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São conhecidos seis tipos de coronavírus humanos, sendo todos zoonóticos, mas somente os de gênero beta necessitam de um reservatório animal para a sua propagação. Eles pertencem à família Coronaviridae e, recentemente, um novo vírus, causador da COVID-19, doença de coronavírus de 2019 foi descrito. Esse é um dos três tipos de coronavírus que causam infecções respiratórias graves, como a SARS, Síndrome Respiratória Aguda Grave, ou pneumonia atípica. Estas têm como agente etiológico, respectivamente, o SARS-CoV-2 e o SARS-CoV-1. O coronavírus tem seu genoma de RNA positivo de fita simples, fazendo com que seja lido diretamente, sendo considerado um tipo de RNA mensageiro, que induz a produção de proteínas. Ambos têm em sua estrutura morfológica uma proteína do nucleocapsídeo (N), uma proteína transmembrana (M), uma proteína do envelope (E) e uma proteína pico (S).

Os interferentes endócrinos (IEs) são substâncias que possuem a capacidade de causar efeitos indesejáveis no organismo de animais e seres humanos. A presença de compostos desta categoria em produtos do cotidiano humano pode causar a contaminação do meio ambiente, principalmente de corpos d’água, devido à ineficiência do tratamento de esgoto. A utilização de água contaminada com IEs pode causar aumento de risco para câncer de mama, testículos e próstata, causar infertilidade e alterações de diversas glândulas do sistema endócrino. O presente artigo traz uma avaliação da presença de interferentes endócrinosem águas subterrâneas e tratadas para abastecimento, coletadas na cidade de Americana – SP. As análises das amostras coletadas forma realizadas utilizando o método de cromatografia gasosa acoplada à espectrometria de massas. Os resultados obtidos permitem concluir que os níveis de interferentes endócrinos presentes nas amostras analisadas não ultrapassam os valores considerados normais. Embora não seja possível detectar quantidades reduzidas dos interferentes, vale ressaltar que doses baixas desses interferentes podem causar os efeitos colaterais na população.

The kidney is an extremely important organ, whose main activity is blood filtration, being responsible for the excretion of undesirable substances in the body. Whenever there is a failure or injury in this organ either due to infections and/ or pathologies, loss of functional capacity may occur, requiring hemodialysis. In more severe cases, transplantation is prioritized. The most common type of graft is allogeneic performed between individuals of the same species, and although there is partial compatibility between the recipient and the donor, there are risks and cases of rejection; so the use of immunosuppressants is essential, involving the use of both conventional and biological therapies. Thus, biological therapies are becoming an alternative treatment for several patients, and the use of monoclonal antibodies is promising, with possible reduction in the dose of immunosuppressant, and reduction of severe side effects. On the market, Muromonab, Basiliximab and Alentuzumab are currently used as therapy in kidney transplanted recipients. The significant decrease in rejection in kidney transplant recipients is partly associated with these biological treatments.

Background: The Precision Medicine’s approach employs the endotype concept as a central feature to personalize medical treatment. Individual immunoreactivity, alongside characteristics such as genetics, environment, and diet, is one of the factors that differentiates the therapeutic-driven endotypes. Objective: To evaluate the opportunity of the Leukocyte Adherence Inhibition test to differentiate the immunoreactivity between two similar therapeutic agents employed on Allergen Immunotherapy. Methods: Side by side Leukocyte Adherence Inhibitions tests were performed with ovalbumin and carbamylated ovalbumin on a population of 33 self-reported egg-allergic individuals. Results: The results showed two endotypes inside the immune response of the studied groups: The first endotype was defined by the 16 individuals that presented a significant decrease in ovalbumin’s immunoreactivity after carbamylation (mean of differences = 35%; p = 0.002). The second endotype was defined by 17 individuals that presented a significant increase in ovalbumin’s immunoreactivity after carbamylation (mean of differences = 32%; p = 0.001). Conclusion: The Leukocyte Adherence Inhibition test was able to differentiate two distinct immunoreactivity patterns when comparing two similar therapeutic agents suggesting, as proof of concept, a potential role to be employed as a Precision Medicine tool.

Made available in DSpace on 2021-06-25T10:24:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Rhipicephalus sanguineus sensu lato (s.l.), popularly known as ‘brown dog tick’, is the primary vector of pathogens affecting dogs worldwide. To enter the host’s organism, these pathogens utilise the anticoagulant, antiplatelet, anti-inflammatory and immunomodulatory actions of compounds present in the tick’s saliva; such compounds are released by the ectoparasite in order to attach and feed on dogs. Nitric oxide (NO) is one of the regulatory factors in inflammation, apoptosis and immunomodulation. Here, we evaluated the in vitro activity of salivary gland extract of female dog ticks on the macrophage-derived J774 cell line, with and without lipopolysaccharide (LPS) stimulation. Cultures were evaluated for possible morphological alterations caused by exposure to the extract. There was no apparent in vitro cytotoxicity of the extract. Also, the NO secretory response in the non-LPS-stimulated cells was not inhibited. On the other hand, the extract presented modulatory action in the cultures of LPS-stimulated cells at a concentration of 0.1 μg/mL, possibly through macrophage activation, and induced a significant decrease in NO secretion. These results confirm the modulatory potential of bioactive molecules in the salivary glands of R. sanguineus ticks. Department of Biology Institute of Biosciences São Paulo State University – UNESP Department of Genetics Evolution and Bioagents Institute of Biology University of Campinas – UNICAMP Department of Biology Institute of Biosciences São Paulo State University – UNESP FAPESP: 2014/02843-8 FAPESP: 2015/20745-6 FAPESP: 2018/2999-9 FAPESP: 2019/02831-3 CNPq: 300625/2012-0

