Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-17T14:35:39Z No. of bitstreams: 1 Sandra Maria Rosa de Aguiar.pdf: 937813 bytes, checksum: 3dc0508cafece93cb858ec9e505c2c38 (MD5) / Made available in DSpace on 2016-08-17T14:35:39Z (GMT). No. of bitstreams: 1 Sandra Maria Rosa de Aguiar.pdf: 937813 bytes, checksum: 3dc0508cafece93cb858ec9e505c2c38 (MD5) Previous issue date: 2008-04-09 / The chemotherapic adjuvant treatment is associated to neutropenia, complications of organic defense, alterations in the nutritional status, and negative impact on quality of life, which compromises the physical, emotional, and social aspects of cancer patients. Not controlled clinical trial, the type before and after, this work is a first attempt to analyze the impact of food supplementation with immunomodulatory nutrients on the hematological profile of patients with breast neoplasia submitted to chemotherapic adjuvant treatment. This research was carried out in HEMOLABOR, an oncology/hematology center in Goiânia, state of Goiás, from November 2007 to January 2008. The dietary supplement was offered before a chemotherapy cycle, using the following formulations: FAC (a combination of fluorouracil, adriblastin, and cyclofosfamide) and CMF (a combination of cyclofosfamide, metotrexate, and 5-fluorouracil). Pre- or postmenopause patients between 18 and 62 years old, presenting Karnofsky index 70 participated in the study. Patients presenting associated neoplasia, chronic transmittable (HIV) or non-transmittable diseases (diabetes, hepatic or renal insufficiency), neurologic or psychiatric problems were excluded. Case inclusion occurred regardless of the number of chemotherapy cycles already performed and according to the appointment scheduling of the institution. The sociodemographic, cultural, and clinic profile of the patients was designed using the WHOQOLbref( WHO) questionnaire, based on the analysis of their medical records and oriented interview. The questions were answered aiming at stratifying the importance of the disease on quality of life according to the patient’s opinion. The food supplement used is described as an enteral formula for oral supplementation, nutritionally complete, adequate for special metabolic situations, enriched with immunomodulatory nutrients (arginine, glutamin, nucleotids, omega-3 fatty acids), which was consumed during the week preceding a new chemotherapy cycle. The hematological profile was designed based on the results of complete blood counts evaluated at three different moments: one cycle before the food supplement intake, one cycle including the offer of the food supplement, and one cycle after the food supplement intake. The difference among these hematological profiles was used as an indicator of food supplementation impact on the patient’s immune status according to the standard of the World Health Organization, in the analysis of hematological toxicity. The results showed recovery of total white blood cells and, specially, of neutrophils after the food supplement intake. An association between the increase or decrease of cellular levels in the complete blood counts and the variables age, Karnofsky index, chemotherapy regime, and quality of life as a whole was not perceived. The data presumably indicate that the gain is lower when the disease staging is more advanced and induces the thought that the positive balance of hematological indicators may represent gain as a function of the nutritional support used. / O tratamento quimioterápico adjuvante está associado à neutropenia com complicações das defesas orgânicas, alterações no estado nutricional e impacto negativo na qualidade de vida, comprometendo os aspectos físico, emocional e social de portadoras de câncer. Ensaio clínico não controlado, do tipo antes e depois, este trabalho é uma primeira aproximação na análise do impacto da suplementação alimentar com nutrientes imunomoduladores sobre o perfil hematológico de pacientes com neoplasia mamária, submetidas à quimioterapia adjuvante. Foi realizado no HEMOLABOR, centro especializado em oncologia e hematologia, em Goiânia, Goiás, no período compreendido entre os meses de novembro de 2007 a janeiro de 2008. A oferta do suplemento dietético foi feita antes de um ciclo de quimioterapia, nas formulações: FAC (combinação de fluorouracil, adriblastina e ciclofosfamida) e CMF (Combinação de ciclofosfamida, metotrexate e 5-fluorouracil). Participaram do estudo pacientes com idade entre 18 e 62 anos, pré ou pós-menopausadas e com índice de Karnofsky igual ou maior que 70. Foram excluídas aquelas que apresentaram neoplasia associada, afecção crônica transmissível (HIV) ou não transmissível (diabetes, insuficiência hepática ou renal), problemas neurológicos ou pisquiátricos. A inclusão dos casos foi independente do número de ciclos quimioterápicos já realizados e foi feita na medida dos agendamentos da instituição. Desenhou-se o perfil sócio–econômico, cultural e clínico das pacientes, utilizando-se o questionário WHOQOL-bref(OMS), a partir da análise de seus prontuários e de entrevista orientada. As questões foram respondidas de forma a estratificar o peso da doença sobre a qualidade de vida na opinião da própria paciente. O suplemento alimentar utilizado é descrito como fórmula enteral para suplementação oral, nutricionalmente completa, própria para situações metabólicas especiais; enriquecida com nutrientes imunomoduladores (arginina, glutamina, nucleotídeos, ácidos graxos ômega 3). Foi ingerido durante a semana que antecedeu um novo ciclo de quimioterapia. O perfil hematológico foi desenhado a partir dos resultados de hemograma completo avaliado em três momentos: um ciclo prévio à suplementação alimentar; um ciclo que incluiu a oferta do suplemento alimentar e um terceiro ciclo posterior à suplementação. A diferença entre estes perfis hematológicos foi utilizada como indicador do impacto da suplementação alimentar sobre o estado imunológico da paciente, de acordo com a padronização da Organização Mundial de Saúde, na análise da toxicidade hematológica. Os resultados mostraram uma recuperação de leucócitos totais e, em particular, de neutrófilos, após a ingestão do suplemento. Não se pressente associação entre aumento ou redução nos níveis celulares do hemograma e as variáveis idade, índice de Karnosfik, regime de quimioterapia e nem mesmo com a qualidade de vida, quando examinada de forma global. Os dados parecem indicar também que os ganhos são menores quando o estadiamento da doença está mais avançado e induz ao pensamento de que o saldo positivo nos indicadores hematológicos possa representar um ganho em função do suporte nutricional utilizado.

