OLIVEIRA, Jefferson Soares de. Caracterização bioquímica e atividade citotóxica in vitro e antitumoral in vivo de proteínas do látex de Calotropis procera. 2011. 149 f. : Tese (Doutorado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-20T14:23:08Z No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:35:56Z (GMT) No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) / Made available in DSpace on 2016-08-02T20:35:56Z (GMT). No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) Previous issue date: 2011 / Latex of Calotropis procera was described as a source of pharmacologically active proteins such as anti-inflammatory and analgesic activities. This study evaluated the cytotoxic activity in vitro of proteins (LP) recovered from the latex of the medicinal plant C. procera against human cancer cells and the in vivo growth inhibition of Sarcoma 180. LP exhibited significant cytotoxicity for cell lines with IC50 values ranging from 0.11 to 1.36 µg/ml for tested cell lines (HL-60, SF295, HCT-8 and MDA-MB-435). There were no visible effects on the viability or morphology of healthy mononuclear cells exposed to PL (10 µg / ml) for 72 h, showing that PL was selective for malignant cells. Fractionation of PL by ion exchange chromatography (pH 5.0) gave rise to three new protein fractions (PI, PII and PIII) and almost all cytotoxicity present in PL was retained in fraction PI. The cytotoxic effects of PL and PI were diminished when pre-treated with pronase or 2-mercaptoethanol, reinforcing the protein nature of active molecules. PI was absent on cysteine protease activity, indicating that this enzyme abundantly found in PL is not involved in cytotoxicity. Mechanistic studies of LP cytotoxicity using HL-60 cells revealed that PL induces apoptosis probably due to changes in DNA topology since PL interfered in the activity of topoisomerase I. The cytotoxic activity present in PI seems to be performed by the synergic action of different proteins. This hypothesis is suggested since PI subjected to gel filtration chromatography produced distinct protein peaks that shared cytotoxic activity, although with lower extent than PI. Studies on growth inhibition of Sarcoma 180 showed that animals treated with PL by oral (10 or 20 mg/kg) or intraperitoneal (2 or 5 mg/kg) rout reduced tumor growth significantly (up 51.83%, po) and increased life span of transplanted animals for up to four days. The inhibitory activity of tumor growth was lost when the LP was subjected to proteolysis, acidic treatment or collected in iodoacetamide. On the other hand, LP maintained its in vivo activity after heat treatment, suggesting that thermo stable proteins are involved in the suppression of tumor growth. Biochemical parameters such as the enzymatic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the content of urea in serum were not affected in animals treated with LP. Treatment of animals with LP induced increasing of leukocyte numbers and protected from leukopenia induced by 5-FU administration. In addition, no significant changes in the histopathology of liver of animals treated with oral LP were seen. In vivo antitumor activity was retained in the PII and this activity was observed even when animals received a single dose of PII. It seems that the in vivo action of latex proteins is related to an immunestimulant event and not to the cytotoxic action of protein on cells Sarcoma 180. PII-3, recovered after PII fractionation on ion exchange column at pH 6.0, retained the tumor growth inhibition activity found in PII. PII-3 was shown to possess cysteine proteinase and papain inhibitor activities; however is not completely clear weather this molecules are involved in the antitumor activity. This study confirms the pharmacological potential of latex proteins from C. procera to control the development of tumor cells. / O látex de Calotropis procera foi descrito como uma fonte de proteínas farmacologicamente ativas como atividade antiinflamatória e analgésica. O presente trabalho avaliou a atividade citotóxica in vitro das proteínas (PL) recuperadas do látex da planta medicinal C. procera contra células de câncer humano e a inibição do crescimento do Sarcoma 180 transplantado em camundongos. PL apresentou significante citotoxicidade para as linhagens celulares com valores de IC50 variando entre 0,11 a 1,36 µg/ml para as linhagens celulares testadas (HL-60, SF295, HCT-8 e MDA-MB-435). Não foram observados efeitos visíveis sobre a viabilidade ou a morfologia de células mononucleares saudáveis expostas a PL (10 µg / ml) por 72 h, mostrando que PL apresentou seletividade para células tumorais. O fracionamento de PL por cromatografia de troca iônica (pH 5,0) deu origem a três novas frações (PI, PII e PIII) e quase toda citotoxicidade presente em PL ficou retida na fração PI. Os efeitos citotóxicos de PL e PI foram diminuídos quando previamente tratados com pronase, ou 2-mercaptoetanol, sugerindo uma natureza protéica de moléculas ativas. PI não apresentou atividade de proteinase cisteínica, indicando que esta enzima, encontrada em abundância em PL, não está envolvida na citotoxicidade. Estudos do mecanismo da ação citotóxica de PL utilizando células HL-60 revelou que PL induz apoptose celular provavelmente devido a alterações na topologia de DNA, já que PL interferiu na atividade de topoisomerase I. A atividade citotóxica presente em PI parece ser desempenhada pela ação combinada de diferentes proteínas uma vez que PI submetida à cromatografia de filtração em gel gerou picos protéicos distintos que compartilharam atividade citotóxica, embora com menor potência que PI. Estudo de inibição do crescimento do Sarcoma 180 revelou que animais tratados com PL por via oral (10 or 20 mg/kg) ou intraperitoneal (2 or 5 mg/kg) reduziram de modo significativo o crescimento do tumor (em até 51,83%; v.o.) e prolongou o tempo de sobrevivência dos animais transplantados por até quatro dias. A atividade inibitória do crescimento do tumor foi perdida quando a fração PL foi submetida à proteólise, tratamento ácido ou com iodoacetamida. No entanto, PL conservou a sua atividade in vivo após o tratamento térmico, sugerindo que proteínas termoestáveis estão envolvidas na supressão do crescimento tumoral. Os parâmetros bioquímicos, como a atividade enzimática da aspartato aminotransferase (AST) e alanina aminotransferase (ALT) e o teor de uréia no soro, não foram afetados nos animais tratados com PL. PL induziu aumento no número de leucócitos de animais tratados e ainda eliminou completamente a leucopenia induzida pela administração do 5-FU. Em adição, não foram observadas mudanças na histopatologia do fígado de animais tratados com PL por via oral. Atividade antitumoral in vivo ficou retida no PII e esta atividade foi observada mesmo quando animais transplantados receberam uma única dose de PII sugerindo que a ação in vivo de proteínas do látex está relacionada a um evento imunoestimunate de proteínas e não à ação citotóxica sobre as células do Sarcoma 180. PII-3, obtido após fracionamento de PII em coluna de troca iônica em pH 6,0 reteve a atividade de inibição do crescimento tumoral de PII. Esta fração possui atividade de proteinase cisteínica e atividade de inibidor de papaína, porém não é completamente claro o envolvimento dessas moléculas na atividade in vivo. Este estudo confirma o potencial farmacológico das proteínas do látex de C. procera para controlar o desenvolvimento de células tumorais.