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This paper deals with stability of solution for a one-dimensional model of Rao–Nakra sandwich beam with Kelvin–Voigt damping and time delay given by 𝜌1ℎ1𝑢𝑡𝑡 − 𝐸1ℎ1𝑢𝑥𝑥 − 𝜅(−𝑢 + 𝑣 + 𝛼𝑤𝑥) − 𝑎𝑢𝑥𝑥𝑡 − 𝜇𝑢𝑥𝑥𝑡( ・ , 𝑡 − 𝜏) = 0, 𝜌3ℎ3𝑣𝑡𝑡 − 𝐸3ℎ3𝑣𝑥𝑥 + 𝜅(−𝑢 + 𝑣 + 𝛼𝑤𝑥) − 𝑏𝑣𝑥𝑥𝑡 = 0, 𝜌ℎ𝑤𝑡𝑡 + 𝐸𝐼𝑤𝑥𝑥𝑥𝑥 − 𝜅𝛼(−𝑢 + 𝑣 + 𝛼𝑤𝑥)𝑥 − 𝑐𝑤𝑥𝑥𝑡 = 0. A sandwich beam is an engineering model that consists of three layers: two stiff outer layers, bottom and top faces, and a more compliant inner layer called “core layer”. Rao–Nakra system consists of three layers and the assumption is that there is no slip at the interface between contacts. The top and bottom layers are wave equations for the longitudinal displacements under Euler–Bernoulli beam assumptions. The core layer is one equation that describes the transverse displacement under Timoshenko beam assumptions. By using the semigroup theory, the well-posedness is given by applying the Lumer–Phillips Theorem. Exponential stability is proved by employing the Gearhart-Huang-Prüss’ Theorem.

In this manuscript, by using the semigroup theory, the well-posedness and exponential stability for a Rao-Nakra sandwich beam equation with internal damping and time delay is proved. The system consists of two wave equations for the longitudinal displacements of the top and bottom layers, and one Euler-Bernoulli beam equation for the transversal displacement. To the best of our knowledge from the literature , by this time, no attention was given to the asymptotic stability for Rao-Nakra model with time delay.

A Kirchhoff equation type with memory term competing with a logarithmic source is considered. By using potential well theory, we obtained the global existence of solution for the initial data in a stability set created from Nehari Manifold and prove blow up results for initial data in the instability set.

In this manuscript we prove the property of growth determined by spectrum of the linear operator associated with the Timoshenko system with two histories.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1β, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

After unilateral lesion of the medial forebrain bundle (MFB) by 6-OHDA rats exhibit lateralized deficits in spontaneous behavior or apomorphine-induced rotations. We investigated whether such lateralization is attenuated by either deep brain stimulation (DBS) or glutamatergic neurotransmission in the inferior colliculus (IC) of Wistar rats. Intracollicular DBS did not affect spontaneous lateralization but attenuated apomorphine-induced rotations. Spontaneous lateralization disappeared after both glutamatergic antagonist MK-801 or the agonist NMDA microinjected in the IC. Apomorphine-induced rotations were potentiated by MK-801 but were not affected by NMDA intracollicular microinjection. After injecting a bidirectional neural tract tracer into the IC, cell bodies and/or axonal fibers were found in the periaqueductal gray, superior colliculus, substantia nigra, cuneiform nucleus and pedunculo-pontine tegmental nucleus, suggesting the involvement of these structures in the motor improvement after IC manipulation. Importantly, the side of the IC microinjection regarding the lesion (ipsi- or contralateral) is particularly important and this effect may not involve the neostriatum directly. Significance Statement The inferior colliculus, usually viewed as an auditory structure, when properly manipulated may counteract motor deficits in Parkinsonian rats. Indeed, the present study showed that 30 Hz deep brain stimulation or glutamatergic neural network in the inferior colliculus reduced body asymmetry induced by medial forebrain bundle unilateral 6-OHDA lesion in rats, an animal model of Parkinsonism. Understanding how glutamatergic mechanisms in the inferior colliculus influence motor control, classically attributed to the basal nuclei circuitry, could be useful in the development of new therapeutics to treat Parkinson's disease and other motor disorders., Competing Interests: Authors report no conflict of interests., (Copyright © 2022 Melo-Thomas et al.)