A bactéria Mycoplasma hyopneumoniae é um patógeno suíno economicamente significante, que se adere às células do epitélio respiratório causando a pneumonia enzoótica suína (PES). A adesão celular e outros processos importantes para infecção dependem de proteínas de membrana, cuja maioria é transportada pela via de exportação Sec-dependente. A sinal-peptidase tipo I (SPase I) é uma protease de membrana que atua na liberação de proteínas translocadas através da via Sec-dependente. Neste contexto, a SPase I possui participação crítica na exportação de proteínas que podem atuar nas relações patógeno-hospedeiro. Este estudo tem como objetivo a caracterização funcional e imunológica da SPase I de M. hyopneumoniae (MhSPase I), a fim de demonstrar sua funcionalidade in vivo e in vitro, sua associação à complexos proteicos de membrana e também seu papel na indução de uma resposta imune. A atividade funcional da MhSPase I recombinante (rMhSPase I) in vivo foi demonstrada através de ensaios de complementação em uma linhagem de Escherichia coli SPase I mutante condicional (E. coli IT89). A rMhSPase foi capaz de complementar parcialmente a atividade da SPase I da linhagem mutante de E. coli, permitindo a sobrevivência bacteriana em condições não permissivas. Entre as proteínas preditas in silico como secretadas por M. hyopneumoniae, dois possíveis substratos da MhSPase I foram selecionados e expressos em E. coli, a fim de realizar ensaios de clivagem de peptídeo-sinal in vitro. A associação da MhSPase I à complexos proteicos de membrana foi analisada através de ensaios de interação entre a proteína recombinante purificada e extratos proteicos de M. hyopneumoniae enriquecidos com proteínas de membrana. As respostas imune inata e adaptativa induzidas pela rMhSPase em camundongos também foram avaliadas, sendo ambas caracterizadas como anti-inflamatórias. Estes resultados sugerem um potencial imunomodulador da MhSPase I, o qual pode ser importante para a proteção de M. hyopneumoniae durante o processo inflamatório induzido pela PES no trato respiratório suíno. / Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory epithelial cells causing the porcine enzootic pneumonia (PEP). Cell adhesion and other processes important for infection depend on membrane proteins, most of which are transported by the Sec-dependent pathway. Type I signal peptidase (SPase I) is a membrane protease which acts in the release of translocated proteins through Sec-dependent pathway. In this context, SPase I is critical for protein export, which may act on pathogen-host relations. The aim of this study is the functional and immunological characterization of the M. hyopneumoniae SPase I (MhSPase I), in order to demonstrate its in vivo and in vitro functionality, its participation into membrane protein complexes and also its role in immune responses induction. The in vivo functional activity of recombinant MhSPase I (rMhSPase I) was demonstrated through complementation assays with an Escherichia coli conditional SPase I mutant (E. coli IT89). The rMhSPase I was able to partially complement SPase I activity of E. coli IT89 in non-permissive conditions, allowing the bacterial to survive. Among the proteins in silico predict as secreted by M. hyopneumoniae, two possible substrates of MhSPase I were selected and expressed in E. coli, in order to perform the in vitro signal peptide cleavage assays. The association of MhSPase I with membrane protein complexes was analyzed through interaction assays between the rMhSPase I and protein extracts of M. hyopneumoniae enriched with membrane proteins. The innate and adaptive immune responses induced in mice by rMhSPase I were also assessed, being both characterized as anti-inflammatories. These results suggest an immunomodulatory potential of MhSPase I, which may protect M. hyopneumoniae from host inflammatory process induced by PEP.