O câncer é uma alteração celular decorrente de alterações multifatoriais, como a idade, fatores genéticos e lesões proliferativas atípicas, podendo atingir um ou vários órgãos do corpo. O tumor de mama se destaca pelo seu risco letal e mutilador, sendo que sua identificação em estágios inicias resulta em uma maior chance de cura. Os marcadores tumorais são substâncias encontrados em tumores e em líquidos biológicos e alterações na expressão dos mesmos estão diretamente ligadas ao crescimento de células neoplásicas. Existem vários marcadores tumorais, sendo que alguns podem ser específicos enquanto outros estão presentes em diversos tipos de tumores. Estes biomarcadores são importantes para o diagnóstico precoce do tumor de mama, favorecendo o tratamento e a sobrevida do paciente. Os marcadores mais utilizados para diagnóstico e acompanhamento clínico de tumores de mama são: antígeno tumoral semelhante à mucina (MCA; mucin like antigen), antígeno do tumor 15.3 (CA15.3), antígeno de câncer 27.29 (CA 27.29), oncogene C-erbB-2, catepsina e antígeno carcinoembrionário (CEA). Embora esses marcadores sejam amplamente utilizados, a avaliação de novos marcadores irá possibilitar o desenvolvimento de novos estudos clínicos, entre eles terapias gênicas específicas para tumores de mama.

As neoplasias de pele do tipo melanoma são consideradas graves devido a sua alta letalidade e ao grande risco de metástase. Existem diversas opções de tratamentos convencionais, porém nenhum deles apresenta um bom prognóstico. Nesse contexto, tem-se estudado terapias biológicas com inibidores de vias de sinalização e anticorpos monoclonais no tratamento de melanoma, buscando resultados mais eficazes. O presente estudo discorre sobre as terapias biológicas mais utilizadas e a ação no tratamento de melanomas. A metodologia utilizada foi revisão literária em bases de dados científicos. Os resultados mostram que o melhor prognóstico para melanoma ainda está relacionado ao diagnóstico precoce. Contudo, o estudo das terapias biológicas no tratamento do melanoma pode proporcionar índices maiores de cura e diminuir os riscos de metástase. Entre os inibidores de vias de sinalização intracelulares para tratamento de melanomas tem destaque os inibidores de quinases como BRAF. Entre os anticorpos monoclonais, destacam-se os anticorpos direcionados para as moléculas PD-1 e CTLA-4.

Objetivo: o presente trabalho teve por objetivo descrever a utilização e os resultados obtidos com o instrumento Medical Outcomes Study Short Form -36 item (SF-36) para avaliação de qualidade de vida de pessoas em situação de uso, abuso ou dependência de substâncias psicoativas (SPA). Método: realizou-se um levantamento bibliográfico em artigos e revistas científicas, dissertações e monografias. Resultados: os estudos mostram que o uso de substâncias psicoativas traz prejuízos para saúde e qualidade de vida das pessoas. Conclusão: observa-se que o instrumento SF-36 se mostra válido e confiável para a avaliação da qualidade de vida entre consumidores de SPA e possui em seus domínios algumas dimensões que são bastante sensíveis a avaliação de aspectos da qualidade de vida desta população.

Made available in DSpace on 2022-04-29T08:37:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1β production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation. Laboratório de Biologia Molecular de Artrópodes—LBMA Instituto de Biologia—IB Universidade Estadual Paulista Câmpus Rio Claro—UNESP-RC, Avenida 24-A, n◦1515, Bela Vista Laboratório de Imunologia Translacional—LIT Departamento de Clínica Médica Faculdade de Ciências Médicas—FCM Universidade Estadual de Campinas—UNICAMP Cidade Universitária “Zeferino Vaz”, Rua Tessália Vieira de Camargo, n◦126 Departamento de Biomedicina Faculdade de Americana—FAM, Avenida Joaquim Bôer, n◦733, Jardim Luciane Faculdade de Medicina Universidade Anhembi Morumbi, Av. Rio das Pedras, 1601-Pompéia Laboratório de Biologia Molecular de Artrópodes—LBMA Instituto de Biologia—IB Universidade Estadual Paulista Câmpus Rio Claro—UNESP-RC, Avenida 24-A, n◦1515, Bela Vista FAPESP: 2017/07988-2 FAPESP: 2017/18422-0 FAPESP: 2019/02298-3

O carcinoma de pulmão é o segundo tipo de tumor de maior incidência em todo o mundo, sendo 85% deles carcinomas de pulmão de não pequenas células (CPNPC). As células tumorais do CPNPC proliferam em razão de um bloqueio da resposta de linfócitos T citotóxicos. Na resposta imune a tumores, a interação do ligante-1 do receptor de morte celular programada (PD-L1), expresso em células tumorais com a proteína de morte celular programada 1 (PD-1), expressa em linfócitos T citotóxicos, promove a supressão da resposta imune, levando à inibição da ativação de linfócitos T citotóxicos. Apesar de as terapias biológicas mostrarem-se eficazes para o tratamento de tumores pulmonares, estudos buscam uma opção de tratamento genético, como o método CRISPR/Cas9. O objetivo desta revisão é fornecer uma atualização do método CRISPR-Cas9 e a aplicação dele como ferramenta terapêutica buscando desativar o gene que codifica a proteína PD-1 em casos de CPNPC. A alteração genética da proteína PD-1 pelo CRISPR-Cas9 pode interromper a interação entre receptor e ligante, permitindo que linfócitos T citotóxicos reconheçam e exerçam uma resposta antitumoral contra tumores de CPNPC.