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No presente trabalho é descrita a obtenção de hidrazonas derivadas de isoniazida e de seus complexos de cobre(II) e gálio(III) candidatos a protótipos de fármacos antituberculose e antitumoral. Para investigar o efeito da modificação química sobre as bioatividades do fármaco isoniazida, foram preparados cinco derivados hidrazônicos: 2-piridinocarboxaldeído isonicotinoil hidrazona (HPCIH, 1), 2-acetilpiridina isonicotinoil hidrazona (HAPIH, 2), 2-benzoilpiridina isonicotinoil hidrazona (HBPIH, 3), 2-piridinoformamida isonicotinoil hidrazona (HPAmIH, 4) e 2-pirazinoformamida isonicotinoil hidrazona (HPzAmIH, 5), sendo o composto HPAmIH (4) inédito. Análises de ponto de fusão, espectroscopia de infravermelho (IV), espectrometria de massas, ressonância magnética nuclear (RMN), análise elementar e termogravimetria confirmaram a obtenção e pureza das hidrazonas. Foi determinada ainda a estrutura de HPCIH (1) por difração de raios X de monocristal. Essas moléculas foram efetivas em inibir o crescimento de cepas de micobactérias Mycobacterium tuberculosis H37Rv (ATCC 27294) nas concentrações testadas, com exceção de HPzAmIH (5). As hidrazonas HAPIH (2) e HBPIH (3) foram os compostos orgânicos mais ativos (concentração inibitória mínima, CIM = 0,625 g/mL), apresentando atividade antimicobacteriana apenas duas vezes inferior à do fármaco isoniazida.Quanto à ação contra células tumorais, as hidrazonas HAPIH (2) e HBPIH (3) foram as mais potentes contra as linhagens OVCAR-8 (tumor de ovário - humano), HCT-116 (tumor de cólon - humano) e SF-295 (glioblastoma humano), com inibições de 34,98 a 98,63% do crescimento celular, na concentração de 5 g/mL, enquanto que a isoniazida não foi efetiva contra as linhagens estudadas. Para avaliar o efeito da coordenação a metais sobre a atividade farmacológica das hidrazonas, foram sintetizados os complexos de cobre(II) e gálio(III), sendo todos inéditos: [Cu(HPCIH)Cl2]∙H2O (6), [Cu(HAPIH)Cl2]∙H2O (7), [Cu2(HBPIH)2Cl2]Cl2∙4H2O(8), [Cu(HPAmIH)Cl2]∙H2O (9), [Cu(HPzAmIH)Cl2]∙H2O (10), [Ga(HPCIH)2](NO3)32H2O (11), [Ga(HAPIH)(APIH)](NO3)22H2O (12), [Ga(HPAmIH)(PAmIH)](NO3)22H2O(13) e [Ga(HPzAmIH)(PzAmIH)](NO3)2H2O (14). Os complexos foram caracterizados por espectroscopia de IV, análise elementar, condutivimetria, RMN e espectroscopia eletrônica. Em geral, os complexos também demonstraram ação contra M. tuberculosis, sendo que apenas para 6, 9, 10 e 14 foi verificada melhor atividade em relação às hidrazonas livres. Os complexos metálicos foram tanto quanto ou mais ativos contra as células tumorais OVCAR-8, HCT-116 e SF-295 do que as hidrazonas livres. Merecem destaque os complexos 79 e 12, que apresentaram inibição de crescimento celular de 72,2100%, na concentração de 5 g/mL. Os resultados demonstram portanto que em geral os compostos 114 são menos ativos do que a isoniazida contra M. tuberculosis, enquanto que a modificação química do fármaco, formando-se hidrazonas com posterior complexação cobre(II) e gálio(III) constituíram uma estratégia interessante na obtenção de compostos mais potentes contra células tumorais / In this work we describe the synthesis of isoniazide-derived hydrazones and their copper(II) and gallium(III) complexes candidates for drug prototypes to treat antituberculosis and cancer. Five hidrazone derivatives have been prepared in order to investigate the effect of chemical modification on the bioactivities of the drug isoniazid: 2-pyridinecarboxaldehyde isonicotinoyl hydrazone (HPCIH, 1), 2-acetylpyridine isonicotinoyl hydrazone (HAPIH, 2), 2-benzoylpyridine isonicotinoyl hydrazone (HBPIH, 3), 2-pyridineformamide isonicotinoyl hydrazone (HPAmIH, 4) and pyrazineformamide 2-isonicotinoyl hydrazone (HPzAmIH, 5), being HPAmIH (4) unpublished. Melting point, infrared spectroscopy (IR), mass spectrometry, nuclear magnetic resonance (NMR), elemental analysis and thermogravimetric analysis has confirmed the formation of hydrazones as well as its purity. All compounds have been effective in inhibiting the growth of Mycobacterium tuberculosis H37Rv (ATCC 27294) mycobacteria strains in the tested concentrations, except HPzAmIH (5). The hydrazones HAPIH (2) and HBPIH (3) have been the most active compounds (minimum inhibitory concentration, MIC = 0.625 g/mL), which have presented the antimycobacterial activity only two times lower than isoniazid drug. In relation to the action against tumor cells, the hydrazones HAPIH (2) and HBPIH (3) have been the most potent compounds against the cell lines OVCAR-8 (ovarian tumor - human), HCT-116 (colon tumor - human) and SF-295 (glioblastoma - human), with inhibitions from 34.98 to 98.63% of cellular growth at a concentration of 5 g/mL. Isoniazid, in turn, hasnt been effective against all cell lines studied. To evaluate the effect of coordinating the metal on the pharmacological activity of hydrazones, complexes of copper (II) and gallium (III) have been synthesized, being all novel compounds: [Cu(HPCIH)Cl2]∙H2O (6), [Cu(HAPIH)Cl2]∙H2O (7), [Cu2(HBPIH)2Cl2]Cl2∙4H2O(8), [Cu(HPAmIH)Cl2]∙H2O (9), [Cu(HPzAmIH)Cl2]∙H2O (10), [Ga(HPCIH)2](NO3)32H2O (11), [Ga(HAPIH)(APIH)](NO3)22H2O (12), [Ga(HPAmIH)(PAmIH)](NO3)22H2O (13) and [Ga(HPzAmIH)(PzAmIH)](NO3)2H2O (14).The complexes have been characterized by IR spectroscopy, NMR, elemental analysis, conductivimetry and electron spectroscopy. In general, these complexes have also shown action against M. tuberculosis, whereas only 6, 9, 10 and 14 have demonstrated better activity than the free hydrazones. The metal complexes have been equally or more active against human the cancer cell lines OVCAR-8, HCT-116 and SF-295 than the free hydrazones. The complexes 79 and 12 deserve to be highlighted, which have shown 72.2100% inhibition of cell growth in the 5 g/mL concentration. The results therefore demonstrate that in general compounds 114 are less active than isoniazid against M. tuberculosis, while chemical modification of the drug, forming hydrazones and subsequent complexation with the copper (II) and gallium(III) metal ions have been an interesting strategy to obtain compounds more potent against tumor cells

