30 research outputs found

    Uso de animais em experimentos no Brasil: práticas culturais, científicas e nova ética para o século XXI

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    From 2018 to 2021, 895 research institutions in Brazil registered at the National Council for the Control of Animal Experiments, Concea, were authorized to use more than 16 million animals in experiments. This historical series is published for the first time and the data were obtained through a Brazilian law known as the Information Access Law (LAI). The purpose of this text is to review the literature and problematize access to data on the use of animals as a fundamental condition to change the paradigm in experimental science in the construction of a world with fewer deaths. The article reconstructs the cultural bases and scientific practices that influenced ethics about animals, especially their use in laboratories. At the same time, it indicates that, since antiquity, there were already thoughts that animals should be treated with dignity and humanity. The text presents the emergence of alternative methods in the late 1950s and questions the role of ethics committees in reducing the use of animals in laboratories.De 2018 a 2021, 895 institui√ß√Ķes de pesquisas no Brasil cadastradas no Conselho Nacional de Controle de Experimenta√ß√£o Animal, o Concea, tiveram a autoriza√ß√£o para utilizar mais de 16 milh√Ķes de animais em experimentos. A s√©rie hist√≥rica est√° sendo publicada com ineditismo por este artigo e os dados foram conseguidos por meio da Lei de Acesso √† Informa√ß√£o, a LAI. A proposta desse texto √© problematizar o acesso aos dados do uso de animais como condi√ß√£o fundamental para mudar o paradigma na ci√™ncia experimental na constru√ß√£o de um mundo com menos mortes. O artigo reconstitui, historicamente, as bases culturais e pr√°ticas cient√≠ficas que influenciaram a √©tica em rela√ß√£o aos animais, em especial ao uso desses em laborat√≥rios. Ao mesmo tempo, indica que, desde a antiguidade, j√° existiam pensamentos de que os animais deveriam ser tratados com dignidade e humanidade. O texto apresenta a emerg√™ncia dos m√©todos alternativos ao uso de animais a partir do final dos anos 50 e questiona o papel dos comit√™s de √©tica para a diminui√ß√£o do uso de cobaias nos laborat√≥rios

    Characterization of neutralizing human anti-tetanus monoclonal antibodies produced by stable cell lines

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    Monocyte Activation Test (MAT) as a possibility of replacement for the rabbit pyrogen test in hyperimmune sera

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    The use of a commercial kit for the monocyte-activation test (MAT) was evaluated for assessing pyrogenic contamination of hyperimmune sera . Three batches of sera, two pyrogen free and one pyrogenic, were tested. Endotoxin spike recover indicated that sample dilutions from 1/2 to 1/10 are suitable. Kit transport and storage conditions were also evaluated, proving that an adequate cold chain must be assured to achieve good results. Furthermore, the commercial MAT kit seemed suitable to replace the rabbit pyrogen test (RPT) for pyrogen testing of hyperimmune sera, although further tests are needed to a full validation

    Strategies to improve productivity of CHO-S cells expressing an anti-TNFőĪ monoclonal antibody with biosimilar potential

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    Tumor necrosis factor alpha (TNFőĪ) is a proinflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFőĪ drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn‚Äôs disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFőĪ adalimumab. This project is based on the first development stage for an adalimumab biosimilar candidate with potential for national production through the generation of a productive and stable cell line and assessment of its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFőĪ and FcRs, tested by surface plasmon resonance, did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFőĪ in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFőĪ activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding kinetics and functional analysis performed suggest a potential for further development of adabut as a biosimilar. The process of the cell line development for adabut generated data consisting of cell growth/viability, volumetric and specific productivity, antigen binding kinetics, functional assays and long-term stability, from which 3 clones were selected. One clone (123) was top ranked and 2 others (70 and 225) showed equivalent performance. To increase the basal productivity of clone 123, 0.6g/L, we run 3 fed-batch experiments to evaluate 6 basal media, 8 supplements and a temperature shift to 32 C on day 6 on some conditions. The experiments were conducted in 250 and 500mL-shaker flasks along 14 days or while cell viability was above 60%. The flasks were sampled daily for cell counting/viability by Vi-Cell XR cell counter and glucose monitoring. Samples were stored for additional metabolites and antibody concentration assessment. By testing 24 different fed-batch strategies we were able to increase up to 6 times the volumetric clone productivity, while maintaining product quality. No significant differences were observed between the mAbs purified from days 14, 16 or 17 fed-batch cultures and the reference commercial product by SDS-PAGE, SEC-HPLC, IEX-HPLC and IEF analyses