Made available in DSpace on 2019-10-06T15:26:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Rhipicephalus sanguineus s. l. is popularly known as the “brown dog tick” since dogs are its preferential hosts, but the species has been reported to parasitize other mammals, including humans, with significant medical-veterinary importance since it transmits several important pathogenic agents during the feeding period. The tick saliva is a complex mixture that has several functions, including the capability to modulate the hemostatic, inflammatory and immunologic systems of the host, allowing pathogens to settle. Despite knowledge about the immunosuppressive action of tick saliva, little is known about the mechanisms involved in this process and the morphophysiological effects caused by exposure to the salivary gland extract, taking into consideration the different periods of the glandular cycle. Thus, the objective of this study was to analyze the in vitro effects of salivary gland extracts obtained from R. sanguineus s. l. females fed on host rabbits for two (SGE2 – Salivary Gland Extracts of 2 days) and four days (SGE4 – Salivary Gland Extracts of 4 days) on J774 cells (monocyte macrophage cell line) and verify the occurrence of morphological and immunomodulatory alterations in these cells when exposed to different concentrations of these extracts. The results showed that: (i) SGE2 and SGE4 at the concentration of 4 μg/mL presented cytotoxicity to the J774 cells exposed for 24 and 48 hours; (ii) SGE2 at the concentrations of 2 μg/mL(48-hour exposure) and 1 μg/mL (24-hour exposure) and SGE4 at the concentrations of 2 and 1 μg/mL (48-hour exposure) showed proinflammatory activity, confirmed by the increased secretion of NO and proinflammatory cytokine (IL-2), and the presence of morphological characteristics detected by microscopy; and (iii) SGE2 and SGE4 at the concentrations of 0.5 and 0.1 μg/mL had immunomodulatory activity, demonstrated by decreases in the secretion of NO and proinflammatory cytokines (IL2, IL-6 and TNF-α) and increase in the synthesis of IL-10, confirmed by the morphophysiological analysis. These unprecedented data are extremely relevant for future research to identify the processes involved in the ectoparasite-host relationship, as well to develop more efficient tick control strategies. Biology Department Biosciences Institute São Paulo State University (UNESP), Avenida 24 A, 1515 Department of Biomedical Science Faculty of Americana FAM Biology Department Biosciences Institute São Paulo State University (UNESP), Avenida 24 A, 1515 FAPESP: 2013/20258-2 FAPESP: 2014/02843-8 FAPESP: 2015/20745-6 FAPESP: 2018/02999-9

Introdução: A fosfoetanolamina sintética (FS), conhecida como pílula do câncer, foi apresentada como promissora do tratamento de tumores. Essa substância tem seu mecanismo de ação voltado para as membranas celulares pela transdução de sinais e metabolismo de lipídeos que resultam na indução da apoptose. Objetivo: O presente trabalho avaliou os artigos da literatura que relacionam o uso da substância fosfoetanolamina sintética (FS) como inibidor da progressão e disseminação de células tumorais no Brasil. Buscou-se também descrever os possíveis mecanismos associados com a ação da molécula para tratamento de tumores. Método: O trabalho é uma revisão bibliográfica, narrativa, exploratória e integrativa, nas bases de dados Biblioteca Virtual de Saúde, Google acadêmico, Pubmed e Scientific Electronic Library Online (SciELO). Critérios de inclusão: artigos completos disponíveis na literatura nacional e internacional, com palavras FS e tumores. Resultados: A partir de resultados de busca com 65 artigos, foram selecionados 19 artigos. Após análises das fontes de informações acima, foram selecionados os artigos que descreveram os efeitos da fosfoetanolamina sintética e os possíveis mecanismos associados com a ação da FS para tratamento de tumores. Conclusão: A fosfoetanolamina é um composto lipídico em elevada concentração em tumores, associada com elevada taxa de apoptose. Pesquisas préclínicas buscam validar a utilização da FS para tratamento tumoral. Até o presente não há dados que comprovem a eficácia da FS em neoplasias. Estudos clínicos relacionados ao uso da FS em tumores são essenciais para validação do uso da FS. Em abril de 2017, A FS não mostrou eficácia clínica em ensaios preliminares e os testes clínicos foram suspensos pela ANVISA.

Diabetes mellitus type 1 (DM1) is an autoimmune disease in which β-cells of the pancreas islet are destroyed by T lymphocytes. Specific T cells are activated by antigen-presenting cells, mainly dendritic cells (DCs). It is already known that the regulation of tryptophan pathway in DC can be a mechanism of immunomodulation. The enzyme indoleamine 2,3-dioxygenase (IDO) is present in many cells, including DC, and participates in the metabolism of the amino acid tryptophan. Recent studies suggest the involvement of IDO in the modulation of immune response, which became more evident after the in vitro demonstration of IDO production by DC and of the ability of these cells to inhibit lymphocyte function through the control of tryptophan metabolism. Current studies on immunotherapies describe the use of DC and IDO to control the progression of the immune response that triggers DM1. The initial results obtained are promising and indicate the possibility of developing therapies for the treatment or prevention of the DM1. Clinical trials using these cells in DM1 patients represent an interesting alternative treatment. However, clinical trials are still in the initial phase and a robust group of assays is necessary.

