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After unilateral lesion of the medial forebrain bundle 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 either the glutamatergic antagonist MK-801 or the agonist NMDA microinjections into 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 matter, 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 (ipsilateral or contralateral) is particularly important, and this effect may not involve the neostriatum directly.

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., Graphical abstract This review summarizes 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.Image 1

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.

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 dmWts 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.

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 18F-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 18F-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.

Mast cell tumor (MCT), or mastocytoma, the second most common skin tumor in dogs, is characterized by neoplastic transformations and abnormal proliferation of mast cells from cutaneous or visceral origins. Mutations in the proto-oncogene c-kit are present in 15% of dogs with MCT and up to 35% in high-grade tumors. In the clinical management of the disease, some characteristics, such as anatomical occurrence, histological grading, clinical staging, age and breed, are important parameters to better drive the treatment and predict the prognosis of each patient. However, it is not clear the correlations between c-kit mutation and clinical/epidemiological data, demonstrating the necessity of new studies. In this study, 78 clinical cases of canine patients with MCT were analyzed in Campinas - SP, Brazil, at Oncovida - Veterinary Oncology Center, in the period of 2017 to 2019, to determine the frequency, prevalence and correlations between molecular, clinical and epidemiological aspects. Results presented here shows that mixed breed dogs (51%) were the most affected, followed by Labrador (17%) and Boxer (16%). Most dogs were female (67%), aged 6 to 10 years (62%); 42% of the animals had tumors located in the pelvic limb and 17% had nodules in the chest. The most used method for diagnosis was histopathology, totalizing 60% of cases, followed by aspirative cytology (26%) and immunohistochemistry (8%). Regarding histological variables, 3 patients had grade I (11.8%), 47 had grade II (61%) and 15 had grade III (19%) and, according to KIUPEL et al. (2011) classification, 77 % of the cases presented low grade and 23% were high grade. The survival of these patients was also analyzed, demonstrating that animals presenting grade I and grade II (low grade) had longer survival (68%) in relation to grade III (high grade) (16%) The variables showed a significant correlation between histological grade and c-kit mutation with breed, age, localization of the tumor, diagnostic, chosen treatment and survival, indicating that they are both decisive to the outcome of the disease. Only sex was not correlated, suggesting that both the grade of the tumor and the c-kit mutation were not found to contribute to a different development of the tumor in male or female. These results demonstrate important data on the clinical behavior of MCT in the region of Campinas-SP, presenting relevant knowledge that can be used in the future in the clinical manage of the patients with this disease and demonstrating the necessity of further studies on the epidemiology of MCT in each region of Brazil.

Gliomas used to be classified mainly based on histopathological criteria. In 2016, the Word Health Organization introduced a new classification system incorporating the molecular profile of gliomas. This has prompted research on the utility of molecular signature of gliomas to predict prognosis and response to therapy. While experimental data appear to be promising, the clinical use of molecular markers to predict prognosis and drive individual treatments is still a challenge. This chapter presents an overview of the major genes and markers associated with the characterization and development of gliomas, and the potential of these molecular markers in clinical decision-making. The current challenges and future directions are discussed.

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.

BackgroundGlioblastoma (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 thatPhoneutria nigriventerspider 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.MethodsHuman (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.ResultsTwo (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.ConclusionAll 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.

Malaria affects more than 200 million people annually around the world, killing a child every 2 min. Artemether (ART) and lumefantrine (LUM) are the gold standard choice to treat uncomplicated Plasmodium falciparum malaria; however, they are hydrophobic compounds with low oral bioavailability. Microneedle (MN) arrays consist of micron-sized needles on one side of a supporting base and have the ability to bypass the skin's stratum corneum barrier in a minimally invasive way, creating temporary channels through which drugs can diffuse, including those with poor water solubility. Herein, we report the development of dissolving MNs (DMNs) containing ART (MN-ART) and LUM (MN-LUM) as an alternative treatment regimen for malaria in low-resource settings. To incorporate the drugs into the MNs, nanosuspensions (NSs) for both molecules were developed separately to enhance drug solubility. The NSs were freeze-dried and the powder form was incorporated directly in an aqueous polymeric blend with poly-vinyl-pyrrolidone for MN-ART and a sodium hyaluronate hydrogel for MN-LUM. The in vivo bioavailability studies were performed using a MN reapplication scheme (1 × a day for 3 days), illustrating that an extended-release profile was achieved for both drugs when MNs were applied intradermally, and when compared to conventional oral treatment. The ART-LUM oral treatment was used as a positive control. For antimalarial activity, studies with animals infected with 106 Plasmodium yoelii 17XNL (12 days) were also conducted using female C57BL/6JUnib mice, demonstrating a 99.5% reduction in parasitemia by day 12 post-infection. By abolishing the infection, MN-ART and MN-LUM may serve as a promising controlled intradermal delivery device for antimalarial drugs to be explored in endemic areas.