    Advances and challenges in therapeutic monoclonal antibodies drug development

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    The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic

    Human recombinant fab fragment neutralizes shiga toxin type 2 cytotoxic effects in vitro and in vivo

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    Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.Fil: Luz, Daniela. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Quintilio, Wagner. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Moro, Ana Maria. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Piazza, Roxane Maria Fontes. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; Brasi

    The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody

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    Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50 ) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.Fil: Luz, Daniela. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Gomez, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ferreira, Raissa L.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Melo, Bruna S.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Guth, Beatriz E. C.. Universidade de Sao Paulo; BrasilFil: Quintilio, Wagner. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Moro, Ana Maria. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Presta, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Chen, Gang. University of Toronto; CanadáFil: Sidhu, Sachdev S.. University of Toronto; CanadáFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fontes Piazza, Roxane María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

    Antigen presentation and controlled release as tools to second generation vaccines improvement

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    Esta tese envolveu o estudo de encapsula√ß√£o de vacina bivalente contra difteria e t√©tano de uso adulto (dT) em microesferas de √°cido poli(l√°ctico-coglic√≥lico) (PLGA), com o objetivo de desenvolver uma formula√ß√£o que induzisse uma resposta protetora com um n√ļmero reduzido de doses, ou preferencialmente de apenas uma dose. Para ter sucesso, uma formula√ß√£o como tal deve ter um perfil de libera√ß√£o de ant√≠geno que simule as m√ļltiplas doses que recebem o indiv√≠duo. Assim, nesta tese s√£o mostrados os resultados dos experimentos de caracteriza√ß√£o bioqu√≠mica e imunol√≥gica da formula√ß√£o citada, sem o uso de estabilizadores prot√©icos, a fim de reduzir a complexidade do sistema em estudo. Do ponto de vista das prote√≠nas encapsuladas, tal caracteriza√ß√£o envolveu estudos de fluoresc√™ncia, de dicro√≠smo circular, de atividade antig√™nica e determina√ß√£o por HPLC da degrada√ß√£o durante o processo de encapsula√ß√£o. Do ponto de vista da formula√ß√£o, foram realizados ensaios de degrada√ß√£o in vitro e de atividade imunog√™nica in vivo, em camundongos e cobaias. Os dados obtidos indicaram que a encapsula√ß√£o em microesferas de PLGA de vacina dT, sem o uso de estabilizadores, permitiu a produ√ß√£o de uma formula√ß√£o vacinal vi√°vel, capaz de estimular uma resposta imunol√≥gica protetora e de mem√≥ria, abrindo caminho para o desenvolvimento da tecnologia de produ√ß√£o de vacinas polivalentes encapsuladas em microesferas de PLGA.The study on encapsulating a bivalent single dose vaccine against diphtheria and tetanus for adults (dT) within PLGA microspheres is described in this thesis. A successful single dose vaccine must show an antigen release profile mimicking the several doses a person should receive during the life. Therefore, results from the immunological and biochemical characterization of the formulation, prepared without protein stabilizers, is showed here. From the encapsulated protein point of view the characterization involved fluorescence, circular dichroism, antigenicity and HPLC analysis. From the formulation point of view, there were performed in vitro release assays and immunogenicity on mice and on guinea-pigs. The results indicated that the dT microencapsulation within PLGA microspheres without protein stabilizers lead to the production of a viable vaccine formulation, able to elicit protective and memory immune response

    Mycobacterium leprae 18kDa protein: a carrier model to second generation vaccines