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which causes acute infectious mononucleosis and is etiologically associated with malignant lymphoproliferative disorders including Burkitt's lymphoma, nasopharyngeal carcinoma, B-cell lymphomas in immunocompromised hosts, Hodgkin's disease, T cell lymphomas, and smooth muscle tumors in allograft recipients. The medical significance of EBV is underscored by its potent growth transforming effects on human B-lymphocytes in-vitro and the potentially oncogenic consequences of infection in-vivo. The majority of EBV-associated malignancies occur in the setting of chronic infection and strong virus-specific humoral immunity, suggesting that cellular immunity is primarily responsible for preventing the outgrowth of EBV-transformed B cells in-vivo. Similarly, primary EBV infection in adolescents and adults stimulates an intense cytotoxic-T-lymphocyte (CTL) response which coincides with a marked reduction in the number of infected B cells in the peripheral blood. Evidence of previous EBV infection can be confirmed by the presence of EBV-specific, HLA-restricted memory T cells in the peripheral blood which inhibit the outgrowth of newly EBV-transformed B cells and efficiently lyse established autologous B-lymphoblastoid cell lines. Worldwide, EBV is responsible for substantial morbidity, comparable to measles, mumps and hepatitis virus, for which vaccines exists. Accordingly, the potential public health impact of an EBV vaccine has reinforced our efforts to identify the immunodominant virus-encoded T-cell epitopes which stimulate naive CTL effectors during acute infection and maintain memory CTL surveillance during convalescence. The EBV-encoded antigens against which the memory CTL response is directed have been partially defined, and include most of the EBV latent proteins (EBNA-2, 3a, 3b, 3c, LP, and LMP-l, 2a, 2b) consistently expressed by in-vitro EBV-transformed B lymphocytes (type-III latency). Importantly, all EBV-associated malignancies express EBNA-1, and as yet no EBNA-1-specific memory CTL have been convincingly demonstrated. Additionally, many EBV-specific CTL lines and clones have been described which do not recognize any of the known latent proteins or other EBV protein antigens tested thus far. Thus while much is known about CTL-mediated immunity against EBV, our knowledge of EBV-derived CTL epitopes remains incomplete. In contrast to the EBV-specific memory CTL response, very little is known about the source of viral epitopes recognized during the primary CTL response to EBV. In this regard, acute infectious mononucleosis represents an ideal model system to study virus-specific, cell-mediated immunity. Acute IM is a self-limited illness characterized by the appearance of "atypical" lymphocytes (CD3+/CD8+/HLADR+), including both virus-specific and alloreactive CTL, which undoubtedly contribute to virus elimination and provide CTL precursors for life-long immunity to EBV. Like other herpesvirus, EBV can undergo either lytic or latent cycle replication. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivoand maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivoactivated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM. Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence. Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memoryCTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. CTL from 7 of the 35 acute IM patients evaluated recognized EBNA1 expressing targets, and in 4 of these 7 patients, EBNA1 was an immunodominant antigen. Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The CD11b "memory marker" reached peaked levels on the first sample day for all patients and gradually declined to baseline levels over a period of several months. In contrast, the CD11b marker was quickly shed from in vitropropogated CTL, over a period of 5-10 days. Target cell lysis by in-vivoactivated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8. Our detection of only one EBNA1-specific memory CTL response among the 32 patients tested supports the theory that latently infected B cells in-vivo, expressing only EBNA1, escape CTL recogition and thus might serve as a reservoir for viral persistence and/or reactivation. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL. The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin's lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. Details about the viral gene products which are involved in stimulating a broadly protective, virus-specific immune response in a large number of individuals is fundamental to the design of an effective EBV vaccine. Since the presence of activated CD8+ T cells correlates with the rapid decline of EBV infected B cells in the peripheral blood, a concise description of the EBV-specific CTL response in the setting of acute infection will be necessary for the rational design of an effective acute IM vaccine. Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.

Les hypersensibilités retardées (HSR) cutanées sont hétérogènes, à la fois par la nature des mécanismes impliqués (allergiques versus non allergiques) mais aussi par les différents degrés de sévérité rencontrés. Seules les HSR allergiques sont dues à la présence de lymphocytes T (LT), mal caractérisés chez l'homme. Le but de ce travail est d'étudier la contribution des LT CD8 cytotoxiques dans le développement et la sévérité des HSR cutanées chez l'homme, à partir de deux pathologies fréquentes : les toxidermies aux béta lactamines et l'eczéma allergique de contact à la para-phénylènediamine (PPD). Tout d'abord, la présence de LT spécifiques de médicament au sein des toxidermies aux béta lactamines a été recherchée in vivo et in vitro. Nous avons montré que les HSR sévères étaient plus souvent d'origine allergique que les HSR bénignes. Nous avons ensuite caractérisé le rôle des LT CD8 dans les HSR allergiques. Dans les toxidermies bénignes à l'amoxicilline, l'étude de la cinétique de recrutement des LT au niveau cutané ainsi que l'analyse des LT spécifiques du sang circulant ont permis de mettre en évidence le rôle essentiel des LT CD8 cytotoxiques dans l'initiation de ces réactions. Ensuite, dans l'eczéma allergique de contact à la PPD, un recrutement épidermique précoce des LT CD8 associés à des marqueurs de cytotoxicité, a été retrouvé, corrélé avec la sévérité des lésions. Ces résultats ont été confortés par ceux obtenus dans un modèle pré-clinique d'HSR allergique à la PPD chez la souris. En conclusion, ce travail montre que les LT CD8 cytotoxiques pourraient être les principales cellules effectrices des HSR cutanées allergiques chez l'homme / Skin delayed hypersensitivity (DHS) are heterogeneous, by the nature of the mechanisms involved (allergic versus non allergic) and also by their different degrees of severity. Only allergic DHS is due to T cells, poorly characterized in humans. The aim of this work is to study the contribution of cytotoxic CD8 T cells in the development and severity of skin DHS in humans, induced by two common diseases: cutaneous adverse drug reactions to beta lactam antibiotics and allergic contact dermatitis to para-phenylenediamine (PPD). First, the presence of drug specific T cells in cutaneous adverse drug reactions to beta lactams was investigated in vivo and in vitro. We showed that severe DHS were more often allergic than benign DHS. Then, we characterized the role of CD8 T cells in allergic DHS. In benign cutaneous adverse drug reactions to amoxicillin, the study of the kinetics of skin T cell recruitment as well as the analysis of circulating specific T cells highlight the essential role of cytotoxic CD8 T cells in the initiation phase of these reactions. In allergic contact dermatitis to PPD, early recruitment of epidermal CD8 T cells associated with cytotoxic markers was found, correlated with the severity of lesions. These results were supported by those obtained in a mouse model of allergic contact dermatitis to PPD. In conclusion, this work showed that cytotoxic CD8 T cells could be the main effector cells of allergic skin DHS in humans

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-22T15:04:46Z No. of bitstreams: 1 DISS_2014_Samantha Soares Ourives.pdf: 1603897 bytes, checksum: 16aacb8659dc1e9902e5038014c2c0ac (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-26T14:26:40Z (GMT) No. of bitstreams: 1 DISS_2014_Samantha Soares Ourives.pdf: 1603897 bytes, checksum: 16aacb8659dc1e9902e5038014c2c0ac (MD5) / Made available in DSpace on 2017-09-26T14:26:40Z (GMT). No. of bitstreams: 1 DISS_2014_Samantha Soares Ourives.pdf: 1603897 bytes, checksum: 16aacb8659dc1e9902e5038014c2c0ac (MD5) Previous issue date: 2014-04-25 / CNPq / A malária é uma das doenças parasitárias de maior importância global e é responsável pelas principais causas de morbidade e mortalidade nas áreas tropicais e subtropicais do mundo. Apesar dos esforços para o controle da infecção em diferentes áreas endêmicas, a malária continua em expansão, e as medidas tradicionais de controle da transmissão são pouco eficazes. A resposta imune na malária é complexa, e os mecanismos de ativação e regulação de linfócitos T e suas citocinas ainda são pouco compreendidos. O objetivo deste trabalho foi avaliar a correlação da parasitemia com o número de plaquetas e leucócitos, e identificar e quantificar as subpopulações específicas de células Th1, Th2, Th17 e Treg, durante a infecção por P. vivax. Avaliando a parasitemia e o número de plaquetas, foi verificado que existe correlação negativa (p<0,0005) entre esses parâmetros e que, dependendo da quantidade de parasitas, os pacientes com malária vivax apresentavam um maior grau de plaquetopenia (p<0,0001). Avaliando o número de parasitas e de leucócitos totais, foi verificada ausência de correlação entre esses parâmetros em pacientes com malária vivax. Além disso, também não foi detectada alteração no número de leucócitos totais quando comparado aos controles sadios. Posteriormente, foi realizada, por meio de citometria de fluxo, a identificação e quantificação das subpopulações de células T: Th1 (CD3+CD4+IFN-γ+), Th2 (CD3+CD4+IL4+), Th17 (CD3+CD4+IL-17+), Treg (CD4+CD25+CD127-) e citotóxica (CD3+CD8+), em pacientes com malária vivax e em controles sadios após cultura de linfócitos previamente isolados do sangue periférico, para verificar a alteração do número dessas subpopulações de linfócitos pela infecção por P. vivax. O percentual da citocina IL-10 também foi avaliado nas células Treg (CD4+CD25+CD127-IL-10+). Os indivíduos infectados por P. vivax apresentaram percentual de células T citotóxica e Th1 aumentadas. Já o percentual de células Th2, Th17 e Tregs não apresentaram diferenças entre os grupos. Porém, o percentual de células Treg que produziam a citocina IL-10 estava aumentado em pacientes com malária vivax, quando comparado aos controles sadios. Finalmente, a avaliação do número de células T CD4+ e T CD8+, indicou que não houve diferenças entre a proporção desses linfócitos em controles sadios e nem durante o processo infeccioso induzido por P. vivax. Em conclusão, pacientes com malária vivax apresentam um aumento no número de células T citotóxicas, Th1 e de células Treg (CD4+CD25+CD127-) produtoras de interleucina-10, indicando que a infecção por P. vivax ativa células específicas que podem participar na imunorregulação contra esse parasita. / Malaria is a parasitic disease of major global importance and is responsible for leading causes of morbidity and mortality in tropical and subtropical areas of the world. Despite efforts to control the infection in different endemic areas, malaria continues to expand, and the traditional transmission control measures are ineffective. The immune response in malaria is complex, and the mechanisms of activation and regulation of T lymphocytes and their cytokines are still poorly understood. The objective of this study was to evaluate the correlation of parasitemia with the number of platelets and leukocytes, and identify and quantify specific subpopulations of Th1, Th2, Th17 and Treg cells in infection by P. vivax. Evaluating the parasitemia and the number of platelets was verified that there is a negative correlation (p<0,0005) between these parameters and that, dependent of the number of parasites, vivax malaria patients showed higher degree of thrombocytopenia (p<0,0001). Evaluating the number of parasites and total leukocytes was observed no correlation between these parameters in patients with vivax malaria. Moreover, it was also not detected change in the number of total leukocytes when compared to healthy controls. Subsequently, was performed by flow cytometry, identification and quantification of T cell subsets: Th1 (CD3+CD4+IFN-γ+), Th2 (CD3+CD4+IL4+), Th17 (CD3+CD4+IL-17+), Treg (CD4+CD25+CD127-) and cytotoxic (CD3+CD8+) in patients with vivax malaria and healthy controls after lymphocyte culture previously isolated from peripheral blood, to verify the change in the number of these subpopulations lymphocytes by infection with P. vivax. The percentage of IL-10 was also evaluated on Treg cells (CD4+CD25+CD127+IL-10). Individuals infected with P. vivax showed increased percentage of Th1 and cytotoxic T cells. The percentage of Th2, Th17 and Treg cells did not differ between groups. However, the percentage of Treg cells that produce IL-10 cytokine was increased in patients with vivax malaria compared to healthy controls. Finally, the evaluation of the number of CD4+ T and CD8+ T cells indicated that there were no differences between the proportion of these lymphocytes in healthy controls or during the infectious process induced by P. vivax. In conclusion, vivax malaria patients show an increase in the number of cytotoxic T cell, Th1 and Treg cells (CD4+CD25+CD127-) producers of interleukin-10, indicating that infection with P. vivax activate specific cells which can participate in the immunoregulation against this parasite.