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. The involvement of RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) was also evaluated. Human (NG97) GB cells were treated with twelve subfractions (SFs - obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound heling 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. 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. 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. Support: FAPESP #2015/04194-0; CNPq #431465/2016–9.

Malignant primary brain tumors remain among the most difficult cancers to treat. In malignant tissues, macrophages are accumulated in a high infiltration being known as tumor-associated macrophages (TAMs). These cells are associated with poor prognostics in many types of cancer. Studies in our group demonstrated that the venom of Phoneutria nigriventer (PnV), a wandering spider from South America, has reduced the development of glioblastoma (GBM) in a murine model, inducing a large infiltrate of TAMs. Subsequently, in vitro results demonstrated that PnV activates macrophages, increasing the ability to kill tumor cells. The aim of this study was to analyze the effects of the peptide SNX-482 presented in the venom of Hysterocrates gigas in macrophages for further correlation with the PnV. Macrophages were differentiated from bone marrow precursors collected from male C57BL6 mice and differentiated for 7 days with M-CSF. These cells were used for polarization and coculture with T cells and analyzed by flow cytometry. PCR Array was also performed (QIAGEN) for the analysis of gene expression. The results showed that SNX-482 could activate macrophages in a not dose-dependent response. There was an increase in the main activation markers (CD40, CD80, CD86, CD68, CD83, and MHCII). The polarization indicated that the peptide potentiated the proinflammatory effect of M1 macrophages (increased MHCII and iNOS). The screening of 86 cancer-related genes showed that the Ccr4, Pdcd1, Gzmb, and IFN-γ genes had an increase in their expression. Furthermore, we developed in C57BL/6 mouse a pre-clinical model of intracranial glioblastoma using the Gl261 cell line. The results showed an applied-easy-safe model that could alter the gene expression of cancer markers. Taken together, all the results contributed to increasing the knowledge about the peptide SNX-482 and the model for further pre-clinical assays of glioblastoma, making a great advance in the development of new treatments.

Immunomodulation has been considered an important approach in the treatment of various types of malignant tumors. However, adaptive immune cells have received more attention, while the modulation of innate immune cells is still an underexplored tool. A recent study by our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the circulating NK cells and monocytes and the infiltration of macrophage in glioblastoma, in addition to decreasing the tumor 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. After differentiation from bone marrow precursors, cells were pre-activated with IFN-γ and treated as follow: Control (untreated); LPS (1 μg/mL); PnV (14 μg/mL); PnV-fractions F1, F2 and F3 (1 μg/mL) or PnV-subfractions (1 μg/mL). Results showed that PnV and the three fractions activated macrophages. Further purification generated twenty-three 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 tumor 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 potential to be used as immunoadjuvants in the treatment of cancer.

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.

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.

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.

Gliomas correspond to approximately 80% of primary malignant brain tumors in adults. Associate histopathological classification to the identification of the molecular profile of these tumors is a great strategy to predict the tumor’s behavior and its responsiveness to the treatments. Studies have shown the potential of using biopharmaceuticals against cancer. Our research group has recently found two main subfractions isolated from Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) that can act decreasing the number of migrating cells during transwell assay. Therefore, the present study aimed to characterize, as regards molecular identity, the responsiveness of glioma samples collected from patients to the molecules purified from the PnV, in order to improve diagnostic and prognostic predictions, as well as contribute to the development of new drugs. After consent of the patients, samples of the tumors clinically diagnosed as glioma were collected during the surgical procedure to cultivation, that were performed until 10 passages to establish a lineage before use. To analyze its responsiveness to PnV in a migration assay, cultured human glioblastoma (1-JA63) cells were treated with PnV-isolated (HPLC) subfractions (SF1 and SF11 at 1 µg/ml) for 12 and 48 h (control stayed in IMDM) in a transwell system. SFs (mainly SF1) treatment induced a significant reduction in the number of migrating cells after 48 hours of exposure, in comparison to the control (untreated). Ongoing trials continue the establishment of another tumor samples of gliomas and migration tests with PnV subfractions will be carried out, to compare their effects in relation to other degrees of the disease also correlating the tumor response to the drug with the molecular profile. The results will contribute to the development of a potential individualized therapy also adding information about the correlation between the molecular identity of the tumor and its prognosis.