A bactéria Mycoplasma hyopneumoniae é um patógeno suíno economicamente significante, que se adere às células do epitélio respiratório causando a pneumonia enzoótica suína (PES). A adesão celular e outros processos importantes para infecção dependem de proteínas de membrana, cuja maioria é transportada pela via de exportação Sec-dependente. A sinal-peptidase tipo I (SPase I) é uma protease de membrana que atua na liberação de proteínas translocadas através da via Sec-dependente. Neste contexto, a SPase I possui participação crítica na exportação de proteínas que podem atuar nas relações patógeno-hospedeiro. Este estudo tem como objetivo a caracterização funcional e imunológica da SPase I de M. hyopneumoniae (MhSPase I), a fim de demonstrar sua funcionalidade in vivo e in vitro, sua associação à complexos proteicos de membrana e também seu papel na indução de uma resposta imune. A atividade funcional da MhSPase I recombinante (rMhSPase I) in vivo foi demonstrada através de ensaios de complementação em uma linhagem de Escherichia coli SPase I mutante condicional (E. coli IT89). A rMhSPase foi capaz de complementar parcialmente a atividade da SPase I da linhagem mutante de E. coli, permitindo a sobrevivência bacteriana em condições não permissivas. Entre as proteínas preditas in silico como secretadas por M. hyopneumoniae, dois possíveis substratos da MhSPase I foram selecionados e expressos em E. coli, a fim de realizar ensaios de clivagem de peptídeo-sinal in vitro. A associação da MhSPase I à complexos proteicos de membrana foi analisada através de ensaios de interação entre a proteína recombinante purificada e extratos proteicos de M. hyopneumoniae enriquecidos com proteínas de membrana. As respostas imune inata e adaptativa induzidas pela rMhSPase em camundongos também foram avaliadas, sendo ambas caracterizadas como anti-inflamatórias. Estes resultados sugerem um potencial imunomodulador da MhSPase I, o qual pode ser importante para a proteção de M. hyopneumoniae durante o processo inflamatório induzido pela PES no trato respiratório suíno. / Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory epithelial cells causing the porcine enzootic pneumonia (PEP). Cell adhesion and other processes important for infection depend on membrane proteins, most of which are transported by the Sec-dependent pathway. Type I signal peptidase (SPase I) is a membrane protease which acts in the release of translocated proteins through Sec-dependent pathway. In this context, SPase I is critical for protein export, which may act on pathogen-host relations. The aim of this study is the functional and immunological characterization of the M. hyopneumoniae SPase I (MhSPase I), in order to demonstrate its in vivo and in vitro functionality, its participation into membrane protein complexes and also its role in immune responses induction. The in vivo functional activity of recombinant MhSPase I (rMhSPase I) was demonstrated through complementation assays with an Escherichia coli conditional SPase I mutant (E. coli IT89). The rMhSPase I was able to partially complement SPase I activity of E. coli IT89 in non-permissive conditions, allowing the bacterial to survive. Among the proteins in silico predict as secreted by M. hyopneumoniae, two possible substrates of MhSPase I were selected and expressed in E. coli, in order to perform the in vitro signal peptide cleavage assays. The association of MhSPase I with membrane protein complexes was analyzed through interaction assays between the rMhSPase I and protein extracts of M. hyopneumoniae enriched with membrane proteins. The innate and adaptive immune responses induced in mice by rMhSPase I were also assessed, being both characterized as anti-inflammatories. These results suggest an immunomodulatory potential of MhSPase I, which may protect M. hyopneumoniae from host inflammatory process induced by PEP.

Glycodelin, a homodimeric glycoprotein belonging to the lipocalin superfamily, is synthesised predominantly by the cells of the reproductive system of certain primates including humans. Of the four different known glycoforms of the molecule, glycodelin A (GdA), secreted by the glandular epithelial cells of the endometrium in response to progesterone, is involved in the immunosuppression of the maternal immune response to the semi-allograft fetus. GdA secretion onsets few days after ovulation. In the absence of fertilization, GdA levels drop, but subsequent to a successful fertilization, the concentrations peak till the 12th week of pregnancy and fall steadily to low levels. The importance of GdA has been implicated in implantation, endometrial receptivity, trophoblast invasion and differentiation, and modulating the functions of almost all immune cells. GdA has profound influence on the activity of T cells. It inhibits the proliferation of T cells, induces apoptosis in activated T cells, inhibits the IL-2 production and leads to skewing of the Th-1/Th-2 balance towards Th-2 type of immune response. Cytotoxic T lymphocytes are more resistant to the induction of apoptosis by GdA, but, it suppresses their cytolytic activity Additionally, GdA induces apoptosis in monocytes and natural killer (NK) cells, inhibits the proliferation of B cells and induces tolerogenic phenotype in dendritic cells. Clinical studies showing that women undergoing recurring spontaneous abortions have low levels of GdA supports its role in prevention of fetus rejection. The immunomodulatory activity of Gd resides in the protein backbone, however, apart from GdA and GdF which have similar oligosaccharide chains, other glycoforms do not possess this activity. Glycosylation seems to dictate the stability, folding