Made available in DSpace on 2014-12-17T14:03:39Z (GMT). No. of bitstreams: 1 RaquelHBA_DISSERT.pdf: 1772501 bytes, checksum: 51cea5d11ac1524fb197874e85c15f64 (MD5) Previous issue date: 2012-07-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents / A heparina ? um agente farmac?utico amplamente utilizado em medicina devido ao seu potente efeito anticoagulante. Al?m disso, tem a capacidade de inibir a prolifera??o, invas?o e ades?o de c?lulas cancerosas ao endot?lio vascular. No entanto, a sua aplicabilidade cl?nica pode ser comprometida por efeitos secund?rios tais como hemorragia. Assim, a busca de compostos naturais com baixo risco hemorr?gico e poss?vel aplicabilidade terap?utica tem sido alvo de v?rios grupos de pesquisa. A partir desta perspectiva, este estudo visa avaliar as atividades hemorr?gica, anticoagulante e efeito citot?xico para as diferentes linhagens de c?lulas tumorais (HeLa, B16-F10, HepG2, HS-5,) e c?lulas de fibroblastos (3T3) proporcionadas pelo composto tipo heparina obtido do caranguejo Chaceon fenneri. Dessa forma, o composto foi purificado ap?s prote?lise, cromatografia de troca i?nica e fracionamento com acetona, e caracterizado por eletroforese em gel de agarose e degrada??o enzim?tica. O composto em estudo mostrou comportamento eletrofor?tico semelhante ? heparina de mam?fero, e alta raz?o de propor??o de dissacar?deos trissulfatado / N-acetilado. Al?m disso, o composto apresentou baixa atividade anticoagulante in vitro usando aPTT e um efeito hemorr?gico menor quando comparado com heparina de mam?fero. O composto tipo heparina obtido do caranguejo Chaceon fenneri mostrou efeito citot?xico significativo (p <0,001) em c?lulas linhagens de c?lulas tumorais HeLa, HepG2 e B16-F10 com valores de IC50 de 1000 ug / mL, ap?s a incuba??o durante 72 horas. Para avaliar a influ?ncia do composto sobre o ciclo celular em c?lulas HeLa, foi realizada uma an?lise por citometria de fluxo. Os resultados desta an?lise mostraram que a influ?ncia do composto sobre o ciclo celular aumenta a fase S e diminui a fase G2. Assim, essas propriedades do composto tipo heparina obtido do caranguejo Chaceon fenneri sugerem este composto como um agente terap?utico em potencial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No presente trabalho é descrita a obtenção de hidrazonas derivadas de isoniazida e de seus complexos de cobre(II) e gálio(III) candidatos a protótipos de fármacos antituberculose e antitumoral. Para investigar o efeito da modificação química sobre as bioatividades do fármaco isoniazida, foram preparados cinco derivados hidrazônicos: 2-piridinocarboxaldeído isonicotinoil hidrazona (HPCIH, 1), 2-acetilpiridina isonicotinoil hidrazona (HAPIH, 2), 2-benzoilpiridina isonicotinoil hidrazona (HBPIH, 3), 2-piridinoformamida isonicotinoil hidrazona (HPAmIH, 4) e 2-pirazinoformamida isonicotinoil hidrazona (HPzAmIH, 5), sendo o composto HPAmIH (4) inédito. Análises de ponto de fusão, espectroscopia de infravermelho (IV), espectrometria de massas, ressonância magnética nuclear (RMN), análise elementar e termogravimetria confirmaram a obtenção e pureza das hidrazonas. Foi determinada ainda a estrutura de HPCIH (1) por difração de raios X de monocristal. Essas moléculas foram efetivas em inibir o crescimento de cepas de micobactérias Mycobacterium tuberculosis H37Rv (ATCC 27294) nas concentrações testadas, com exceção de HPzAmIH (5). As hidrazonas HAPIH (2) e HBPIH (3) foram os compostos orgânicos mais ativos (concentração inibitória mínima, CIM = 0,625 &#61549;g/mL), apresentando atividade antimicobacteriana apenas duas vezes inferior à do fármaco isoniazida.Quanto à ação contra células tumorais, as hidrazonas HAPIH (2) e HBPIH (3) foram as mais potentes contra as linhagens OVCAR-8 (tumor de ovário - humano), HCT-116 (tumor de cólon - humano) e SF-295 (glioblastoma humano), com inibições de 34,98 a 98,63% do crescimento celular, na concentração de 5 &#61549;g/mL, enquanto que a isoniazida não foi efetiva contra as linhagens estudadas. Para avaliar o efeito da coordenação a metais sobre a atividade farmacológica das hidrazonas, foram sintetizados os complexos de cobre(II) e gálio(III), sendo todos inéditos: [Cu(HPCIH)Cl2]&#8729;H2O (6), [Cu(HAPIH)Cl2]&#8729;H2O (7), [Cu2(HBPIH)2Cl2]Cl2&#8729;4H2O(8), [Cu(HPAmIH)Cl2]&#8729;H2O (9), [Cu(HPzAmIH)Cl2]&#8729;H2O (10), [Ga(HPCIH)2](NO3)3&#61655;2H2O (11), [Ga(HAPIH)(APIH)](NO3)2&#61655;2H2O (12), [Ga(HPAmIH)(PAmIH)](NO3)2&#61655;2H2O(13) e [Ga(HPzAmIH)(PzAmIH)](NO3)2&#61655;H2O (14). Os complexos foram caracterizados por espectroscopia de IV, análise elementar, condutivimetria, RMN e espectroscopia eletrônica. Em geral, os complexos também demonstraram ação contra M. tuberculosis, sendo que apenas para 6, 9, 10 e 14 foi verificada melhor atividade em relação às hidrazonas livres. Os complexos metálicos foram tanto quanto ou mais ativos contra as células tumorais OVCAR-8, HCT-116 e SF-295 do que as hidrazonas livres. Merecem destaque os complexos 7&#61485;9 e 12, que apresentaram inibição de crescimento celular de 72,2&#61485;100%, na concentração de 5 &#61549;g/mL. Os resultados demonstram portanto que em geral os compostos 1&#61485;14 são menos ativos do que a isoniazida contra M. tuberculosis, enquanto que a modificação química do fármaco, formando-se hidrazonas com posterior complexação cobre(II) e gálio(III) constituíram uma estratégia interessante na obtenção de compostos mais potentes contra células tumorais / In this work we describe the synthesis of isoniazide-derived hydrazones and their copper(II) and gallium(III) complexes candidates for drug prototypes to treat antituberculosis and cancer. Five hidrazone derivatives have been prepared in order to investigate the effect of chemical modification on the bioactivities of the drug isoniazid: 2-pyridinecarboxaldehyde isonicotinoyl hydrazone (HPCIH, 1), 2-acetylpyridine isonicotinoyl hydrazone (HAPIH, 2), 2-benzoylpyridine isonicotinoyl hydrazone (HBPIH, 3), 2-pyridineformamide isonicotinoyl hydrazone (HPAmIH, 4) and pyrazineformamide 2-isonicotinoyl hydrazone (HPzAmIH, 5), being HPAmIH (4) unpublished. Melting point, infrared spectroscopy (IR), mass spectrometry, nuclear magnetic resonance (NMR), elemental analysis and thermogravimetric analysis has confirmed the formation of hydrazones as well as its purity. All compounds have been effective in inhibiting the growth of Mycobacterium tuberculosis H37Rv (ATCC 27294) mycobacteria strains in the tested concentrations, except HPzAmIH (5). The hydrazones HAPIH (2) and HBPIH (3) have been the most active compounds (minimum inhibitory concentration, MIC = 0.625 &#61549;g/mL), which have presented the antimycobacterial activity only two times lower than isoniazid drug. In relation to the action against tumor cells, the hydrazones HAPIH (2) and HBPIH (3) have been the most potent compounds against the cell lines OVCAR-8 (ovarian tumor - human), HCT-116 (colon tumor - human) and SF-295 (glioblastoma - human), with inhibitions from 34.98 to 98.63% of cellular growth at a concentration of 5 &#61549;g/mL. Isoniazid, in turn, hasnt been effective against all cell lines studied. To evaluate the effect of coordinating the metal on the pharmacological activity of hydrazones, complexes of copper (II) and gallium (III) have been synthesized, being all novel compounds: [Cu(HPCIH)Cl2]&#8729;H2O (6), [Cu(HAPIH)Cl2]&#8729;H2O (7), [Cu2(HBPIH)2Cl2]Cl2&#8729;4H2O(8), [Cu(HPAmIH)Cl2]&#8729;H2O (9), [Cu(HPzAmIH)Cl2]&#8729;H2O (10), [Ga(HPCIH)2](NO3)3&#61655;2H2O (11), [Ga(HAPIH)(APIH)](NO3)2&#61655;2H2O (12), [Ga(HPAmIH)(PAmIH)](NO3)2&#61655;2H2O (13) and [Ga(HPzAmIH)(PzAmIH)](NO3)2&#61655;H2O (14).The complexes have been characterized by IR spectroscopy, NMR, elemental analysis, conductivimetry and electron spectroscopy. In general, these complexes have also shown action against M. tuberculosis, whereas only 6, 9, 10 and 14 have demonstrated better activity than the free hydrazones. The metal complexes have been equally or more active against human the cancer cell lines OVCAR-8, HCT-116 and SF-295 than the free hydrazones. The complexes 7&#61485;9 and 12 deserve to be highlighted, which have shown 72.2&#61485;100% inhibition of cell growth in the 5 &#61549;g/mL concentration. The results therefore demonstrate that in general compounds 1&#61485;14 are less active than isoniazid against M. tuberculosis, while chemical modification of the drug, forming hydrazones and subsequent complexation with the copper (II) and gallium(III) metal ions have been an interesting strategy to obtain compounds more potent against tumor cells

