory transduction and/or specific cellular differentiation.

miR-153 silencing induces apoptosis in the MDA-MB-231 breast cancer cell line.

MicroRNAs (miRNAs) are small, non-coding RNAs (18-25 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In this context, the present study aimed to evaluate the in vitro effects of miR-153 inhibition in the breast carcinoma cell line MDA-MB-231. Forty-eight hours after MDA-MB-231 cells were transfected with the miR-153 inhibitor, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to determine the effects of miR-153 on cell viability. Flow cytometry analysis and assessment of caspase 3/7 activity were adopted to determine whether miR-153 affects the proliferation rates and apoptosis levels of MDA-MB-231 cells. Our results showed that silencing of miR-153 significantly inhibited growth when compared to controls at 48 hours, reducing proliferation by 37.6%, and inducing apoptosis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future diagnostic and therapeutic interventions.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

Leptin increases L-type Ca2+ channel expression and GnRH-stimulated LH release in LbetaT2 gonadotropes.

Leptin, a mediator of long-term regulation of energy balance, has been implicated in the release of adenohypophyseal gonadotropins by regulating gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. However, a direct effect of leptin on hormone release from gonadotropes remains virtually unexplored. In the current report, we assessed the long-term (48 h) actions of leptin on voltage-gated channel activity and luteinizing hormone (LH) production in mouse pituitary gonadotrope LbetaT2 cells. Electrophysiological recordings showed that leptin treatment significantly increased whole-cell patch-clamp Ba(2+) current through L-type Ca(2+) channels. Quantitative RT-PCR analysis revealed increased levels of L-type (alpha(1D)) Ca(2+) channel mRNA. Likewise, radioimmunoassays using specific antibodies provided evidence that leptin alone had no effect on LH release but did enhance GnRH-induced secretion of the hormone. Leptin had no apparent effects on LH gene transcription in absence of GnRH, as measured by transient transfection assays using a LH promoter-reporter gene and real-time RT-PCR. These observations suggest that leptin might affect LH release by acting directly on the gonadotropes, favoring hormone production by enhancing responsiveness to GnRH as a result of increased Ca(2+) channel expression.

An improved method for long-term measuring of hemolymph fluctuations of non-essential amino acids, GABA and histamine from freely moving crayfish.

The microdialysis method was adapted to obtain long-term hemolymph dialysates from the pericardial cavity of freely moving Procambarus clarkii crayfish, to measure fluctuations of non-essential amino acids, GABA and histamine by high-performance liquid chromatography using off-line fluorometric derivatization. Asp, Ala, Tau, GABA and histamine (HA) reached its maximal concentrations at the daybreak, whereas glutamate (Glu), Gln and Gly peaked at the end of the light period. The minimum and maximal detected amounts for each substance along the 24h cycle were (in microM): 20-300Asp, 100-200Glu, 400-700Gln, 400-600Gly, 100-200Tau, 150-300Ala, 2-10 GABA and 25-250HA. Cocktails containing the relative concentration of each amino acid, GABA and histamine resulted in a hyperpolarization that reduced the spontaneous firing of cultured peptidergic X organ neurons. Glu, GABA and histamine evoked a long-lasting hyperpolarization that suppressed the spontaneous firing, whereas Asp, Gly and Tau evoked a depolarization accompanied with neuronal firing. Finally, neither Ala nor Gln modified the resting membrane potential.

Expression and functional characterization of GABA transporters in crayfish neurosecretory cells.

The effect of GABA on membrane potential and ionic currents of X-organ neurons isolated from the crayfish eyestalk was investigated. Under voltage-clamp conditions, GABA elicited an inward Na+ current followed by a sustained outward chloride current. Sodium current was partially blocked in a dose-dependent manner by antagonists of GABA plasma membrane transporters such as beta-alanine, nipecotic acid, 1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711), and SKF89976-A at concentrations between 1 and 100 microm. This current was totally blocked by the combined application of NO 711 (5 microm) and beta-alanine (50 microm). We obtained an EC(50) of 5 microm and a Hill coefficient of 0.97 for the GABA transport mediated response. These results together with studies of immunolocalization using antibodies against neuronal vertebrate GABA transporters (GATs) indicate the presence of GAT-1- and GAT-3-like proteins in X-organ neurons. To isolate the sustained outward Cl- current, extracellular free sodium solution was used to minimize the contribution of GAT activity. We concluded that this current was caused by the activation of GABA(A)-like receptors with an EC50 of 10 microm and a Hill number of 1.7. To assign a functional role to the GATs in the X-organ sinus gland system, we determine the GABA concentration (0.46-0.15 microm) in hemolymph samples using HPLC. In summary, our results suggest that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the excitability of the X-organ neurons, maintaining an excitatory tone that is dependent on the circulating GABA level.