and activity of Gd. In absence of glycosylation, the expression of the recombinant Gd is compromised and the protein is improperly folded while over-mannosylation of Gd impairs its immunomodulatory function. Additionally, sialylation seen on the glycan chain regulates the activity. Therefore, in order to obtain adequate amounts of active recombinant Gd (rGd), expression of the protein was attempted in three different systems, insect, yeast and bacteria (Chapter 1). In all of the described systems, the rGd protein was found apoptotically active. The protein expressed in the Sf21 insect cells was demonstrated to be differentially glycosylated compromising the activity. Hence, a genetically modified yeast strain, Pichia pastoris SuperMAN5 was explored for expression. Though presence of a single glycosylated protein species was observed in small-scale cultures, similar to the case of Sf21 cell expression, differentially glycosylated proteins were detected in large-scale fermentation and even the yield was low. Eventually, mutant Gd, modified to increase the stability and aid in proper protein folding, was expressed in E.coli and demonstrated to be able to induce apoptosis in Jurkat cells (T cell leukemia cell line). This active rGd was used for further studies. The immunomodulatory function of GdA during pregnancy protects the semi-allograft fetus from rejection by the maternal immune system. In the process, GdA tweaks the T cell immune response from pro-inflammatory to anti-inflammatory in a specific and localized manner. Allograft rejection seen during mis-match transplantations is basically a pro-inflammatory condition which is mediated by the activation of cellular immune response, NK cell cytotoxicity and antibody-dependent immune response, the same processes that are suppressed for a successful pregnancy. Chapter 2 discusses whether it is feasible to use Gd to prevent allograft rejection. Killing of target graft cells by the cytotoxic T lymphocytes (CTLs) predominantly presides acute graft rejection. GdA treatment has been shown to suppress the cytotoxicity of in vitro generated CTLs. On this basis, the earlier study was translated to in vivo conditions by establishing an allograft nude mouse model. The tumor rejection mediated by the action of in vitro generated cytotoxic alloactivated PBMCs in the nude mouse imitated the allograft rejection. A heterogenous population of immune cells with the predominance of CTLs was chosen to accommodate a more interactive immune response in the tumor microenvironment and enabled the study of other cells which may contribute to the rejection. Reactivation and proliferation of CD4+ and CD8+ T cells following their infiltration in the tumor validated our hypothesis. On treatment with rGd, the cytotoxicity of the alloactivated PBMCs was suppressed, thereby inhibiting the tumor rejection in the nude mouse. Real time PCR analysis showed that rGd treatment was able to affect the functions of the immune cells in vivo. It decreased the T cell population most probably by inducing apoptosis. As expected, the reduction was more prominent in case of CD4+ T cells than CD8+ T cells. The their expression of key molecules responsible for the cytotoxicity such as IL-2, granzyme B and EOMES, was observed to be downregulated by rGd. Concomitantly, decreased levels of pro-inflammatory cytokines, TNFα and IL-6 were also seen. Expression of Foxp3, marker for regulatory T cells, was upregulated in the tumor infiltrating immune cells suggesting an expansion of the concerned population upon rGd treatment. Overall, rGd seems to suppress the cellular immune response to the tumor by modulating the T cell population and their functions. Since, T cell-dependent immune response is central to allograft rejection, the ability of rGd to regulate it could be of therapeutic use in the management of allograft rejection. NK cells are essential for the maintenance of pregnancy, evident from their abundance (70% of total leukocytes) at the first trimester decidua. The third chapter focuses on how Gd regulates the NK cell function. The cytokine production from CD56bright subset of NK cells and their interaction with the HLA antigens expressed by the trophoblast cells helps in creating a favourable environment for the growth of the fetus. It is important to note that the NK cell population present in the decidua exclusively express Gd, implicating a role of Gd in their differentiation from the peripheral CD56bright cells. However, an increased number of CD56dimCD16+ cells in the peripheral blood dictates a negative outcome for the pregnancy. The study, presented in Chapter 3, demonstrated that rGd treatment induces caspase-dependent apoptosis in the activated CD56dimCD16+ cells and reduces their cytotoxicity by downregulating granzyme B and IFNγ production. Similar effect of rGd is also seen on the NKT cells characterised as CD3+CD56dimCD16-. Furthermore, in YT-Indy cells, an activated NK cell line, it was shown that the induction of apoptosis by rGd involves Ca2+ signalling which could explain why Gd affects activated immune cells only. This study therefore reinforces the role of Gd in modulating the NK cell activity during pregnancy. Cytotoxicity of NK and NKT cells also plays an important role during allograft rejection. Decrease in the mRNA levels of CD56 upon rGd treatment in the allograft mouse model indicates that the effect of Gd on NK cells observed in cell culture system can be translated to in vivo conditions. In conclusion, suppression of the cellular immune response and NK cell mediated cytotoxicity by rGd could potentiate its’ probable use in the management of allograft rejection. .