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.

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.

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.

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.

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.

Background Atherosclerotic cardiovascular disease is a chronic inflammatory condition. Thiazolidinediones (TZDs) are used to enhance sensitivity to insulin and have demonstrated a protective effect over a variety of cardiovascular markers and risk factors. Controversially, the TZDs are associated with the development of heart failure. Thus, lines of research have invested in the search for new molecules in order to obtain more selective and less harmful treatment alternatives for the pathogenesis of atherosclerosis and its risk factors. Methods Animals were fed a diet rich in fat for 10 weeks. In the last 2 weeks, animals received either pioglitazone, LPSF/GQ-02, or LPSF/GQ-16 daily through gavage. At the end of the treatment, blood was collected for biochemical analysis and the aortas were dissected for subsequent analyses. Results No changes in the blood lipid profile were found following the use of the drugs in comparison to the control. However, the new thiazolidine derivatives were more efficient in improving insulin resistance in comparison to pioglitazone and the control group. Morphometric analyses revealed that neither pioglitazone nor LPSF/GQ16 led to satisfactory effects over atherosclerosis. However, LPSF/GQ-02 led to a reduction in area of the atherosclerotic lesions. Ultrastructural analyses revealed extensive degeneration of the endothelium and an increase in apoptotic cells in the subendothelial space following the use of pioglitazone and LPSF/GQ-16. However, LPSF/GQ-02 caused minimal cell alterations in the aortic endothelium. Regarding markers, endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase 9 (MMP-9), LPSF/GQ-16, and pioglitazone exerted similar effects, increasing the expression of MMP-9, and had no effect on the expression of eNOS compared with the control group. On the other hand, LPSF/GQ-02 was effective in reducing the expression of MMP-9 and increased eNOS significantly. Conclusions The results suggest that the new thiazolidine derivative LPSF/GQ-02 is a promising candidate for the treatment of atherosclerosis.

Made available in DSpace on 2018-11-26T16:33:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Edit Jacobson's Foundation in Gothenburg, Sweden Sahlgrenska University Hospital in Gothenburg, Sweden AFA Insurance, Stockholm, Sweden 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 Ca2+ 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 (TM) 488-conjugated phalloidin probe), the G-actin/F-actin ratio was determined, and the expression level of connexin 43 (Cx43) was assessed. Incubation in Ca2+-free buffer did not change the Ca2+ responses. However, pre-incubation in thapsigargin/caffeine completely abolished these responses, suggesting that PNV-evoked Ca2+ transients were from intracellular Ca2+ stores. Pretreatment with a Na+/K+-ATPase antagonist (ouabain) or a TLR4 antagonist (LPS-RS) decreased or increased the Ca2+-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. (C) 2016 Elsevier Ltd. All rights reserved. State Univ Campinas UNICAMP, Inst Biol, Dept Biochem & Tissue Biol, BR-13083970 Campinas, SP, Brazil Univ Gothenburg, Sahlgrenska Acad, Inst Neurosci & Physiol, Dept Clin Neurosci, SE-41345 Gothenburg, Sweden Univ Fed Minas Gerais, Inst Biol Sci, Dept Gen Biol, BR-31270901 Belo Horizonte, MG, Brazil Univ Gothenburg, Sahlgrenska Acad, Inst Clin Sci, Dept Anaesthesiol & Intens Care Med, SE-41345 Gothenburg, Sweden Paulista State Univ UNESP, Dept Anim Physiol & Morphol, BR-14884900 Jaboticabal, SP, Brazil UNESP, Dept Morphol, BR-14801903 Araraquara, SP, Brazil Paulista State Univ UNESP, Dept Anim Physiol & Morphol, BR-14884900 Jaboticabal, SP, Brazil UNESP, Dept Morphol, BR-14801903 Araraquara, SP, Brazil FAPESP: 2012/19245-0 FAPESP: 2011/08005-6 CNPq: 305099/2011-6 CNPq: 486142/2012-4 Sahlgrenska University Hospital in Gothenburg, Sweden: LUA/ALF GBG-11587