Glioblastomas (GBs) are responsible for a higher mortality rate among gliomas, corresponding to more than 50% of them and representing a challenge in terms of therapy and prognosis. Peptide-based antineoplastic therapy is a vast and promising field, and these molecules are one of the main classes present in spider venoms. Recently, our research group demonstrated the cytotoxic effects of Phoneutria nigriventer spider venom (PnV) in GBs. The present study aimed to select the purified PnV-components with potential antineoplastic effects, as well as to compare different metabolic conditions. Human GB (NG97) cells were treated with the PnV fractions: F1 (less than 3 kDa), F2 (between 3 and 10 kDa), and F3 (greater than 10 kDa). After treatments, viability (MTT), proliferation (CFSE), death (Annexin V/propidium iodide-PI), and cell cycle (PI) assays were performed. The F1 and F2 fractions in acute periods (1 and 5 h) and low concentrations (0.1 and 1 μg/ml) showed more relevant effects and were repurified in subfractions (SF1-SF11); from these, SF3 and SF4 showed the most significant effects. The previous inhibition of mTOR by rapamycin had a synergistic effect with SFs, reducing cell viability even more significantly than the untreated control. Taken together, the results point to components present in SF3 and SF4 as potential prototypes for the development of new drugs for GB treatment and stimulate studies to use these compounds in combination therapy with a rapamycin-like activity. Future studies will be conducted to characterize, synthesize the molecules, and to evaluate the efficacy and safety in preclinical models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caballero, Barreto, Bonfanti, Munhoz, Rocha e Silva, Sutti, Verinaud, Pinheiro de Mato, Lanfredi and Rapôso.)

Peptides are molecules that have emerged as crucial candidates for the development of anticancer drugs. Spider venoms are a rich source of peptides (venom peptides - VPs) with biological effects. VPs have been tested as adjuvants in the activation of cells of the immune system with the aim of improving immunotherapies for the treatment of neoplasms. In the present study, the effects of SNX-482, a peptide from the African tarantula Hysterocrates gigas, on macrophages were described. The results showed that the peptide activated M0-macrophages, increasing costimulatory molecules (CD40, CD68, CD80, CD83, CD86) involved in antigen presentation, and also augmenting the checkpoint molecules PD-L1, CTLA-4 and FAS-L; these effects were not concentration-dependent. SNX-482 also increased the release of IL-23 and upregulated the expression of ccr4, ifn-g, gzmb and pdcd1, genes important for the anticancer response. The pretreatment of macrophages with the peptide did not interfere in the modulation of T cells, and macrophages previously polarized to M1 and M2 profile did not respond to SNX-482. These findings represent the expansion of knowledge about the use of VPs in drug discovery, pointing to a potential new candidate for anticancer immunotherapy. Considering that most immunotherapies target the adaptive system, the modulation of macrophages (an innate immune cell) by SNX-482 is especially relevant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

New approaches to cancer immunotherapy have been developed, showing the ability to harness the immune system to treat and eliminate cancer. For many solid tumors, therapy with checkpoint inhibitors has shown promise. For hematologic malignancies, adoptive and engineered cell therapies are being widely developed, using cells such as T lymphocytes, as well as natural killer (NK) cells, dendritic cells, and potentially others. Among these adoptive cell therapies, the most active and advanced therapy involves chimeric antigen receptor (CAR)-T cells, which are T cells in which a chimeric antigen receptor is used to redirect specificity and allow T cell recognition, activation and killing of cancers, such as leukemia and lymphoma. Two autologous CAR-T products have been approved by several health authorities, starting with the U.S. Food and Drug Administration (FDA) in 2017. These products have shown powerful, inducing, long-lasting effects against B cell cancers in many cases. In distinction to the results seen in hematologic malignancies, the field of using CAR-T products against solid tumors is in its infancy. Targeting solid tumors and trafficking CAR-T cells into an immunosuppressive microenvironment are both significant challenges. The goal of this review is to summarize some of the most recent aspects of CAR-T cell design and manufacturing that have led to successes in hematological malignancies, allowing the reader to appreciate the barriers that must be overcome to extend CAR-T therapies to solid tumors successfully., Competing Interests: Stephan A. Grupp receives study support from 10.13039/100004336Novartis, Kite, 10.13039/501100011725Servier, and Vertex. He consults for Novartis, Roche, GSK, Humanigen, CBMG, and Janssen/JnJ. He is on study steering committees or scientific advisory boards for Novartis, Allogene, Jazz, Adaptimmune, TCR2, Cellectis, Juno, and Vertex/CRISPR. He has a patent (Toxicity management for anti-tumor activity of CARs, WO 2014011984 A1) that is managed according to the University of Pennsylvania patent policy. The other authors declare no conflicts of interests in this work., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Background: Multiple sclerosis (MS) is a disease of the central nervous system where an autoimmune response leads to chronic inflammation. It represents the second leading cause of non-traumatic disability in the world, affecting mainly young adults and with high female to male incidence. At present, the causative agent in MS is unknown, preventing the development of prophylaxis policies and the understanding of how the human system copes with this complex inflammation. Tetracyclines (Tet) have attracted great attention due to their anti-inflammatory effects. Minocycline and doxycycline represent the second-generation Tet that have been largely used to treat acne and to suppress inflammation. In addition, they are safer and cheaper than other drugs currently used to treat MS., Aim: This study aims to review recent data involving the Tet minocycline and doxycycline and their therapeutic potential in MS., Relevance for Patients: Many of the drugs used to treat MS have severe side effects and are costly. Tet, on the other hand, are a safe and inexpensive class of drugs that can modulate the immune response in MS patients., Competing Interests: The authors have no conflict of interest regarding the publication of this paper. Furthermore, the authors thank the financial support from the Brazilian National Council for Scientific and Technological Development (CNPq # 131292/2019-6) and The São Paulo Research Foundation (FAPESP #2015/04194-0)., (Copyright: © Whioce Publishing Pte. Ltd.)