A bactéria Mycoplasma hyopneumoniae é um patógeno suíno economicamente significante, que se adere às células do epitélio respiratório causando a pneumonia enzoótica suína (PES). A adesão celular e outros processos importantes para infecção dependem de proteínas de membrana, cuja maioria é transportada pela via de exportação Sec-dependente. A sinal-peptidase tipo I (SPase I) é uma protease de membrana que atua na liberação de proteínas translocadas através da via Sec-dependente. Neste contexto, a SPase I possui participação crítica na exportação de proteínas que podem atuar nas relações patógeno-hospedeiro. Este estudo tem como objetivo a caracterização funcional e imunológica da SPase I de M. hyopneumoniae (MhSPase I), a fim de demonstrar sua funcionalidade in vivo e in vitro, sua associação à complexos proteicos de membrana e também seu papel na indução de uma resposta imune. A atividade funcional da MhSPase I recombinante (rMhSPase I) in vivo foi demonstrada através de ensaios de complementação em uma linhagem de Escherichia coli SPase I mutante condicional (E. coli IT89). A rMhSPase foi capaz de complementar parcialmente a atividade da SPase I da linhagem mutante de E. coli, permitindo a sobrevivência bacteriana em condições não permissivas. Entre as proteínas preditas in silico como secretadas por M. hyopneumoniae, dois possíveis substratos da MhSPase I foram selecionados e expressos em E. coli, a fim de realizar ensaios de clivagem de peptídeo-sinal in vitro. A associação da MhSPase I à complexos proteicos de membrana foi analisada através de ensaios de interação entre a proteína recombinante purificada e extratos proteicos de M. hyopneumoniae enriquecidos com proteínas de membrana. As respostas imune inata e adaptativa induzidas pela rMhSPase em camundongos também foram avaliadas, sendo ambas caracterizadas como anti-inflamatórias. Estes resultados sugerem um potencial imunomodulador da MhSPase I, o qual pode ser importante para a proteção de M. hyopneumoniae durante o processo inflamatório induzido pela PES no trato respiratório suíno. / Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory epithelial cells causing the porcine enzootic pneumonia (PEP). Cell adhesion and other processes important for infection depend on membrane proteins, most of which are transported by the Sec-dependent pathway. Type I signal peptidase (SPase I) is a membrane protease which acts in the release of translocated proteins through Sec-dependent pathway. In this context, SPase I is critical for protein export, which may act on pathogen-host relations. The aim of this study is the functional and immunological characterization of the M. hyopneumoniae SPase I (MhSPase I), in order to demonstrate its in vivo and in vitro functionality, its participation into membrane protein complexes and also its role in immune responses induction. The in vivo functional activity of recombinant MhSPase I (rMhSPase I) was demonstrated through complementation assays with an Escherichia coli conditional SPase I mutant (E. coli IT89). The rMhSPase I was able to partially complement SPase I activity of E. coli IT89 in non-permissive conditions, allowing the bacterial to survive. Among the proteins in silico predict as secreted by M. hyopneumoniae, two possible substrates of MhSPase I were selected and expressed in E. coli, in order to perform the in vitro signal peptide cleavage assays. The association of MhSPase I with membrane protein complexes was analyzed through interaction assays between the rMhSPase I and protein extracts of M. hyopneumoniae enriched with membrane proteins. The innate and adaptive immune responses induced in mice by rMhSPase I were also assessed, being both characterized as anti-inflammatories. These results suggest an immunomodulatory potential of MhSPase I, which may protect M. hyopneumoniae from host inflammatory process induced by PEP.

Lectinas são proteínas cuja principal característica é a de se ligar específica e reversivelmente a carboidratos. BanLec é a lectina presente na polpa de bananas, que se liga especificamente a manose e glicose, e é capaz de induzir a proliferação de células T, podendo estimular a resposta imune. Existem indícios de que o teor de BanLec pode variar dependendo do estádio de amadurecimento e do tipo de cultivar, o que pode afetar a quantidade de BanLec existente na fruta quando consumida in natura e a possível resposta imune frente ao consumo de banana. Por este motivo, um dos objetivos desse trabalho foi determinar os teores e a atividade hemaglutinante de BanLec em extratos de farinha de banana verde, além de bananas das cultivares Pacovan, Figo, Terra, Mysore e Nanicão, nos estádios de maturação verde e maduro, e submetidas a tratamento com 1-MCP e baixa temperatura (para cv. Nanicão). Com vista a atender ao objetivo de avaliar seus efeitos imunomoduladores in vivo, a BanLec foi purificada da cultivar Nanicão e administrada por via oral a camundongos BALB/c. Os ensaios de atividade hemaglutinante dos extratos de banana apontaram para maior quantidade de BanLec no fruto maduro, quando comparado ao verde, e ausência dessa proteína na cultivar Figo. Os parâmetros imunológicos analisados após administração de BanLec aos camundongos demonstram que a resposta imune gerada após ingestão de BanLec é dose dependente, além disso, a administração de 50 µg de BanLec aos animais foi capaz de modular citocinas importantes na resposta imunológica, provavelmente causando um efeito que pode ser interpretado como mais protetor do que patogênico. Com base nos resultados obtidos, podemos concluir que existem diferenças nos teores de BanLec dependendo da cultivar e estádio de maturação analisado, sendo que essa proteína não está presente na polpa de todas as variedades de banana e finalmente, que ela tem grande potencial imunomodulador in vivo, uma vez que ativou citocinas de resposta anti-inflamatória. / Lectins are proteins which bind specifically and reversibly to carbohydrates. BanLec is the lectin present in banana pulp, and it binds to mannose and glucose, being capable of inducing T-cell proliferation, and to stimulate the immune response. There are some evidence that the amount of BanLec may vary depending on the maturation stage of the fruit and the cultivar (cv.), which may affect the amount of BanLec and the possible immune response after consumption of banana. Thus, this study aimed