Colorectal cancer is considered a disease of the elderly population. Since the number of geriatric patients continues to rise, monoclonal antibody therapy is the most promising therapy in the recent research. Presently, the monoclonal antibodies most frequently used in the treatment of colorectal tumors are bevacizumab, cetuximab, panitumumab, and ramucirumab. Bevacizumab is a monoclonal antibody that acts on VEGF. Cetuximab and panitumumab act on EGFR. Ramucirumab binds directly to the ligand-binding pocket of VEGFR-2 to block the binding of VEGF-A, VEGF-C, and VEGF-D. These monoclonal antibodies, alone or in association with radiotherapy or chemotherapy, are presenting good results and are increasing patient survival, despite the side effects. Due to the limited number of molecules available, several studies are trying to develop new monoclonal antibodies for the treatment of colorectal tumors. Among those being studied, some recent molecules are in phase I and/or II trials and are yielding advantageous results, such as anti-DR5, anti-Fn14, anti-IGF-1R, anti-EGFR, anti-NRP1, and anti-A33 antibodies. This has been successful in reducing side effects and in treating nonresponsive patients.

Dendritic cells (DCs) play a crucial role in balancing immune responses, and in that sense the interactions between the B7-1 and B7-2 molecules expressed on DCs and CD28 and CTLA-4 on helper T cells are fundamental. While coupling of B7 and CD28 molecules activates immune responses, binding of B7 to CTLA4 results in its blockade. CTLA4-Ig fusion protein, a competitor molecule of the B7-CD28 interaction, has been used for the development of immunological tolerance both experimentally and in patients. Here, we evaluated the effects of adoptive transfer of bone marrow-derived dendritic cells (BMDCs) pulsed with CTLA4-Ig in TNBS-induced colitis. CTLA4-Ig-modulated BMDCs or naïve BMDC were administered intravenously to BALB/c mice prior to TNBS rectal instillation. Five days later, spleens and colon segments were removed for immunological and histological analysis. Our results showed that the adoptive transfer of CTLA4-Ig-modulated BMDCs was able to reduce the severity of inflammation caused by the administration of TNBS, in view of tissue integrity and reduced leukocyte infiltration in the colon segments of the treated mice compared to controls. Non-specific spleen cell activation in vitro showed a reduction in the frequency of CD4+ IL-17+ T cells and CD4+ IFN-γ+ T cells as well as IL-9 secretion in cultures. To our knowledge, this is the first description of the beneficial effects of treatment with CTLA4-Ig modulated BMDC in experimental colitis.

Objetivo: Descrever os dados de coinfecção de tuberculose/Vírus da Imunodeficiência Humana (HIV) notificados entre 2000 a 2015 em Americana (SP). Método: Trata-se de um estudo epidemiológico descritivo. Foram utilizados os registros de notificação fornecidos pelo Sistema de Informação de Agravos de Notificação (SINAN), da Vigilância Epidemiológica, e de Boletins Epidemiológicos do Ministério da Saúde do Estado de São Paulo. Não foram incluídos dados de outras coinfecções. Os dados coletados de 2000 a 2015 foram analisados estatisticamente em programa GraphPad Prism. Resultados: Foi observado uma diminuição nos casos de coinfecção TB/HIV de 2014 a 2015. Os casos de tuberculose foram mais elevados na população masculina. Também se observou que, nos dois últimos anos, houve queda no número de casos de tuberculose notificados e aumento de casos de cura da doença. Conclusão: A análise dos dados permitiu concluir que ocorreu uma redução de número de casos de coinfecção no período avaliado.

Although systemic reactions caused by allergenic proteins present in venoms affect a small part of the world population, Hymenoptera stings are among the main causes of immediate hypersensitivity responses, with risk of anaphylactic shock. In the attempt to obtain therapeutic treatments and prophylaxis to hypersensitivity responses, interest in the molecular characterization of these allergens has grown in the scientific community due to the promising results obtained in immunological and clinical studies. The present review provides an update on the knowledge regarding the immune response and the therapeutic potential of Antigen 5 derived from Hymenoptera venom. The results confirm that the identification and topology of epitopes, associated with molecular regions that interact with antibodies, are crucial to the improvement of hypersensitivity diagnostic methods.

Background: Methylphenidate is the first-choice drug to the treatment of patients with attention deficit/hyperactivity disorder (ADHD). However, there was an increase in the indiscriminate use of this drug. Objectives We aim to describe the mechanisms of action as well as the side effects of this drug and indiscriminate use of this medicine in Brazil and worldwide. Methods: We performed a bibliographic review on electronic scientific site as PubMed, Scielo and Google Scholar. Results: Forty-five articles were found and, among them, 28 were selected. Full article available in the international and national literature were selected based on key words: methylphenidate; action mechanism; adverse effects and ADHD. Articles published before 2003 were excluded. Discussion/Conclusions: Methylphenidate acts directly on three distinct regions of the central nervous system by blocking the reuptake of the hormone dopamine and noradrenaline. Nervousness, nasopharyngitis, insomnia, nausea are the most common adverse effects caused using methylphenidate. The less frequent reactions are headache, alteration in blood pressure and metabolic, hematological, immune changes. Further studies are necessary for an in-depth assessment of the mechanism of action and side effects of this drug on ADHD.