Made available in DSpace on 2015-05-14T13:00:07Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 927562 bytes, checksum: 636b817d8fa21526e39f3df100a21156 (MD5) Previous issue date: 2009-03-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Solanum paludosum Moric. (Solanaceae) is an herbaceous species, known popularly as "jurubeba-roxa" in the Northeast of Brazil (AGRA; BHATTACHARYYA, 1999). The chemical and pharmacological studies with root bark of this species showed the presence of glycoalkaloids, and that the ethanol extract and aqueous phase obtained the same parts showed spasmolytic activity (ATAÍDE, 1982; BARBOSA-FILHO et al., 1991; BASÍLIO, 2008). As glycoalkaloids are known to present cytotoxicity and many species of Solanum present spasmolytic activity, decided to study the total alkaloids fraction from root bark of S. paludosum (FAT-SP) on the cytotoxic potential in rat s erythrocytes and to investigate the spasmolytic activity in rat uterus, rat aorta and guinea-pig ileum and trachea. FAT-SP did not show hemolytic activity but presented non-selective spasmolytic effect. The preliminary pharmacological screening showed that FAT-SP on rat uterus, did not inhibit the oxytocin (10-2 IU/mL)-induced phasic contractions, but significantly inhibited the CCh (10-5 M)-induced phasic contractions (IC50 = 178.8 ± 7.1 μg/mL). On guinea-pig trachea, was about 2.2 times more potent in relax the trachea pre-contracted with CCh 10-6 M in the absence (EC50 = 159.4 ± 23.0 μg/mL) than in the presence (EC50 = 353.2 ±15.2 μg/mL) of functional epithelium; on guinea-pig ileum, inhibited the CCh (10-6 M)-induced phasic contractions (IC50 = 129.8 ± 3.7 μg/mL) and was more potent in relation to histamine (10-6 M)-induced contractions (Emax = 18.4 ± 2.3%) and on rat aorta, we observed that FAT-SP relaxes the rat aorta pre-contracted with phenylephrine 3 x 10-7 M of manner more potent in the presence (EC50 = 75.4 ± 6.2 μg/mL) than absence (EC50 = 242.8 ± 11.7 g/mL) of functional endothelium. According with the parameter of potency FAT-SP was more potent on guinea-pig ileum and rat aorta. Thus, we decided to investigate a possible mechanism of action in these organs. FAT-SP relaxed in a significant and concentration dependent manner the guinea-pig ileum pre-contracted with 10-6 M CCh (EC50 = 37.4 ± 3.2 μg/mL) or histamine (EC50 = 54.2 ± 2.9 μg/mL), but not with 40 mM KCl (Emax = 28.6 ± 2.8%). Since, FAT-SP was more potent to relax the ileum pre-contracted with carbachol, this is suggestive the involvement of muscarinic receptors in this effect. This hypothesis was confirmed in functional level by the observation that the FAT-SP shifted to the right the cumulative concentration-response curves to CCh without changing the Emax, which is characteristic of competitive antagonism to the CCh. The relaxation produced by FAT-SP on rat aorta in the presence of L-NAME 10-5 M (EC50 = 147.0 ± 22.1 g/mL), the NO synthase inhibitor, was attenuated, but not in the presence of atropine 10-6 M (EC50 = 93.3 ± 10.0 g/mL), a non-selective muscarinic receptor antagonist, or indomethacin 10-5 M (EC50 = 90.7 ± 13.2 g/mL), a non-selective COX inhibitor, thus suggesting the involvement of the endothelial NO synthase. Furthermore, in the presence of hydroxocobalamin 3 x 10-5 M (EC50 = 294.9 ± 20.4 μg/mL), a scavenger of NO, or ODQ 10-5 M (EC50 = 228.2 ± 30.9 μg/mL), a selective blocker of soluble GC, the relaxation curve of FAT-SP was attenuated, suggesting the involvement NO/CG pathway in the spasmolytic effect of the FAT-SP. / Solanum paludosum Moric. (Solanaceae) é uma espécie herbácea, conhecida popularmente como jurubeba-roxa no Nordeste do Brasil (AGRA; BHATTACHARYYA, 1999). O estudo químico e farmacológico com a casca da raiz desta espécie mostrou a presença de glicoalcalóides, e que o extrato etanólico e a fase aquosa obtidos da mesma parte mostraram atividade espasmolítica (ATAÍDE, 1982; BARBOSA-FILHO et al., 1991; BASÍLIO, 2008). Como muitos glicoalcalóides são conhecidos por apresentarem citotoxicidade e muitas espécies de Solanum apresentam atividade espasmolítica, decidiu-se estudar a fração de alcalóides totais obtida da casca da raiz de S. paludosum (FAT-SP) em relação ao potencial citotóxico em eritrócitos de rato e investigar uma possível atividade espasmolítica em útero de rata, aorta de rato, íleo e traquéia de cobaia. A FAT-SP não mostrou atividade hemolítica, mas apresentou efeito espasmolítico não seletivo. A triagem farmacológica preliminar mostrou que a FAT-SP, em útero de rata, não inibiu as contrações fásicas induzidas por 10-2 UI/mL de ocitocina, mas inibiu de maneira significante as contrações induzidas por 10-5 M de CCh; em traquéia de cobaia, foi cerca de 2,2 vezes mais potente em relaxar a traquéia pré-contraída com 10-6 M de CCh na ausência (CE50 = 159,4 ± 23,0 μg/mL) do que na presença (CE50 = 353,2 ± 15,2 μg/mL) de epitélio funcional; em íleo de cobaia, inibiu as contrações fásicas induzida por 10-6 M de CCh (CI50 = 129,8 ± 3,7 μg/mL) de maneira mais potente em relação às contrações induzidas por 10-6 M de histamina (Emax = 18,4 ± 2,3%) e em aorta de rato, foi observado que a FAT-SP relaxa a aorta de rato pré-contraída com 3 x 10-7 M de fenilefrina de maneira mais potente na presença (CE50 = 75,4 ± 6,2 ug/mL) do que na ausência (CE50 = 242,8 ± 11,7 ug/mL) de endotélio funcional. De acordo com o parâmetro de potência a FAT-SP foi mais potente em íleo de cobaia e aorta de rato. Diante disso, decidiu-se investigar um possível mecanismo de ação nestes órgãos. A FAT-SP relaxou o íleo de cobaia de maneira significante e dependente de concentração pré-contraído com 10-6 M de CCh (CE50= 37,4 ± 3,2 μg/mL) ou de histamina (CE50 = 54,2 ± 2,9 μg/mL), mas não com 40 mM de KCl (Emax = 28,6 ± 2,8%). Visto que, a FAT-SP foi mais potente em relaxaro íleo pré-contraido com carbacol, isto é sugestivo do envolvimeto dos receptores muscarínicos neste efeito. Esta hipótese foi confirmada em nível funcional pela observação de que a FAT-SP deslocou para direita as curvas concentrações-resposta cumulativas ao CCh sem alteração do Emax, o que é característico de antagonismo competitivo ao CCh. O relaxamento produzido pela FAT-SP em aorta de rato na presença de 10-5 M de L-NAME (CE50 = 147,0 ± 22,1 ug/mL), inibidor da sintase do NO endotelial, foi atenuado, mas não na presença de 10-6 M de atropina (CE50 = 93,3 ± 10,0 ug/mL), um antagonista não seletivo dos receptores muscarínicos, ou de 10-5 M de indometacina (CE50 = 90,7 ± 13,2 ug/mL), um inibidor não seletivo da COX, sugerindo assim, a participação da sintase do NO endotelial. Além disso, na presença de 3 x 10-5 M de hidroxocobalamina (CE50 = 294,9 ± 20,4 μg/mL), sequestrador do NO, ou de 10-5 M de ODQ (CE50 = 228,2 ± 30,9 ug/mL), inibidor seletivo da GC solúvel, a curva de relaxamento da FAT-SP foi atenuada, sugerindo a participação da via NO/CG no efeito espasmolítico da FAT-SP.