to evaluate the amount of BanLec and its hemagglutinating activity in crude extracts of bananas from cultivars Pacovan, Figo, Terra, Mysore and Nanicão, in both unripe and ripe maturation stage, and also fruits which were treated with 1-MCP and low temperature. In addition, in order to access their immunomodulatory effects in vivo, BanLec was purified by affinity chromatography and administered orally to BALB/c mice. The hemagglutinating activity assays indicate higher amount of BanLec in ripe fruit. Moreover, the possible was undetectable in the pulp of banana Figo. The immunological parameters of mice orally fed with BanLec showed that the immunological response is dependent on the amount of protein administrated, in agreement to previous in vitro studies. Besides, 50 µg of BanLec, were able to modulate some cytokines in immune response, causing an effect that seems to be more protective than pathogenic. We conclude that there are important differences in amount of BanLec depending on the cultivar and the maturation stage, and BanLec has a dose-dependent immunomodulatory effect in vivo.

Introduction: Multiple sclerosis (MS) is an autoimmune disorder of the CNS that typically affects young women of childbearing age. Due to the international published data, safety for pregnant women with MS can be assumed. However, no study has been published in the Czech Republic to address the effect of MS on pregnancy and perinatal outcomes. Objective: Analysis of the clinical course of patients with MS during and after pregnancy, and perinatal outcomes in comparison with healthy pregnant women. Methods: A single centre prospective observational study in the period 2006-2015 was conducted. Complete data from 68 patients with MS were analyzed (85 deliveries) and were compared with a control cohort of 68 age- and parity - matched healthy pregnancies. Results: The comparison between relapse rate and EDSS before, during and after delivery showed no statistically significant difference (relapse in 7.4% and 9.5%, EDSS 1.27 and 1.49). Perinatal outcomes were comparable in both cohorts. The weight of newborns differed by 159 g, (p = 0.295), complications in pregnancy were represented in 16.2% in the group with MS and in 27.9% in controls (p = 0.295), caesarean section was performed in 16.2% in patients with MS and in 23.5% of controls (p = 0.629), 79.4% of patients with MS were breast-feeding. In the MS...

Introdução: Com o crescente aumento de hipersensibilidades alimentares e doenças do trato gastrintestinal, o mercado de lácteos vem ampliando a variedade de produtos com diferentes matrizes alimentares. A expansão da criação bubalina no país incentivou o desenvolvimento de produtos derivados do leite bubalino, os quais contém propriedades nutricionais e físico-químicas diferentes da matriz bovina, ganhando maior participação no atual mercado consumidor. As bactérias probióticas são capazes de modular a resposta imune inata e adaptativa de acordo com a cepa, matriz e tecnologia empregada no desenvolvimento do produto. Assim como a modulação imune também pode ser alterada de maneira epigenética por interferência da matriz alimentar empregada. Método: Sendo os microRNAs de leite e leite bubalino homólogos aos do leite humano, foram preparados leites fermentados em matriz bovina e bubalina usando a cepa Bifidobacterium animalis subsp. lactis HN019 com o intuito de identificar a diferença entre o perfil físco-químico, viabilidade da cepa probiótica utilizada, e presença de microRNAs imunomoduladores homólogos com o intuito de verificar se as modificações sofridas nas matrizes bovina e bubalina durante o processo de fermentação por bactérias probióticas teria interferência na presença de miRNAs imunomoduladores e na resposta imune de mucosa. Conclusão: Os resultados obtidos mostraram maior teor de proteínas, sólidos totais e gordura na matriz bubalina em relação a matriz bovina. Enquanto na matriz bubalina fermentada apresentou maior teor de ácidos graxos comparado a matriz bovina fermentada, o perfil de acidificação e pós acidificação foi semelhante em ambas as matrizes. O processo de fermentação em matrizes lácteas distintas, proporcionou modificação do perfil celular imunológico na mucosa intestinal, porém levou a destruição dos miRNAs do processamento térmico, identificando que esta modificação da resposta imune independe dos microRNAs homólogos estudados. / Introduction: With the increasing increase of food hypersensitivity and diseases of the gastrointestinal tract, the dairy market has been increasing the variety of products with different food matrices. The expansion of buffalo breeding in the country encouraged the development of products derived from buffalo milk, which contain different nutritional and physicochemical properties of the bovine matrix, gaining greater participation in the current consumer market. Probiotic bacteria are able to modulate the innate and adaptive immune response according to the strain, matrix and technology employed in product development. Just as immune modulation can also be altered epigenetically by interference of the food matrix employed. Method: Since the microRNAs of milk and buffalo milk were homologous to those of human milk, fermented milks were prepared in bovine matrix and buffalo using the strain Bifidobacterium animalis subsp. Lactis HN019 in order to identify the difference between the physico-chemical profile, viability of the probiotic strain used, and the presence of homologous immunomodulatory microRNAs in order to verify if the modifications suffered in the bovine and buffalo matrices during the fermentation process by probiotic bacteria would interfere with the presence of immunomodulatory miRNAs and the mucosal immune response. Conclusion: The results showed higher protein content, total solids and fat in the buffalo matrix in relation to the bovine matrix. While in the fermented buffalo matrix presented higher content of fatty acids compared to fermented bovine matrix, the profile of acidification and post acidification was similar in both matrices. The fermentation process in distinct milk matrices provided a modification of the immunological cell profile in the intestinal mucosa but led to the destruction of the miRNAs of the thermal processing, identifying that this modification of the immune response is independent of the homologous microRNAs studied.