Made available in DSpace on 2018-12-11T16:43:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-06 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Sahlgrenska University Hospital 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 Ca2+ 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 24 h; A3: sildenafil 1 or 10 μM for 4 h and then sildenafil 1 or 10 μM + LPS for 24 h. B1: control; B2: LPS for 24 h; B3: LPS for 24 h and then LPS + sildenafil 1 or 10 μM for 24 h. Cytoskeleton integrity was analyzed through GFAP immunolabeling and actin labeling with an Alexa 488-conjugated phalloidin probe. Calcium responses were assessed through a Ca2+-sensitive fluorophore Fura-2/AM. The results show that both preventive and restorative treatments with sildenafil (in both concentrations) reduced the Ca2+ 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. Laboratory of Ultrastructure Aggeu Magalhães Research Center (CPqAM) Oswaldo Cruz Foundation (Fiocruz) Federal University of Pernambuco (UFPE) Department of Morphology Paulista State University (UNESP) Department of Clinical Neuroscience Institute of Neuroscience and Physiology The Sahlgrenska Academy University of Gothenburg Department of Biochemistry and Tissue Biology Institute of Biology State University of Campinas (UNICAMP) Department of Morphology Paulista State University (UNESP) CAPES: 99999.005966/2014-00

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.

Some pharmacological studies showed that diethylcarbamazine (DEC) interferes with the arachidonic acid metabolism, acting as an anti-inflammatory drug. The chronic alcohol consumption activates the hepatic inflammatory response associated to T-cell activation and overproduction of pro-inflammatory cytokines. The present work analyzed the anti-inflammatory effect of DEC on hepatic cells of alcoholic mice. Thirty-two male C57BL/6 mice were equally divided in the following groups: (a) control group (C), which received only water, (b) DEC-treated group, which received 50 mg/kg for 12 day (DEC50), (c) the alcoholic group (EtOH), submitted to only alcohol and (d) the alcohol-DEC treated group (EtOH50), submitted to alcohol plus DEC treatment after the induction of chronic alcoholism for 5 weeks. Biochemical analyses were performed and liver fragments were processed for light microscopy, transmission electron microscopy, immunohistochemical and western blot. The level of AST increased significantly in alcoholic group whereas a significant reduction of serum AST was detected in the EtOH50 group. Histological and ultrastructural analysis of alcoholic group showed evident hepatocellular damage, which was strikingly reduced in the alcoholic DEC-treated group. Immunohistochemistry results revealed highly expression of inflammatory markers as MDA, NF-κB, TNF-α, IL-6, VCAM and ICAM by the hepatic cells of the EtOH group; however no immunoreactivity for any of these cytokines was detected after DEC treatment. Western blot analyses showed increased MCP-1 and iNOS expression in EtOH group, which was significantly inhibited by DEC treatment. According to the present results, DEC can be a potential drug for the treatment of chronic inflammation induced by chronic alcoholism.

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.

Phoneutria nigriventer spider venom (PNV) causes uneven BBB permeability throughout different cerebral regions. Little is known about cellular and molecular responses which course with the PNV-induced BBB opening. We investigate by immunohistochemistry (IHC) and Western blotting (WB), the GFAP, S100, IFN-gamma and TNF-alpha proteins expression in hippocampus and cerebellum after different time-points from venom or saline intravenous injection. All proteins variably altered its expression temporally and regionally. WB showed increased GFAP content at 15-45 min followed by a shift below the control level which was less pronounced in hippocampus. IHC showed reactive gliosis during all the trial period. In cerebellum, GFAP was mostly immunodetected in astrocytes of the molecular layer (Bergmann glia), as was S100 protein. The maximum S100 immunolabeling was achieved at 5h. IFN-gamma and TNF-alpha, expressed mostly by hippocampal neurons, increased along the trial period, suggesting a role in BBB permeability. In envenomed animals, closer contacts astrocyte-astrocyte, granule cells-granule cells and astrocytes-Purkinje cells were observed in cerebellum. Closer contacts between neurons-neurons-astrocytes-astrocytes were also seen in hippocampus. PNV contains serotonin, histamine, Ca(2+) channels-blocking toxins, some of which affect glutamate release. The hypothesis that such substances plus the cytokines generated, could have a role in BBB permeability, and that calcium homeostasis loss and disturbance of glutamate release are associated with the marked GFAP/S100 reaction in Bergmann glia is discussed. The existence of a CNS mechanism of defense modulated differentially for fast synthesis and turnover of GFAP, S100, IFN-gamma and TNF-alpha proteins was evident. A clear explanation for this differential modulation is unclear, but likely result from regional differences in astrocytic/neuronal populations, BBB tightness, and/or extent/distribution of microvasculature and/or ion channels density/distribution. Such differences would respond for transient characteristics of BBB disruption. This in vivo model is useful for studies on drug delivery throughout the CNS and experimental manipulation of the BBB.