Background: Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na + /K + -ATPase β2 (AMOG) involvement., Methods: Human (NG97) GB cells were treated with twelve subfractions (SFs-obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test., Results: Two (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na + /K + -ATPase β2., Conclusion: All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

Studies that show an overview of the peripheral immune response in a model of Paracoccidioides brasiliensis (Pb) infection in females are scarce in the literature. We sought to characterize the innate and adaptive immune responses in female C57BL/6 mice infected with Pb through two distinct routes of administration, intranasal and intravenous. In addition to the lung, P. brasiliensis yeast cells were observed in liver and brain tissues of females infected intravenously. To our knowledge, our study is the first to prove the presence of this pathogenic fungus in the cerebral cortex of female mice. During the initial stages of infection, augmented expression of both MHCII and CD86 was observed on the surface of CD11c + pulmonary antigen-presenting cells (APCs) in intranasally and intravenously infected females. However, CD40 expression was downregulated in these cells. Concomitantly with increasing serum IL-10 levels, we noted that splenic dendritic cells (DCs) from both intravenously- and intranasally-infected female mice had acquired an immature phenotype. Further, increased T regulatory cell counts were observed in female mice infected via both routes, along with an increase in the infiltration of IL-10-producing CD8 + T cells into the lungs. Moreover, we noted that P. brasiliensis infection resulted in enhanced IL-10 production - by CD11c + APCs in the lung tissue - and induction of Th17 polarization. Taken together, our results suggest that P. brasiliensis could modulates the immune response in female mice by influencing the balance between regulatory T cells (Tregs) and Th17 polarization., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG -/- mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18 F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18 F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

Hippo Tumor Suppressor Pathway is the main pathway for cell growth that regulates tissue enlargement and organ size by limiting cell growth. This pathway is activated in response to cell cycle arrest signals (cell polarity, transduction, and DNA damage) and limited by growth factors or mitogens associated with EGF and LPA. The major pathway consists of the central kinase of Ste20 MAPK ( Saccharomyces cerevisiae ), Hpo ( Drosophila melanogaster ) or MST kinases (mammalian) that activates the mammalian AGC kinase dm Wts or LATS effector (MST and LATS). YAP in the nucleus work as a cofactor for a wide range of transcription factors involved in proliferation (TEA domain family, TEAD1-4), stem cells (Oct4 mononuclear factor and SMAD-related TGFβ effector), differentiation (RUNX1), and Cell cycle/apoptosis control (p53, p63, and p73 family members). This is due to the diverse roles of YAP and may limit tumor progression and establishment. TEAD also coordinates various signal transduction pathways such as Hippo, WNT, TGFβ and EGFR, and effects on lack of regulation of TEAD cancerous genes, such as KRAS, BRAF, LKB1, NF2 and MYC, which play essential roles in tumor progression, metastasis, cancer metabolism, immunity, and drug resistance. However, RAS signaling is a pivotal factor in the inactivation of Hippo, which controls EGFR-RAS-RAF-MEK-ERK-mediated interaction of Hippo signaling. Thus, the loss of the Hippo pathway may have significant consequences on the targets of RAS-RAF mutations in cancer., Competing Interests: The authors declare no conflict of interests., (© 2019 Chongqing Medical University. Production and hosting by Elsevier B.V.)