Ce travail a pour objet de déterminer les conditions optimales de production de substituts allogéniques capables de combler les lésions cartilagineuses dans le cadre du traitement de l’arthrose. Il s’oriente particulièrement sur la composante cellulaire de ces substituts. L’usage de cellules souches mésenchymateuses issues de cordons ombilicaux (CSM-GW) implique de déterminer quels facteurs obstétricaux, liés à l’environnement direct et indirect des CSM-GW, peuvent influencer leur prolifération ainsi que leur différenciation chondrocytaire. Dans une première partie de ce travail, trois types de facteurs ont été étudiés : les facteurs liés à l’enfant donneur, au déroulement de l’accouchement et de la délivrance, à la grossesse et à la mère. Nos données montrent que les CSM-GW ont des capacités prolifératives améliorées lorsque l’accouchement s’est déroulé à terme et sans complication, avec utilisation de Syntocinon® pendant le travail. Sur la base de ces résultats, nous avons utilisé les CSM-GW les plus efficaces dans le cadre de l’ingénierie du cartilage. Il a ensuite été essentiel d’élucider le profil d’action des CSM-GW dans un contexte allogénique. Le deuxième temps de ce travail a donc consisté à chercher le profil de stimulation le plus performant, au regard de la viabilité des cellules et de l’évolution de la sécrétion des facteurs solubles responsables des propriétés immunomodulatrices des CSM-GW au cours de la différenciation chondrocytaire. Nous avons alors mimé, in vitro et en biomatériaux d’Alginate/Acide hyaluronique (Alg/HA) une telle situation en stimulant les CSM-GW avec différentes doses d’IFN-γ et de TNF-α. Selon nos résultats, la stimulation par IFN-γ et TNF-α sur les CSM-GW en biomatériaux d’Alg/HA est plus efficace lorsque ces deux cytokines sont utilisées conjointement et n’est pas délétère pour la viabilité cellulaire aux concentrations respectives de 20 et 30 ng/mL. Cette double stimulation induit une augmentation de la sécrétion d’IL-6 et de PGE-2 par les CSM-GW, ne modifie pas leur sécrétion de TGF-β, et diminue la sécrétion de VEGF. Nous avons confirmé ces données lors d’une mise en situation fonctionnelle : des cocultures avec des cellules mononucléées de sang périphérique (PBMC) de donneurs sains nous ont permis d’évaluer la réponse des CSM-GW lors d’une situation allogénique. Ces mises en situations allogéniques ont été étudiées à différents temps afin d’évaluer les propriétés immunologiques des CSM-GW au cours du temps passé en biomatériaux. Nos résultats montrent que les CSM-GW peuvent exprimer des molécules HLA-G ainsi qu’IDO, mais ces expressions sont limitées en biomatériaux d’Alg//HA. Les CSM-GW en biomatériaux d’Alg/HA en situation allogénique ne sont pas immunogènes, quel que soit le temps de différenciation. En revanche, leurs capacités immunomodulatrices décroissent au cours du temps et sont plus fortes à J0 et J3 de la différenciation chondrocytaire, ce qui oriente vers une utilisation précoce de ces cellules. Les conclusions de ce travail permettent de (i) sélectionner les cordons idoines à l’ingénierie cellulaire et l’ingénierie du cartilage, (ii) définir les conditions permettant de mimer une situation allogénique in vitro, (iii) connaitre les propriétés immunomodulatrices des CSM-GW au cours de la culture en biomatériaux d’Alg/HA, y compris en situation allogénique / The purpose of this work is to determine the optimal conditions for allogeneic substitutes production, adapted to filling the cartilaginous lesions in osteoarthritis treatment. It focuses on the cellular component of these substitutes. The use of mesenchymal stem cells from umbilical cords (WJ-MSC) involves determining which factors, related to direct and indirect environment of the WJ-MSC, can influence their proliferation and chondrogenic differentiation. In a first part of our work, three types of factors were studied: related to the donor child, the course of labor and delivery, pregnancy and the mother. Our results show that WJ-MSC have enhanced proliferative capacities when coming from full-term birth and without complications, with the use of Syntocinon® during labor. On this basis, we used the most effective WJ-MSC for cartilage engineering. It was then essential to elucidate their action profile in allogeneic context. We stimulated WJ-MSC embedded in Alginate/Hyaluronic Acid (Alg/HA) scaffolds with different concentrations of IFN-γ and TNF-α in order to determine the most effective stimulation profile, with regard to viability of the cells and evolution of immunomodulatory soluble factors secretion. According to our