Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli. We believe that the knowledge about NG97 cell line may be useful for a deeper understanding of biological and immunological characteristics of gliomas

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model.

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

Artesunate ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte migration to the central nervous system

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOExperimental autoimmune encephalomyelitis (EAE) is T-celldependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE. Methods and Results: C57BL6 mice were immunized with MOG(35-55) peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS. Conclusions: Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.Experimental autoimmune encephalomyelitis (EAE) is T-celldependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellula228707714FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2014/19492-3; 2014/02631-0; 2014/11588-

Violacein treatment modulates acute and chronic inflammation through the suppression of cytokine production and induction of regulatory T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOInflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 mu g of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naive mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+ Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacteriumChromobacterium vio105116FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [2011/17965-3]CNPq [471066/2012-5]FAPESP [2014/02631-0, 2011/23664-6, 2012/01892-0]2011/17965-3; 471066/2012-5; 2014/02631-0; 2011/23664-6; 2012/01892-0sem informaçã

Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. the goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. the aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. the number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. the highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. the kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)National Institute of Health (US NIH)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Microbiol & Immunol, Alfenas, MG, BrazilFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Biochem, Alfenas, MG, BrazilState Univ Campinas UNICAMP, Inst Biol, Dept Struct & Funct Biol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilMassachusetts Gen Hosp, Wellman Ctr Photomed, Boston, MA 02114 USAHarvard Univ, Sch Med, Dept Dermatol, Boston, MA 02115 USAMIT, Harvard Mit Div Hlth Sci & Technol, Cambridge, MA 02139 USAFed Univ Alfenas UNIFAL MG, Inst Biomed Sci, Dept Pathol & Parasitol, Alfenas, MG, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, SP, BrazilCNPq: 486135/2012-8CNPq: 304827/2012-6FAPEMIG: CBB-PPM-00119-14National Institute of Health (US NIH): R01AI050875CAPES: AEX-9765-14-0Web of Scienc

Estudo da imogenicidade de uma vacina contra carcinoma renal humano

Orientador: Fanzi Ahmed Monstafa DawoodDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A partir de massa tumoral, obtida através de remoção cirúrgica, de um Adenocarcinoma Renal estágio IV obeteve-se vários fragmentos que foram submetidos a um processo de extração e polimerização de proteínas associada à membrana. Esse extrato foi denominado de PATT. Uma amostra de tecido renal normal também foi submetida a este processo, recebendo a denominação de PATN. Para efeito de analise comparativa as duas frações, PATT e PATN, foram submetidas: 1 Análise imunoquímica, através de imunodifusão dupla e imunoeletroforese. 2 À análise eletroforética em gel de policacrilamida, na presença de SDS. 3 A uma gel filtração em Sephadex G-100. Através da análise por imunodifusão e imunoeletroforese não foi possível detectar diferenças entre as frações PATT e PATN. Entretanto, análise eletroforética em gel de poliacrilamida na presença de SDS demonstrou a existência de um componente na fração PATT praticamente ausente na fração PATN. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Not informed.MestradoImunologiaMestre em Ciências Biológica

Alterações da resposta imune a infecção experimental pelo trypanosoma cruzi, induzidos pela infecção concomitante com um coronavirus murino

Orientador: Humberto de Araujo RangelTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A manutenção de formas sanguícolas do Trypanosoma cruzi nos laboratórios que desenvolvem pesquisas com o parasita é realizada através de repiques de sangue do hospedeiro previamente infectado. Entretanto, até recentemente estes animais eram criados de modo convencional, ou seja, em condições favoráveis à infecção com diferentes patógenos naturais, inclusive vírus de difícil detecção e controle. Com a implantação do CEMIB-UNICAMP, capaz de fornecer animais controlados do ponto de vista genético e sanitário, resolvemos investigar se algum destes patógenos murinos, estaria sendo transmitido junto com o parasita e se algumas características da Doença de Chagas experimental estariam sendo alteradas por este(s) contaminante(s). MAP testes realizados com soro de animais infectados com Trypanosoma cruzi indicaram a presença de coronavírus em estoques do parasita mantidos exclusivamente por passagem de animal para animal. Estoques previamente mantidos em meios de cultura de células ou em meios axênicos não indicaram a presença de contaminantes. Os resultados obtidos mostraram que camundongos infectados com o estoque contaminado apresentavam níveis de parasitemia e taxa de mortalidade mais elevados que os animais infectados os estoques não contaminados. Além disso, o plasma filtrado de animais infectados com o estoque contaminado foi capaz de elevar os níveis de parasitemia e de mortalidade de animais infectados com os estoques não contaminados. O fator, presente no plasma dos animais infectados com o estoque contaminado era sem dúvida um vírus, denominado vírus X, uma vez que podia ser seriadamente transferido de animal para animal, era completamente inativado quando aquecido a 56° C por 45 minutos e tinha sua virulência aumentada provocando a morte de animais de quatro semanas de idade após a transferência seriada. Anticorpos anti-X, preparados com a décima passagem seriada do vírus, ou anticorpos anti-MHV3 (Murine Virus Hepatitis-3) foram capazes de proteger os animais jovens da morte, quando estes eram desafiados com o vírus X, e de diminuir a parasitemia quando os animais eram infectados com o estoque contaminado. Estes dados indicavam que cepas de Trypanosoma cruzi, mantidas apenas por transferência de animal para animal, poderiam estar contaminadas com um vírus capaz de aumentar a patogenicidade do parasita ou de alterar a resposta imune do hospedeiro. Animais infectados com o estoque contaminado ou com o vírus isolado, apresentaram uma marcada linfopenia, no segundo dia após a infecção, enquanto que nenhuma alteração foi observada nos animais infectados com os outros estoques do parasita. Experimentos de neutralização, utilizando anticorpos anti-X, mostraram que esta linfopenia podia ser completamente abolida. Alterações do peso e da celularidade de diferentes órgãos linfóides também foram observadas, sendo evidenciada perda da arquitetura tímica e alterações no processo de maturação de células da medula óssea quando os animais eram infectados com o estoque de parasitas contaminado ou com o vírus isolado. As populações de células, apresentando marcadores específicos para linfócitos B e T, mostraram alterações em diferentes tecidos linfóides. Observou-se uma profunda queda na porcentagem de células T e B, quando os animais eram infectados com o vírus isolado ou com o estoque do parasita contendo este contaminante. Estes resultados confirmam e estendem, também para a Doença de Chagas experimental, os dados obtidos na literatura de que este vírus é capaz de alterar as respostas do hospedeiro, através da imunomodulação, e indicam que alguns dados obtidos com a infecção experimental pelo Trypanosoma cruzi devem ser interpretados com cautelaDoutoradoImunologiaDoutor em Ciências Biológica

Chloroquine: Modes Of Action Of An Undervalued Drug.

For more than two decades, chloroquine (CQ) was largely and deliberately used as first choice drug for malaria treatment. However, worldwide increasing cases of resistant strains of Plasmodium have hampered its use. Nevertheless, CQ has recently been tested as adjunct therapy in several inflammatory situations, such as rheumatoid arthritis and transplantation procedures, presenting intriguing and promising results. In this review, we discuss recent findings and CQ mechanisms of action vis-à-vis its use as a broad adjunct therapy.15350-