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules., (© 2018 Wiley Periodicals, Inc.)

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background and Aim: Animal venoms comprise a mix of bioactive molecules with high affinity for multiple targets in cells and tissues. Scorpion and spider venoms and purified peptides exhibit significant effects on cancer cells, encompassing four potential mechanisms: 1) induction of cell cycle arrest, growth inhibition, and apoptosis; 2) inhibition of angiogenesis; 3) inhibition of invasion and metastasis; and 4) blocking of specific transmembrane channels. Tumor biology is complex and entails many intertwined processes, as reflected in the putative hallmarks of cancer. This complexity, however, gives rise to numerous (potential) pharmacological intervention sites. Molecules that target multiple proteins or pathways, such as components of animal venoms, may therefore be effective anti-cancer agents. The objective of this review was to address the anti-cancer properties and in vitro mechanisms of scorpion and spider venoms and toxins, and highlight current obstacles in translating the preclinical research to a clinical setting. Relevance for patients: Cancer is a considerable global contributor to disease-related death. Despite some advances being made, therapy remains palliative rather than curative for the majority of cancer indications. Consequently, more effective therapies need to be devised for poorly responding cancer types to optimize clinical cancer management. Scorpion and spider venoms may occupy a role in the development of improved anti-cancer modalities.

Astrocytes are effectively involved in the pathophysiological processes in the central nervous system (CNS) and may contribute to or protect against development of inflammatory and degenerative diseases. Sildenafil is a potent and selective phosphodiesterase-5 (PDE-5) inhibitor, which induces cyclic GMP accumulation. However, the mechanisms of actions on glial cells are not clear. The aim of the present work is to evaluate the role of sildenafil in lipopolysaccharide (LPS)-stimulated astrocytes. The cytoskeleton integrity and Ca(2+) waves were assessed as indicators of inflammatory state. Two main groups were done: (A) one prevention and (B) one restoration. Each of these groups: A1: control; A2: LPS for 24h; A3: sildenafil 1 or 10μM for 4h and then sildenafil 1 or 10μM+LPS for 24h. B1: control; B2: LPS for 24h; B3: LPS for 24h and then LPS+sildenafil 1 or 10μM for 24h. Cytoskeleton integrity was analyzed through GFAP immunolabeling and actin labeling with an Alexa 488-conjugated phalloidin probe. Calcium responses were assessed through a Ca(2+)-sensitive fluorophore Fura-2/AM. The results show that both preventive and restorative treatments with sildenafil (in both concentrations) reduced the Ca(2+) responses in intensity and induced a more organized actin fiber pattern, compared to LPS treated cells. This work demonstrated for the first time that astrocytes are a key part of the sildenafil protective effects in the CNS., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Bites from genus Phoneutria (Ctenidae, Araneomorpha) are the second most frequent source of spider accidents in Southeast Brazil. Severe envenoming from Phoneutria nigriventer produces vision disturbance, tremor and convulsion, suggesting that the CNS is involved; however, the mechanisms by which P. nigriventer venom (PNV) affects the CNS remain poorly understood. The present study aimed to investigate whether PNV directly impairs astrocytes. Cultured astrocytes were exposed to PNV, and intracellular Ca(2+) release and signaling were measured (Fura-2/AM), Na(+)/K(+)-ATPase and Toll-like receptor 4 (TLR4) involvement were investigated, actin filaments were stained (Alexa™ 488-conjugated phalloidin probe), the G-actin/F-actin ratio was determined, and the expression level of connexin 43 (Cx43) was assessed. Incubation in Ca(2+)-free buffer did not change the Ca(2+) responses. However, pre-incubation in thapsigargin/caffeine completely abolished these responses, suggesting that PNV-evoked Ca(2+) transients were from intracellular Ca(2+) stores. Pretreatment with a Na(+)/K(+)-ATPase antagonist (ouabain) or a TLR4 antagonist (LPS-RS) decreased or increased the Ca(2+)-evoked transients, respectively. Astrocytes showed altered actin filament structure after PNV exposure. PNV treatment increased the expression levels of Na(+)/K(+)-ATPase and Cx43 but decreased those of TLR4. The present results suggest that PNV directly affects astrocytes. Na(+)/K(+)-ATPase may thus represent a more specific drug target for controlling the neurotoxicity of PNV., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