results, the stimulation by IFN-γ and TNF-α on WJ-MSC in Alg/HA scaffolds is more effective when these two cytokines are used together and is not deleterious for cell viability at the concentrations of 20 and 30 ng/mL, respectively. This double stimulation induces an increase in the secretion of IL-6 and PGE-2 by the WJ-MSC, a decrease in the secretion of VEGF and does not modify the secretion of TGF-β. We confirmed these data during a functional study: cocultures with peripheral blood mononuclear cells (PBMC) from healthy donors allowed us to evaluate the response of WJ-MSC in an allogeneic situation. These allogeneic situations have been studied at different times to evaluate the immunological properties of WJ-MSC during the time of chondrogenic differentiation. Our results show that WJ-MSC can express HLA-G molecules as well as IDO, but these expressions are limited in Alg/HA biomaterials. Finally, the WJ-MSC in Alg/HA biomaterials in allogeneic conditions are not immunogenic, regardless of the time of differentiation. On the other hand, their immunomodulatory capacities decrease over time and are stronger at day 0 and day 3 of chondrogenic differentiation, which leads to an early use of these cells. Finally, this work allows us to (i) select the umbilical cords suitable for cellular and cartilage engineering, (ii) define the conditions mimicking in vitro an allogeneic situation, (iii) elucidate the immunomodulatory properties of WJ-MSC during Alg/HA biomaterials chondrogenic differentiation, including allogeneic situations

Made available in DSpace on 2015-05-14T13:00:09Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1776418 bytes, checksum: 744ac3ac5030bc552fceb05b70d06ca5 (MD5) Previous issue date: 2015-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer antileishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro, in vivo and in silico antileishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10, interleukin-12, IFN-γ were obtained in vitro and expression of p38 mitogen activated protein kinase (p38 MAPK), NF-κB (p50 and p65) was studied by western blotting.in vivo and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages in contrast of IL-12 and IFN-γ levels increased at concentrations ranging from 1·6 to 40·0 μg/ml. Fhf and Skm showed expression of p38 and NF-κB, pathways involved in the production of Th1 cytokines and nitric oxide. In vivo testing showed reduction in lesion size in mice infected with L. braziliensis, as well as reduction in parasite burden in linfonode and spleen. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2.Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity. / A leishmaniose é uma doença negligenciada cujo tratamento disponibilizado está associado a uma série de efeitos tóxicos, alto custo e diminuição da eficácia terapêutica devido ao aumento da resistência pelos parasitas. Assim, a pesquisa de novas drogas mais eficazes e seguras torna-se de grande importância. Compostos derivados de produtos naturais pode ser uma fonte mais eficaz e econômica para o tratamento desta parasitose. Este estudo avaliou a atividade leishmanicida da espécie Spiranthera odoratíssima (Rutaceae) testando suas frações e isolados in vitro, in vivo e in silico utilizando formas promastigota e amastigota de diferentes espécies de Leishmania em macrófagos J774 A.1. Os ensaios in vitro avaliaram citotoxicidade, produção de óxido nítrico (NO), produção de citocinas interleucina 10, interleucina 12 e IFN-γ. A expressão da proteína quinase p38 ativada por mitógeno (p38 MAPK) e NF-κB (p50 e p65) foi avaliada in vitro por western blotting. Os resultados observados in vitro da fração hexânica do fruto (Fhf) e seu alcaloide isolado esquimianina (Skm) demostraram uma significante ação leishmanicida (P<0·001) contra L. braziliensis. Esta ação foi associada ao aumento da produção de óxido nítrico e diminuição de IL-10 em macrófagos assim como aumento da produção de citocinas Th1 como Il-12 e IFNγ para as concentrações estabelecidas entre 1.6 a 40 μg/ml. Fhf e Skm demostraram induzir a expressão de p38 e NFκB, vias estas envolvidas na produção de citocinas Th1 e na indução de óxido nítrico. Os ensaios in vivo demostraram reduzir lesão em camundongos Swiss infectados com L. braziliensis, assim como foi reduzido à carga parasitária em linfonodos e baço destes animais. O ensaio in silico demonstrou interação significativa entre Skm e resíduos de aminoácidos de NOS-II. Estes resultados em conjunto sugerem que o alcaloide esquimianina é um candidato promissor contra L. braziliensis devido sua potente atividade imunomoduladora.