Influenza A virus infections are a major cause of morbidity and mortality in the United States and throughout the world. The current vaccine elicits primarily a humoral response that is specific for the external glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, these are the viral proteins that are most susceptible to antigenic shift and drift, and can evade the humoral response. Cytotoxic T lymphocytes (CTL) recognize and lyse virus-infected cells and are important in clearing influenza A virus infections. CTL can recognize epitopes on both the external glycoproteins and the more conserved internal viral proteins. This thesis investigates the hypothesis that there is a broad CTL memory response in humans, and, if boosted by vaccines, these CTL may help clear influenza A virus strains of different subtypes. The CTL repertoire specific for influenza A viruses reported in inbred mice is extremely limited and has focused on a few immunodominant epitopes. We perfonned preliminary bulk culture chromium release assays using human peripheral blood mononuclear cells (PBMC) stimulated with influenza virus strain A/PR/8/34 (H1N1) in vitro. CTL activity was observed against autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia constructs that expressed several influenza A viral proteins, including nucleoprotein (NP), matrix (M1), nonstructural 1 (NS1) and polymerase (PB1). This was more diverse than the limited response reported in inbred mice. To further characterize the CTL repertoire in humans, PBMC from healthy adult donors were stimulated and CTL were cloned by limiting dilution. Isolated cell lines were further characterized by their CD4/CD8 surface expression, histocompatibility leukocyte antigen (HLA) restriction, cross-reactive or subtype-specific influenza A subtype recognition, and epitope recognition. CTL lines isolated from three donors recognized epitopes on many different influenza virus proteins. The ELISPOT assay was used to identify the number of IFN-γ- secreting cells and determine the precursor frequency of the CTL specific for the epitopes that were mapped. The precursor frequency of IFN-γ producing CTL ranged from 1 in 4,156 PBMC to 1 in 31,250 PBMC. The precursor frequency for one epitope was below the level of detection of this assay, but most of the memory CTL were readily detected. The cross-reactive or subtype-specific recognition of various human influenza A subtypes by these T cell lines was determined by chromium release assays. Most of the CTL lines recognized B-LCL infected with any of the three influenza A subtypes that have caused epidemics in the last century (H1N1, H2N2, and H3N2) and recognized epitopes on conserved internal influenza viral proteins. Most of the subtype-specific cell lines recognized the surface HA or NA glycoproteins, which are not well conserved between influenza subtypes. Although most of the T cell lines that were characterized were cross-reactive with influenza viruses of human origin, infection of humans with a divergent swine or avian derived strain could cause a global pandemic. To study the human CTL responses to non-human influenza viruses, B-LCL were infected with an Hsw1N1 influenza A virus of swine origin, and cell lines were tested for recognition of these targets in a chromium release assay. Most cell lines lysed the targets infected With the Hsw1N1 subtype to the same degree as targets infected with the human H1N1 strain. Two influenza viruses of duck origin were also tested and were recognized by many of the cell lines. The subtypes of these duck strains were Hav1N1 and H5N2. The isolates of influenza A virus from the Hong Kong outbreak of 1997 were also used to infect targets and analyze recognition by these CTL. We found that approximately 50% of the human T cell lines tested recognized both of the Hong Kong isolates, 25% recognized at least one isolate, and 25% recognized neither isolate to the same degree as the A/PR/8/34 (H1N1) virus. We analyzed the amino acid (aa) changes in the epitopes of the T cells lines from the 25% of cell lines that did not recognize either Hong Kong virus isolate. Non-conservative mutations were found in all of the epitopes that lost recognition by the human CTL lines. Bulk cultures of PBMC from three donors that were stimulated with A/PR/8/34 (H1N1) influenza A virus of human origin recognized all of the non-human virus strains tested. Thus, humans have memory CTL that recognize influenza viruses of avian and swine species. This may provide a second line of defense against influenza infection in case of exposure to a novel influenza A virus derived from these species. These results made it clear that humans have broad CTL memory to influenza A virus. In order to determine whether these T cells could be boosted in a vaccine, immune-stimulatory complexes (Iscom) incorporating inactivated influenza particles were tested in vitro. Iscoms containing inactivated influenza A vaccine (Flu-Iscom) were used to pulse autologous B-LCL overnight that were then used as targets in chromium release assays with human CTL lines as effectors. A CD8+ HA-specific CTL line lysed these targets, but not targets pulsed with Iscoms alone or with inactivated influenza A vaccine alone. An NS1-specific cell line recognized targets pulsed with NS1 protein and Iscoms, but not targets pulsed with Iscoms or NS1 protein alone. Therefore, CTL could recognize in vitrotarget cells that were exposed to the Iscom vaccines containing their specific epitope. Flu-Iscom and Iscom mixed with inactivated influenza virus particles (Flu-Iscomatrix) were then used as vaccines in a clinical trial to test CTL and neutralizing antibody induction against influenza. Fifty-five donors were bled pre-vaccination, and on days 14 and day 56 post-vaccination. Bulk culture chromium release assays were performed using targets infected with live vaccine strain viruses. There were significantly more increases in the influenza A specific CTL activity in the PBMC of donors that were vaccinated with the Flu-Iscom and Flu-Iscomatrix vaccines than in recipients of the standard vaccine. In order to determine whether these increases in cytotoxicity were due to an increase in the precursor frequency of influenza specific CTL, the PBMC were used in ELISPOT assays to assess the changes pre-and post-vaccination. When there was an increase in the level of cytotoxicity