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    O principal problema no desenvolvimento de vacinas de segunda gera√ß√£o √© a baixa resposta contra os ant√≠genos, geralmente extremamente purificados. Isto tem levado √† busca de novos adjuvantes que sejam mais eficientes e menos t√≥xicos. Este trabalho objetivava o estudo de uma prote√≠na de choque t√©rmico de Mycobacterium leprae - 18kDa-hsp - recombinante, produzida em levedura, veiculada em lipossomos, como modelo de prote√≠na carregadora para ant√≠genos fracos. Al√©m de um estudo de estabiliza√ß√£o de lipossomos por liofiliza√ß√£o. A 18kDa-hsp apresenta certas caracter√≠sticas peculiares que levaram ao desenvolvimento de um m√©todo de dosagem pr√≥prio e extens√≠vel a outras prote√≠nas com baixo teor em amino√°cido crom√≥foros. Tendo uso potencial como prote√≠na carregadora, a 18kDa-hsp √© est√°vel, suportando bem aquecimento, liofiliza√ß√£o, acila√ß√£o e associa√ß√£o com lipossomos de fosfol√≠pides, sem perda significativa de sua atividade. Estudos paralelos de estabiliza√ß√£o de lipossomos unilamelares mostraram que se pode usar trealose como crioprotetora no processo de liofiliza√ß√£o-reidrata√ß√£o, sem perdas significativas do conte√ļdo dos lipossomos e com manuten√ß√£o das estruturas das ves√≠culas. Finalmente, o sistema lipossomos-18kDa-hsp mostra-se promissor como um sistema est√°vel para veicula√ß√£o de imun√≥genos fracos.The major problem on development of second generation vaccines is the poor response against some immunogens. It has leading an intense research to find other substances non toxic and more efficient on enhancement of the immunological response. The aim of this work was the study of a heat-shock protein from Mycobacterium leprae - 18kDa-hsp - produced in yeast and vehiculated in liposomes, as a protein carrier model for weak immunogens. Furthermore, there was a study on stabilization of liposomes by lyophilization. The peculiar protperties of the 18kDa-hsp lead us to develop a new method for quantification that can be extended to other proteins with low chromophore content. Potentially a carrier protein, the 18kDa-hsp is very stab1e: it has no significant loss of its antigenic reactivity when heated, lyophilized, acylated and associated to liposomes. Parallel studies on liposomes stabilisation show trehalose as a good cryoprotector. The sugar can avoid the leakage of the entrapped marker (protein) and maintain the vesicles structures on the lyophilisation-rehydration processo Finally, the system liposomes-18kDa-hsp is promising for vehiculation of weak immunogens

    Antigen presentation and controlled release as tools to second generation vaccines improvement

    No full text
    Esta tese envolveu o estudo de encapsula√ß√£o de vacina bivalente contra difteria e t√©tano de uso adulto (dT) em microesferas de √°cido poli(l√°ctico-coglic√≥lico) (PLGA), com o objetivo de desenvolver uma formula√ß√£o que induzisse uma resposta protetora com um n√ļmero reduzido de doses, ou preferencialmente de apenas uma dose. Para ter sucesso, uma formula√ß√£o como tal deve ter um perfil de libera√ß√£o de ant√≠geno que simule as m√ļltiplas doses que recebem o indiv√≠duo. Assim, nesta tese s√£o mostrados os resultados dos experimentos de caracteriza√ß√£o bioqu√≠mica e imunol√≥gica da formula√ß√£o citada, sem o uso de estabilizadores prot√©icos, a fim de reduzir a complexidade do sistema em estudo. Do ponto de vista das prote√≠nas encapsuladas, tal caracteriza√ß√£o envolveu estudos de fluoresc√™ncia, de dicro√≠smo circular, de atividade antig√™nica e determina√ß√£o por HPLC da degrada√ß√£o durante o processo de encapsula√ß√£o. Do ponto de vista da formula√ß√£o, foram realizados ensaios de degrada√ß√£o in vitro e de atividade imunog√™nica in vivo, em camundongos e cobaias. Os dados obtidos indicaram que a encapsula√ß√£o em microesferas de PLGA de vacina dT, sem o uso de estabilizadores, permitiu a produ√ß√£o de uma formula√ß√£o vacinal vi√°vel, capaz de estimular uma resposta imunol√≥gica protetora e de mem√≥ria, abrindo caminho para o desenvolvimento da tecnologia de produ√ß√£o de vacinas polivalentes encapsuladas em microesferas de PLGA.The study on encapsulating a bivalent single dose vaccine against diphtheria and tetanus for adults (dT) within PLGA microspheres is described in this thesis. A successful single dose vaccine must show an antigen release profile mimicking the several doses a person should receive during the life. Therefore, results from the immunological and biochemical characterization of the formulation, prepared without protein stabilizers, is showed here. From the encapsulated protein point of view the characterization involved fluorescence, circular dichroism, antigenicity and HPLC analysis. From the formulation point of view, there were performed in vitro release assays and immunogenicity on mice and on guinea-pigs. The results indicated that the dT microencapsulation within PLGA microspheres without protein stabilizers lead to the production of a viable vaccine formulation, able to elicit protective and memory immune response
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