Made available in DSpace on 2018-12-11T16:53:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Introduction In addition to conventional therapies, several new strategies have been proposed for modulating autoimmune diseases, including the adoptive transfer of immunological cells. In this context, dendritic cells (DCs) appear to be one of the most promising treatments for autoimmune disorders. The present study aimed to evaluate the effects of adoptive transfer of DCs obtained from both naïve and ovalbumin (OVA)-tolerant mice on the severity of TNBS induced colitis and analyze the eventual protective mechanisms. Methods and results To induce oral tolerance, BALB/c mice were fed 4mg/mL OVA solution for seven consecutive days. Spleen DCs were isolated from tolerant (tDC) and naïve (nDC) mice, and then adop-tively transferred to syngeneic mice. Three days later, colitis was induced in DC treated mice by intrarectal instillation of 100μg2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol. Control subjects received only intrarectal instillation of either TNBS solution or a vehicle. Five days later, mice from all groups were euthanized and examined for physiological and immunological parameters. Regarding the phenotype, we observed that the frequencies of CD11+ MHC II+ and CD11+ MHCII+ CD86+ cells were significantly lower in DCs isolated from tolerant mice than in those from naive mice. However, pretreatment with both types of DCs was able to significantly reduce clinical signs of colitis such as diarrhea, rectal prolapse, bleeding, and cachexia, although only treatment with tDCs was able to prevent weight loss from instillation of TNBS. In vitro proliferation of spleen cells from mice treated with either type of DCs was significantly lower than that observed in splenic cell cultures of naïve mice. Institute of Biosciences Universidade Estadual Paulista UNESP Faculty of Food Engineering University of Campinas UNICAMP Department of Food School of Nutrition Federal University of Ouro Preto Department of Biochemistry and Tissue Biology Institute of Biology University of Campinas UNICAMP Department of Genetics Evolution Microbiology and Immunology Institute of Biology University of Campinas UNICAMP Department of Biomedical Science Faculty of Americana FAM Institute of Biosciences Universidade Estadual Paulista UNESP FAPESP: 2013/20258-2 FAPESP: 2014/08591-0 FAPESP: 2014/086192 FAPESP: 2014/167010 FAPESP: 2015/09326-1

Made available in DSpace on 2018-12-11T16:46:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-01 Dendritic cells (DC) are potential tools for therapeutic applications and several strategies to generate tolerogenic DCs are under investigation. When activated by cytokines and microbial products, DCs express mediators that modulate immune responses. In this regard, the metabolites generated by the activities of inducible nitric oxide synthase (iNOS) and arginase in DCs seem to play important roles. Here, we evaluated the effects of adoptive transfer of DCs generated in vitro from bone marrow precursors (BMDC) modulated with L-NAME (Nω-nitro-L-arginine methyl ester) and NOHA (NG-Hydroxy-L-arginine), inhibitors of iNOS and arginase, respectively, upon the immune response of the wild type (BALB/c) and OVA-TCR transgenic (DO11.10) mice. The modulation with L-NAME increased CD86 expression in BMDC, whereas treatment with NOHA increased both CD80 and CD86 expression. Adoptive transfer of either L-NAME- or NOHA-modulated BMDCs to BALB/c mice reduced the plasma levels of ovalbumin-specific antibody as well as proliferation and cytokine secretion in cultures of spleen cells in comparison adoptive transfer of non-modulated DCs. Conversely, transfer of both modulated and non-modulated BMDCs had no effect on immune response of DO11.10 mice. Together, these results show that the treatment with iNOS and Arg inhibitors leads to increased expression of co-stimulatory molecules in DCs, and provides evidences that L-arginine metabolism may be an important therapeutic target for modulating immune responses in inflammatory disorders. Department of Genetics Evolution and Bioagents Institute of Biology University of Campinas UNICAMP Department of Biomedical Science Faculty of Americana FAM Institute of Biosciences Universidade Estadual Paulista UNESP Medical School University of Campinas UNICAMP Institute of Biosciences Universidade Estadual Paulista UNESP

Made available in DSpace on 2018-12-11T16:45:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Introduction Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease. Methods and Results Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-ã. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-ã) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher. Conclusion Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression. Department of Genetics Evolution and Bioagents Institute of Biology State University of Campinas UNICAMP Institute of Biosciences Universidade Estadual Paulista UNESP Faculty of Food Engineering University of Campinas UNICAMP Department of Histology and Embryology Institute of Biology State University of Campinas UNICAMP Department of Biomedical Science Faculty of Americana FAM Institute of Biosciences Universidade Estadual Paulista UNESP FAPESP: 2013/20258-2#2014/086192#2015/09326-1#2014/1670102014/08591-0

A deficiência total ou parcial da enzima denominada lactase, responsável por hidrolisar em glicose e galactose a lactose presente no leite, é popularmente conhecida como intolerância à lactose. No presente trabalho foram revisadas as causas e tratamentos para intolerância à lactose. Foi realizada uma revisão retrospectiva e integrativa da literatura nas bases SciELO, MedLine e PubMed. A intolerância possui três classificações: primária, secundária e congênita. A intolerância ontogenética à lactose ou hipolactasia primária adulta é a forma mais comum. Já a deficiência secundária consiste em um quadro fisiopatológico que tem como consequência a má absorção de lactose. Por fim, a intolerância congênita à lactose é uma deficiência de herança genética, que acomete recém-nascidos nos primeiros dias de vida. Na hipolactasia, o agravamento surge na vida adulta, justamente com perda da função gradativa da enzima que degrada a lactose. Isso ocorre pelo fato de essa enzima, presente e ativa durante a amamentação em mamífero, perder sua função no início do desmame. Em pacientes não intolerantes, essa mesma enzima passa por um processo de mutação, permanecendo ativa ao longo da vida adulta. O tratamento mais comum para pacientes com intolerância à lactose envolve a retirada total ou parcial do leite e seus derivados, já que a supressão total causa alguns danos à nutrição e à manutenção do organismo.

Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary antigens. In the present study, we determined the influence of aging and oral tolerance on the secretion of co-stimulatory molecules by dendritic cells (DC), and on the ability of DC to induce proliferation and cytokine secretion by naive T cells from BALB/c and OVA transgenic (DO11.10) mice. We observed that oral tolerance could be induced in BALB/c mice (N = 5 in each group) of all ages (8, 20, 40, 60, and 80 weeks old), although a decline in specific antibody levels was observed in the sera of both tolerized and im munized mice with advancing age (40 to 80 weeks old). DC obtained from young, adult and middle-aged (8, 20, and 40 weeks old) tolerized mice were less efficient (65, 17 and 20%, respectively) than DC from immunized mice (P < 0.05) in inducing antigen-specific proliferation of naive T cells from both BALB/c and DO11.10 young mice, or in stimulating IFN- γ, IL-4 and IL-10 production. However, TGF-β levels were significantly elevated in co-cultures carried out with DC from tolerant mice (P < 0.05). DC from both immunized and tolerized old and very old (60 and 80 weeks old) mice were equally ineffective in inducing T cell proliferation and cytokine production (P < 0.05). A marked reduction in CD86+ marker expression was observed in DC isolated from both old and tolerized mice (75 and 50%, respectively). The results indicate that the aging process does not interfere with the establishment of oral tolerance in BALB/c mice, but reduces DC functions, probably due to the decline of the expression of the CD86 surface marker.

Background Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. Methods Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts. Results Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages. Conclusions Amongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.

Dendritic cells (DCs), the most important professional antigen-presenting cells (APC), play crucial role in both immunity and tolerance. It is well known that DCs are able to mount immune responses against foreign antigens and simultaneously tolerate self-antigens. Since DCs can be modulated depending on the surrounding microenvironment, they can act as a bridge between innate and adaptive immunity. However, the mechanisms that support this dual role are not entirely clear. Recent studies have shown that DCs can be manipulatedex vivoin order to trigger their tolerogenic profile, what can be a tool to be used in clinical trials aiming the treatment of various diseases and the prevention of transplant rejection. In this sense, the blockage of costimulatory molecules on DC, in the attempt of inhibiting the second signal in the immunological synapse, can be considered as one of the main strategies under development. This review brings an update on current therapies using tolerogenic dendritic cells modulated with costimulatory blockers with the aim of reducing transplant rejection. However, although there are current clinical trials using tolerogenic DC to treat allograft rejection, the actual challenge is to modulate these cells in order to maintain a permanent tolerogenic profile.

In this work, we evaluated the effects of administration of OVA on phenotype and function of intraepithelial lymphocytes (IELs) from small intestine of transgenic (TGN) DO11.10 and wild-type BALB/c mice. While the small intestines from BALB/c presented a well preserved structure, those from TGN showed an inflamed aspect. The ingestion of OVA induced a reduction in the number of IELs in small intestines of TGN, but it did not change the frequencies of CD8+and CD4+T-cell subsets. Administration of OVA via oral + ip increased the frequency of CD103+cells in CD4+T-cell subset in IELs of both BALB/c and TGN mice and elevated its expression in CD8β+T-cell subset in IELs of TGN. The frequency of Foxp3+cells increased in all subsets in IELs of BALB/c treated with OVA; in IELs of TGN, it increased only in CD25+subset. IELs from BALB/c tolerant mice had lower expression of all cytokines studied, whereas those from TGN showed high expression of inflammatory cytokines, especially of IFN-γ, TGF-β, and TNF-α. Overall, our results suggest that the inability of TGN to become tolerant may be related to disorganization and altered proportions of inflammatory/regulatory T cells in its intestinal mucosa.

The limited amount of information on the primary age-related deficiencies in the innate immune system led us to study the production of inducible nitric oxide synthase (iNOS), arginase, and cytokines in macrophages of young (8 weeks old) and old (72 weeks old) female BALB/c mice. We first evaluated iNOS and arginase inducers on peritoneal (PMΦ) and bone marrow-derived (BMMΦ) macrophages of young BALB/c and C57BL/6 mice, and then investigated their effects on macrophages of old mice. Upon stimulation with lipopolysaccharide (LPS), resident and thioglycolate-elicited PMΦ from young mice presented higher iNOS activity than those from old mice (54.4%). However, LPS-stimulated BMMΦ from old mice showed the highest NO levels (50.1%). Identical NO levels were produced by PMΦ and BMMΦ of both young and old mice stimulated with interferon-γ. Arginase activity was higher in resident and elicited PMΦ of young mice stimulated with LPS (48.8 and 32.7%, respectively) and in resident PMΦ stimulated with interleukin (IL)-4 (64%). BMMΦ of old mice, however, showed higher arginase activity after treatment with IL-4 (46.5%). In response to LPS, PMΦ from old mice showed the highest levels of IL-1α (772.3 ± 51.9 pg/mL), whereas, those from young mice produced the highest amounts of tumor necrosis factor (TNF)-α (937.2 ± 132.1 pg/mL). Only TNF-α was expressed in LPS-treated BMMΦ, and cells from old mice showed the highest levels of this cytokine (994.1 ± 49.42 pg/mL). Overall, these results suggest that macrophages from young and old mice respond differently to inflammatory stimuli, depending on the source and maturity of the cell donors.

Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary antigens. In the present study, we determined the influence of aging and oral tolerance on the secretion of co-stimulatory molecules by dendritic cells (DC), and on the ability of DC to induce proliferation and cytokine secretion by naive T cells from BALB/c and OVA transgenic (DO11.10) mice. We observed that oral tolerance could be induced in BALB/c mice (N = 5 in each group) of all ages (8, 20, 40, 60, and 80 weeks old), although a decline in specific antibody levels was observed in the sera of both tolerized and immunized mice with advancing age (40 to 80 weeks old). DC obtained from young, adult and middle-aged (8, 20, and 40 weeks old) tolerized mice were less efficient (65, 17 and 20%, respectively) than DC from immunized mice (P < 0.05) in inducing antigen-specific proliferation of naive T cells from both BALB/c and DO11.10 young mice, or in stimulating IFN-g, IL-4 and IL-10 production. However, TGF-β levels were significantly elevated in co-cultures carried out with DC from tolerant mice (P < 0.05). DC from both immunized and tolerized old and very old (60 and 80 weeks old) mice were equally ineffective in inducing T cell proliferation and cytokine production (P < 0.05). A marked reduction in CD86+ marker expression was observed in DC isolated from both old and tolerized mice (75 and 50%, respectively). The results indicate that the aging process does not interfere with the establishment of oral tolerance in BALB/c mice, but reduces DC functions, probably due to the decline of the expression of the CD86 surface marker.

Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4+ and CD8+ T cells in the spleen increased until 8 weeks of age but there was no change in the CD4+/CD8+ ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFN-gamma: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA-T-cell receptor (OVA-TCR)-specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose-dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naive cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2-dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1- and Th2-antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2 (IL-4 and IL-10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T-cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)-beta secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF-beta as a regulatory cytokine in oral tolerance in the murine model.

Orientador: Wirla Maria da Silva Cunha Tamshiro Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A administração de uma proteína solúvel por via oral é um dos meios mais efetivos de induzir a supressão da resposta imune sistêmica a antígenos protéicos, conhecida como tolerância oral ou de mucosas. No presente estudo, a tolerância oral à ovalbumina (OVA) foi analisada em camundongos BALB/c, selvagens e transgênicos (DO11.10), jovens, adultos, de meia-idade e velhos, utilizando-se protocolos de ingestão forçada (gavagem) ou voluntária (adicionada à água de beber) da proteína, aplicados por tempos variados. Tanto a gavagem como a ingestão voluntária de OVA induziram a tolerância sistêmica em camundongos BALB/c selvagens, de todas as faixas etárias avaliadas, de modo dose-dependente, conforme constatado pela produção reduzida de anticorpos séricos específicos, após o desafio antigênico por via parenteral. Entretanto, o tratamento oral com OVA não produziu o mesmo efeito nos camundongos transgênicos DO11.10, de qualquer das idades examinadas, muito embora esses animais tenham apresentado uma redução significativa no número de células do clonotipo KJ1-26, observada tanto no baço quanto no sangue periférico, após a ingestão da proteína. Nos camundongos BALB/c, o esquema de imunização parenteral com OVA, utilizando o hidróxido de alumínio como adjuvante, gerou preferencialmente a produção de anticorpos da classe IgG1, dependentes de linfócitos Th2, cujos níveis foram significativamente reduzidos pelo pré-tratamento oral com OVA. Nos camundongos transgênicos DO11.10, o mesmo esquema de imunização induziu uma resposta humoral mista, com a produção de anticorpos IgG2a e IgG1, dependentes das citocinas produzidas por linfócitos Th1 e Th2, respectivamente, cujos níveis não foram afetados pela prévia ingestão de OVA. Uma vez que camundongos BALB/c jovens tornaram-se tolerantes pela ingestão voluntária de OVA (4mg/mL, por 7 dias consecutivos), esse estado foi mantido por mais de 44 semanas e pôde ser revertido pela transferência adotiva de 6x10 células esplênicas naive, isoladas de camundongos jovens da linhagem selvagem. Entretanto, nos camundongos transgênicos DO11.10, a transferência adotiva do mesmo número de células esplênicas da linhagem selvagem, seguida por novo tratamento oral com OVA por ingestão voluntária, resultou em níveis ainda mais elevados de anticorpos séricos específicos. As células dendríticas (DCs) de camundongos BALB/c jovens tolerantes à OVA foram menos eficientes em sustentar a proliferação in vitro de células T naive, isoladas tanto de camundongos selvagens como transgênicos, do que as DCs de camundongos BALB/c imunizados apenas por via intraperitoneal (i.p.). As co-culturas de células T naive e DCs de camundongos tolerantes apresentaram níveis significativamente menores de citocinas liberadas por células com perfil Th1 (IL-2 e IFN-g) e Th2 (IL-4 e IL-10) do que as culturas realizadas na presença de DCs obtidas de camundongos BALB/c imunizados com OVA por via i.p. Por outro lado, as DCs de camundongos transgênicos DO11.10, alimentados ou não com OVA e desafiados com o antígeno por via i.p., foram igualmente eficientes em induzir a proliferação de células T específicas, assim como a secreção das citocinas IL-2, IFN-g, IL-4 e IL-10. Entretanto, a secreção de TGF- 'beta' foi significativamente mais elevada nos sobrenadantes de cultura de células T naive co-cultivadas com as DCs de camundongos BALB/c tolerantes do que com as DCs de camundongos DO11.10 ou dos BALB/c imunizados por via parenteral. Os resultados obtidos no presente trabalho indicam que, em um sistema imune integro, a tolerância às proteínas da dieta se estabelece às custas das interações entre células apresentadoras de antígeno e células T, nas quais as proporções celulares e as moléculas estimuladoras resultantes estão em delicado equilíbrio. Alterações nesse balanço podem provocar, como observado no camundongo DO11.10, o desencadeamento de respostas imunes dirigidas contra o antígeno relacionado com a população linfocitária predominante, podendo resultar em doenças inflamatórias e alérgicas. Estudos mais aprofundados são necessários para entender a dinâmica dessas populações celulares na indução e manutenção da tolerância, para eventualmente utilizar esse conhecimento na reversão de manifestações autoimunes ou de hipersensibilidades ou ainda na prevenção da rejeição de transplantes Abstract: The oral administration of soluble protein is one of the most effective ways of inducing the suppression of the systemic immune response to proteic antigens, otherwise known as oral tolerance. In this study, oral tolerance to ovalbumin (OVA) was analyzed in wild type BALB/c and DO11.10 transgenic mice of four ages: young, young adult, middle-aged and old, using protocols of forced ingestion (gavage) and continuous feeding (OVA added to the drinking water). Both gavage and voluntary ingestion (continuous feeding) of OVA induced dosedependent systemic tolerance in the BALB/c mice of all ages evaluated, as indicated by reduced production of specific antibodies in the sera after antigenic challenge. However, neither treatment affected the DO11.10 transgenic mice, since these present a significant reduction in the number of clonotype KJ1-26 cells after the ingestion of the protein, as observed in the spleen and peripheral blood. In BALB/c mice, a schedule of parenteral immunization with OVA using alumen as adjuvant preferentially leads to IgG1 antibody production, which is dependent on Th2 lymphocytes, with levels significantly reduced by oral pre-treatment with OVA. In DO11.10 transgenic mice, however, the same immunization schedule induced not only the production of the IgG1 antibodies related to cytokines produced by Th2 lymphocytes, but also IgG2a, related to Th1-producing cytokines, which had not been affected in the BALB/c mice. Once young BALB/c mice became tolerant by voluntary ingestion of OVA (4mg/mL, for 7 consecutive days), the state of tolerance was maintained for more than 44 weeks, although reversed by adoptive transference of 6 x 10 (x6) naive spleen cells, isolated from young wild type mice However, in the DO11.10 transgenic mice, the adoptive transfer of the same number of BALB/c spleen cells had to be followed by a new oral treatment with OVA by continuous feeding; this resulted in higher specific antibody levels in the sera. The dendritic cells (DCs) from tolerant young BALB/c mice were less efficient in supporting in vitro proliferation of isolated naive T cells (whether from BALB/c or DO11.10 mice) than were the DCs from BALB/c mice immunized intraperitoneally (i.p.). Cocultures of naive T cells and DCs from tolerant BALB/c mice revealed lower levels of cytokines from Th1 (IL-2 and IFN -) and Th2 (IL-4 and IL-10) than did those including DCs from i.p. immunized individuals. On the other hand, the DCs from DO11.10 transgenic mice, whether or not they had been fed with OVA, challenged intraperitoneally with the antigen were all equally efficient in the induction of the proliferation of specific T cells, as well as the secretion of IL-2, IFN -g, IL-4 and IL-10. However, TGF- 'beta' secretion was significantly higher in the supernatants of T cell cultures from naive BALB/c mice co-cultivated with DCs from tolerant BALB/c mice than in the presence of DCs from DO11.10 or immunized BALB/c mice. The results indicate that tolerance to dietary proteins is dependent of interactions between antigen presenting cells and T cells, with the ratio of these cells and the resultant stimulatory molecules in delicate balance. As observed in DO11.10 mice, alterations in this balance can cause immune responses against the antigen and generate inflammatory and allergic illnesses. Other studies are necessary to understand the dynamics of these cell populations in the induction and maintenance of tolerance, in the hopes that autoimmune and hypersensitivity manifestations can be reversed and transplant rejection prevented. Doutorado Imunologia Doutor em Genética e Biologia Molecular