While it has recently been shown that sildenafil (Viagra®) has a protective effect on myelination/remyelination, the mechanism of this protection is still unknown. In general, cytokines, chemokines and metalloproteinases have a pro-inflammatory action, but can also exert a role in modulating glial cell activation, contributing to the balance of cell response. Investigating these molecules can contribute to clarifying the mechanisms of sildenafil neuroprotection. In addition, it is not known whether sildenafil is able to restore an already installed neurodegenerative process or if the treatment period is critical for its action. The aim of the present study was to evaluate, in a cuprizone (CPZ)-induced demyelination model, the effects and mechanisms of time-dependent treatment with sildenafil (beginning 15 days after neurodegeneration and continuing for 15 days, or starting concomitantly with neurodegeneration and continuing for 30 days) on neuroinflammation and remyelination. Neuroinflammation and demyelination induced by CPZ in rodents has been widely used as a model of multiple sclerosis (MS). In the present study, five male C57BL/6 mice aged 7-10 weeks were used per group. For four weeks, the groups received either cuprizone (CPZ) 0.2% mixed in feed or CPZ combined with the administration of sildenafil (Viagra®, Pfizer, 25 mg/kg) orally in drinking water, starting concurrently with (sild-T0) or 15 days (sild-T15) after the start of CPZ treatment. Control animals received pure food and water. The cerebella were dissected and processed for immunohistochemistry, immunofluorescence (frozen), Western blotting, Luxol fast blue staining and transmission electron microscopy. Magnetic resonance was performed for live animals, after the same treatment, using CPZ 0.3%. CPZ induced an increase in the expression of IL-1β and a decrease in MCP-1, CCR-2, MBP and GST-pi, as well as promoting damage in the structure and ultra-structure of the myelin sheath. Interestingly, the administering of sild-T0 promoted a further increase of MMP-9, MCP-1, and CCR-2, possibly contributing to changes in the microglia phenotype, which becomes more phagocytic, cleaning myelin debris. It was also observed that, after sild-T0 treatment, the expression of GST-pi and MBP increased and the myelin structure was improved. However, sild-T15 was not efficient in all aspects, probably due to the short treatment period and to starting after the installation of the degenerative process. Therefore, the present study shows that sildenafil modulates inflammation, with the involvement of MMP-9, MCP-1, and CCR-2, and also contributes to myelin repair. These protective effects were dependent on the therapeutic strategy used. This clarification can strengthen research proposals into the mechanism of action of sildenafil and contribute to the control of neurodegenerative diseases such as MS., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКβα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1β TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKβα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1β and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKβα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group was more effective than sild-T15 in improving clinical status and promoting the control of neuroinflammation. The present study offers evidence that sildenafil has anti-inflammatory and neuroprotective effects, which are probably achieved through modulation of AMPK-IKβα-NFκB signaling. In addition, eNOS may play a role in the sildenafil neuroprotective mechanism, contributing to the activation of AMPK. However, other pathways such as MAPK-NFkB and the downstream proteins AMPK (AMPK-SIRT1-NFκB) should also be further investigated. An understanding of these mechanisms of action is critical for the clinical use of sildenafil to control neuroinflammation in neurodegenerative diseases such as MS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P. berghei NK65 and treated with chloroquine (CQ), with MOG35-55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35-55-immunized mice after adoptive transfer of P. berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.

Dendritic cells (DCs) are professional antigen-presenting cells specifically targeted during Plasmodium infection. Upon infection, DCs show impaired antigen presentation and T-cell activation abilities. In this study, we aimed to evaluate whether cellular extracts obtained from Plasmodium berghei-infected erythrocytes (PbX) modulate DCs phenotypically and functionally and the potential therapeutic usage of PbX-modulated DCs in the control of experimental autoimmune encephalomyelitis (EAE, the mouse model for human multiple sclerosis). We found that PbX-treated DCs have impaired maturation and stimulated the generation of regulatory T cells when cultured with naive T lymphocytes in vitro. When adoptively transferred to C57BL/6 mice the EAE severity was reduced. Disease amelioration correlated with a diminished infiltration of cytokine-producing T cells in the central nervous system as well as the suppression of encephalitogenic T cells. Our study shows that extracts obtained from P. berghei-infected erythrocytes modulate DCs towards an immunosuppressive phenotype. In addition, the adoptive transfer of PbX-modulated DCs was able to ameliorate EAE development through the suppression of specific cellular immune responses towards neuro-antigens. To our knowledge, this is the first study to present evidence that DCs treated with P. berghei extracts are able to control autoimmune neuroinflammation., (© 2014 John Wiley & Sons Ltd.)