Drugs and neurotoxins activate specific neural circuits by increasing or decreasing the formation and release of neurotransmitters, such as nitric oxide (NO), and by inducing immediate early genes, such as FOS. We have previously shown that Phoneutria nigriventer spider venom (PNV) impairs the microtubule-dependent transcellular barrier of the blood-brain interface and causes structural alterations in perivascular astrocytic end-feet without producing morphological changes in central neuronal cells. In the present study, we used FOS and neuronal nitric oxide synthase (n-NOS) immunolabeling to investigate the ability of PNV to activate the central nervous system. Three groups of rats were used: the first group received a sublethal dose of PNV (850 microg/kg, via a tail vein), the second received an equal volume of 0.9% saline (sham group) and the third group received no injection. Envenomed rats showed salivation, lachrymation, tremors and flaccidity followed by spastic paralysis of the hind limbs and convulsions. Cryosections (30 microm thick) were serially collected at 600 microm intervals for free-floating immunohistochemical analysis. FOS-like positive neurons predominated in motor-related areas such as dorsolateral (dlPAG) and ventral periaqueductal gray matter (vPAG), frontal (FCM) and parietal motor cortex (PCM), and periventricular thalamic nucleus (PTN) and in acute stress-related areas (rhinal cortex and lateral septal nuclei). The greatest relative increases in FOS-like positive neurons occurred in the vPAG, PCM and PTN motor-related areas. n-NOS-positive neurons predominated in the periventricular thalamic nuclei, followed by the dorsolateral periaqueductal gray matter and parietal cortex motor area. The marked activation of motor areas and, to a lesser extent, of acute stress-related areas suggested the involvement of neuronal pathways in these regions in the response to envenoming by PNV. In addition, the occurrence of n-NOS immunolabeling in some anatomical regions with FOS-like positive neurons suggests that NO may modulate the response to PNV in these regions.

A highly controlled transport of substances at the interface between blood and brain characterizes the blood-brain barrier (BBB), fundamental for maintenance of the homeostasis of the cerebral milieu. In this study, we investigated the time course (15 min, 1, 2, and 5 h) of BBB opening induced by intravenous (i.v.) injection of Phoneutria nigriventer spider venom (PNV) using quantitative and morphological approaches on cerebellum and hippocampus vessels for assessment of BBB permeability. The results showed vasogenic edema and tracer extravasation faster and severalfold higher in hippocampus than in cerebellum. Reactive astrocytes with swollen perivascular end-feet processes were found only in cerebellum. An immediate and total degradation of laminin in capillaries occurred resulting in the disappearance of the basement membrane. In medium-sized vessels, this effect was less prominent. The changes were transient, with cerebellum in general presenting a faster recovery. However, at 5 h laminin was overexpressed, principally in hippocampus. The rapid and abrupt shift of laminin expression in capillaries (at 15 min) coincided with the immediate and severe signs of intoxication shown by the animals, but not with the peak of leakage of vessels and vasogenic edema, which occurred later (1-2 h). The findings suggest a complex regulatory mechanism, since the extension of BBB impairment caused by PNV depends on the region of the SNC, and on the vessels types. It is suggested that the components of the BBB (gliovascular unit) have a critical role in these differences. P. nigriventer venom can be a useful tool to explore the mechanisms of BBB.

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.

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.

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS−/−mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF-α, COX-2, IL-1β, and IFN-γexpression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS−/−mice. Sildenafil reduced Iba-1, IFN-γ, and IL-1βlevels but had no effect on the expression of GFAP, TNF-α, and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS−/−mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS−/−mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS−/−mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.