The anti-inflammatory properties of some flavonoids have been attributed to their ability to inhibit the production of NO by activated macrophages. Soybean cotyledons accumulate certain flavonoids following elicitation with an extract of the fungal pathogen Diaporthe phaseolorum f. sp. meridionalis (Dpm). Sodium nitroprusside (SNP), a nitric oxide donor, can substitute for Dpm in inducing flavonoid production. In this study, we investigated the effect of flavonoid-containing diffusates obtained from Dpm- and SNP-elicited soybean cotyledons on NO production by lipopolysaccharide (LPS)- and LPS plus interferon-γ (IFNγ)-activated murine macrophages. Significant inhibition of NO production, measured as nitrite formation, was observed when macrophages were activated in the presence of soybean diffusates from Dpm- or SNP-elicited cotyledons. This inhibition was dependent on the duration of exposure to the elicitor. Daidzein, genistein, luteolin and apigenin, the main flavonoids present in diffusates of elicited cotyledons, suppressed the NO production by LPS + IFNγ activated macrophages in a concentration-dependent manner, with IC50 values of 81.4 μM, 34.5 μM, 38.6 μM and 10.4 μM respectively. For macrophages activated with LPS alone, the IC50 values were 40.0 μM, 16.6 μM, 10.4 μM and 2.8 μM, respectively. Western blot analysis showed that iNOS expression was not affected by daidzein, was reduced by genistein, and was abolished by apigenin, luteolin and Dpm- and SNP-soybean diffusates at concentrations that significantly inhibited NO production by activated macrophages. These results suggest that the suppressive effect of flavonoids on iNOS expression could account for the potent inhibitory effect of Dpm- and SNP-diffusates on NO production by activated macrophages. Since the physiological concentration of flavonoids in plants is normally low, the treatment of soybean tissues with SNP may provide a simple method for substantially increasing the concentration of metabolites that are beneficial for the treatment of chronic inflammatory diseases associated with NO production.

Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3(+) and IL-10(+) cells were higher, whereas IFNγ(+) cells and IL-17(+) cells were lower, among gated CD4(+) spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.