Severe accidents caused by the "armed" spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca(2+), K(+) and Na(+) channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.

Nitric oxide (NO) is the main activator of the soluble guanylate cyclase (sGC)-guanosine 3'5' cyclic monophosphate (cGMP) pathway. The level of cGMP is regulated by phosphodiesterases (PDEs), which break down cGMP. It has been reported that levels of NO in the central nervous system (CNS) can greatly increase during demyelination and/or neuroinflammation. Controversially, in demyelination models, mice without iNOS may develop more severe cases of disease. Furthermore, cGMP accumulation caused by PDE inhibitors has an anti-inflammatory/neuroprotective effect in MS-models. The role of the NO-cGMP pathway in the nervous tissue is, therefore, complex and not fully understood. The aim of the present study was to contribute to existing knowledge of the role of this pathway in the CNS. Wild type (WT - C57BL/6) and iNOS(-/-) animals were treated with sildenafil (25mg/kg) for 8 weeks. Control animals were not treated. VCAM and ICAM (adhesion proteins), GFAP and Iba-1 (astrocyte and microglia markers, respectively), PKG (cGMP-dependent protein kinase), sGC, eNOS (constitutive endothelial NO sinthase) and GSTpi (a marker of mature oligodendrocytes) were evaluated in the cerebellum using immunohistochemistry or western blotting. Myelin was assessed by luxol fast blue staining and electron transmission microscopy. Treatment with sildenafil reduced ICAM and VCAM levels (anti-inflammatory effect) and increased GFAP and Iba-1 expression (clearance phenotype) in WT animals. The expression of VCAM, ICAM, GFAP, PKG and sGC was lower in iNOS(-/-) mice than in WT control animals. The treatment of iNOS(-/-) animals with sildenafil resulted in an increase of all proteins (pro-inflammatory effect). There was overexpression of eNOS in untreated iNOS(-/-) mice. The myelin structure of iNOS(-/-) animals was damaged in comparison with WT control. Sildenafil increased GSTpi and resulted in an improved myelin structure in iNOS(-/-) mice. In conclusion, NO-cGMP signaling plays a role in the regulation of inflammation and myelination processes. The accumulation of cGMP produced opposite effects in WT and iNOS(-/-) mice. This can be explained by the overexpression of eNOS in iNOS(-/-) mice, unbalancing cGMP signaling, or cGMP has a dual role in inflammation. Drugs that modulate the NO-sGC-cGMP pathway may be clinically beneficial in the treatment of neuroinflammatory/demyelinating disorders, but further studies of the regulation of this pathway are required., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.

Sildenafil induces cGMP accumulation through phosphodiesterase-5 (PDE5) inhibition. cGMP-pathways protect oligodendrocytes and modulate astroglial and microglial reactions. Microglia and astrocytes play an important role in perpetuating multiple sclerosis (MS), a chronic inflammatory disease characterized by demyelination. Therefore, sildenafil can be a potential tool for MS treatment. The present study investigated the effects of sildenafil on the myelin structure and astrocyte/microglia-mediated neuroinflammation in an animal model of MS. Cuprizone-induced demyelination and neuroinflammation in rodents has been widely used as a model for MS. Herein, five male C57BL/6 mice (7-10 weeks old) were used per group. Over a 4-week period, the different groups received the following: (1) cuprizone (0.2%) mixed into the chow; (2) cuprizone in the chow and sildenafil (Viagra®; 3, 25 or 50mg/kg) in the drinking water; or (3) pure chow and water (control group). Cerebella were analyzed using transmission electron microscopy, western blotting, immunohistochemistry and luxol fast blue staining. Cuprizone induced tissue damage, with an increase in GFAP, Iba-1 and COX-2 and demyelination in comparison to the control group. However, cuprizone did not affect the expression of cytokines (TNF-α, IFN-γ, IL-1β and IL-2). Sildenafil reduced GFAP (25 and 50mg/kg) and Iba-1 expression (25mg/kg) in comparison to the cuprizone group, indicating the modulation of astrocytes and microglia, respectively. Sildenafil preserved myelin and axons ultrastructure and strongly reduced IFN-γ, TNF-α, IL-1β, IL-2 and COX-2 expression in comparison to the control and/or cuprizone groups. The results demonstrate a protective effect of sildenafil in the cerebellum. Thus, well-designed clinical trials may demonstrate that the oral administration of sildenafil can be suitable for individuals with MS and other neuroinflammatory/neurodegenerative diseases, providing additional benefits to current treatments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.