detected in bulk culture CTL, there was often also an increase in the precursor frequency of influenza-specific CTL. Peptide-specific increases in the number of CTL that recognize epitopes such as M1 aa 58-66 were detected in several donors confirming the increase in influenza-specific CTL post-vaccination. Another type of T cell that may be involved in defense against viruses is the γδ T cell. T cells expressing the γδ T cell receptor (TCR) have been found extensively in mucosal tissues in mice and humans. Influenza A viruses enter via the airway tract, infecting the epithelial cells at the mucosal surface. These epithelial cells have been shown in vitro to be targets for influenza-specific cytolytic recognition of αβ T cells. To analyze whether γδ T cells can respond to influenza A-infected APCs, PBMC were stimulated with influenza A virus. Intracellular IFN-γ staining was used to determine whether γ/δ T cells can secrete IFN-γ in response to the influenza A virus infection. We observed an increase in the percentage of γ/δ T cells secreting IFN-γ post-influenza A virus infection of PBMC compared to uninfected or allantoic fluid-stimulated cultures. These T cells also upregulated CD25 and CD69 in response to live influenza A virus. We focused on the responses in the CD8- population of γδ T cells, which are the majority of γδ T lymphocytes. Furthermore, the increases in IFN-γ production and activation marker expression were much more clear in the CD8- γδ+ T cells. The level of CD8- γδ T cell activation with inactivated influenza A virus was much less, and in some cases no higher than uninfected PBMC. The CD8+ αβ and γδ responses could be partially blocked by anti-class I antibodies, but the CD8- γδ responses could not. Vaccinia virus infection did not activate the CD8- γδ T cells to the same degree as influenza virus infection. γδ T cells are thought to have a regulatory role that includes the secretion of cytokines and epithelial growth factors to help restore tissue back to health. Humans have broad multi-specific T lymphocyte responses by αβ T cells to influenza A viruses and those responses are cross-reactive with human, avian, and swine virus strains. These CTL can be activated in vitro and boosted in number in vivo by Iscom incorporating vaccines. There is also a population of γδ+ T lymphocytes in humans that responds to influenza virus infection by producing cytokines and becoming activated. Increasing memory CTL as a second line of defense against influenza A viruses may be important in future vaccine development.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / This present work reports the chemical and biological analysis of the stem and leaves from Bauhinia pulchella. In this study, the ethanol extract from stems was obtained by maceration, subjected to chromatographic fractionation, leading to isolation of three flavonoids: (+)-3â,4â-dihydroxyphenyl-chroman-7-ol (BP-2), (-)-fisetinidol (BP-3) and (+)-epicatechin (BP-4); a mixture of triterpenes taraxerone and &#946;-amyrenone (BP-1); a mixture of steroids sitosterol and stigmasterol (BP-5); and a bibenzyl named 2-hydroxy-3â,5â-dimethoxybibenzyl (BP-6). It is notewhorthy to mention that BP-1 and BP-4 substances are unprecedented in the genus, while BP-2 is unpublished. Chemical structures of secondary metabolites obtained were elucidated by 1H and 13C NMR; IR and MS associated with comparison of data described in the literature. Chemical composition of the essential oil from leaves of B. pulchella, obtained by hydrodistillation, was determined and quantified by gas chromatography-mass spectroscopy (GC/MS) and gas chromatography-flame ionization detector (GC/FID), which identified 95,68% of all constituents. &#945;-pinene (23.89%); caryophyllene oxide (22.43%) and &#946;-pinene (12.19%) were the major components. The essential oil was tested against Aedes aegypti larvae and showed LC50 value of 105.93  1.48 &#956;g/mL. The cytotoxic activity of essential oil was evaluated on human tumor cell lines (HL-60; MCF-7; NCI-H292 and HEP-2) was evaluated, showing IC50 values with confidence intervals of 9.941 (8.238 to 12.00), 53.05 (41.39 to 67.99), 48.98 (44.22 to 54.25) and 50.42 (42.47 to 59.87) &#956;g/mL, respectively and the cell line HL-60 the most sensitive among the cells tested. This is the first report of the chemical study of Bauhinia pulchella, as well the investigation of larvicidal activity and cytotoxicity of the essential oil from its leaves. / O presente trabalho relata o estudo quÃmico e biolÃgico do caule e das folhas de Bauhinia pulchella. Nesse estudo, o extrato etanÃlico do caule, obtido por maceraÃÃo, foi submetido a fracionamento cromatogrÃfico levando ao isolamento de trÃs flavonoides (+)-3â-4âdiidroxifenil-cromano-7-ol (BP-2), (-)-fisetinidol (BP-3) e (+)-epicatequina (BP-4); da mistura de triterpenos taraxerona e &#946;-amirenona (BP-1); da mistura de esteroides sitosterol e estigmasterol (BP-5) e de um bibenzil denominado 2-hidrÃxi-3â-5â-dimetoxibibenzila (BP-6). Cabe ressaltar que as substÃncias BP-1 e BP-4 sÃo inÃditas no gÃnero, enquanto BP-2 à inÃdita na literatura. As estruturas dos metabÃlitos secundÃrios isolados foram elucidadas por RMN 1H e 13C; IV e EM, juntamente com a comparaÃÃo com os dados descritos na literatura. A composiÃÃo quÃmica do Ãleo essencial das folhas de B. pulchella, obtido por hidrodestilaÃÃo, foi determinada e quantificada por cromatografia gasosa acoplada à espectrometria de massas (CG-EM) e detector de ionizaÃÃo por chama (CG-DIC), sendo, portanto, identificados 95,68% dos seus constituintes: &#945;-pineno (23,89%), Ãxido de cariofileno (22,43%) e &#946;-pineno (12,19%) foram os constituintes majoritÃrios. O Ãleo essencial teve sua atividade larvicida sobre Aedes aegypti avaliada, sendo obtido um valor de CL50 igual a 105,93  1,48 &#956;g/mL. O poder citotÃxico do Ãleo essencial foi avaliado sobre as linhagens tumorais humanas HL-60, MCF-7, NCI-H292 e HEP-2, sendo obtidos valores de CI50 e intervalos de confianÃa iguais a 9,941 (8,238 a 12,00); 53,05 (41,39 a 67,99); 48,98 (44,22 a 54,25) e 50,42 (42,47 a 59,87) &#956;g/mL, respectivamente, sendo a linhagem celular HL-60 a mais sensÃvel dentre as cÃlulas testadas. Este à o primeiro relato do estudo quÃmico de Bauhinia pulchella, bem como da investigaÃÃo da atividade larvicida e citotÃxica do Ãleo essencial de suas folhas.