Orientadores : Sonia Maria Alves Bueno, Ricardo de Lima Zollner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T11:50:08Z (GMT). No. of bitstreams: 1 Duarte_IsaSantos_D.pdf: 749498 bytes, checksum: 83d66753c75e7828fd4a8b858aecf473 (MD5) Previous issue date: 2006 / Resumo: A alergia é uma enfermidade do sistema imunológico que afeta aproximadamente de 20 a 30% da população mundial. Dentre as reações alérgicas, a reação de hipersensibilidade imediata é mediada pelas imunoglobulinas E (IgE). Os indivíduos geneticamente predispostos a manifestar reações por hipersensibilidade imediata e polissensibilizados aos alérgenos ambientais são considerados atópicos e, geralmente, possuem teores de IgE total até 10.000 vezes mais elevados do que as pessoas não-atópicas. O conhecimento das interações entre a IgE e ligantes de afinidade pode levar ao desenvolvimento de novos tratamentos da hipersensibilidade imediata, por exemplo a terapia de adsorção seletiva através de circulação extracorpórea, assim como ao desenvolvimento de métodos de obtenção de IgE purificada, para aplicação nas áreas de diagnóstico, pesquisa molecular, dentre outras. Os adsorventes empregados na terapia de adsorção seletiva, bem como na purificação de IgE, geralmente são anticorpos anti-IgE imobilizados em agarose, os quais são de alto custo e difícil obtenção. Este trabalho avaliou o desempenho de adsorventes alternativos ao Sepharose-anti-IgE, visando a remoção de IgE total e específica aos ácaros Dermatophagoides pteronyssinus e Blomia tropicalis de amostras plasmáticas e a preparação de soluções enriquecidas em IgE, como uma das etapas do processo de purificação de IgE. Os adsorventes estudados constituíram-se de lectinas (concanavalina A e Lens culinaris), aminas (poli-L-lisina e aminohexil) e o aminoácido D-triptofano, imobilizados em agarose. Dentre eles, o gel agarose-Lens culinaris mostrou-se o mais promissor para aplicação na terapia de adsorção seletiva de IgE e o gel Sepharose-concanavalina A mostrou-se o mais adequado para ser usado na obtenção de soluções enriquecidas em IgE. Experimentos cromatográficos foram realizados visando estabelecer condições experimentais (velocidade superficial, número de passagens de plasma pela coluna, temperatura e razão entre volume de plasma e volume de leito) mais favoráveis à adsorção de IgE em agarose-Lens culinaris. Posteriormente, essas condições foram utilizadas nos experimentos de simulação in vitro de circulação extracorpórea, nos quais o gel agarose-Lens culinaris removeu de 40,7 a 42,8% de IgE¿s total e específicas. A obtenção da solução enriquecida em IgE foi realizada por meio de duas etapas cromatográficas, empregando-se os princípios de afinidade (colunas agarosejacalina e Sepharose-concanavalina A) e de exclusão por tamanho (permeação em gel). A solução final enriquecida em IgE obtida, continha como principais impurezas, IgA e IgG. Como resultado das duas etapas, 36,6% de IgE foi recuperada e o fator de enriquecimento em IgE, em relação a IgA, IgG, IgM e albumina, foi de 75,8. Apesar do gel Sepharose-anti- IgE apresentar desempenho melhor tanto na remoção quanto na purificação de IgE, os adsorventes agarose-Lens culinaris e Sepharose-concanavalina A apresentam custos mais atrativos / Abstract: Allergy is a disorder of the imune system, affecting approximately 20%-30% of the general population. Among allergic reactions, immediate hypersensitivity is mediated by immunoglobulin E (IgE). Individuals that have a genetic predisposition for responses to immediate hypersensitivity are named atopic and generally have elevated serum IgE concentration, up to 10,000-fold higher than in the normal population. The knowledge of the interactions between IgE and affinity ligands may lead to the development of new methods of treatment for immediate hypersensitivity, for example, IgE selective adsorption therapy through extracorporeal circulation, as well as to new methods for obtaining purified IgE, which is employed in diagnostic and in molecular research. The adsorbents employed in IgE selective adsorption therapy, as well as in IgE purification, are usually antibodies anti-IgE immobilized on agarose, which have high costs and are difficult to obtain. This work assessed the performance of adsorbents (alternative to Sepharose-anti-IgE) for the removal of total IgE and IgE specific for the airbone allergens Dermatophagoides pteronyssinus and Blomia tropicalis from plasma samples, as well as in the production of IgE enriched solutions, considered as a step of IgE purification. The adsorbents studied were lectins (concanavalina A and Lens culinaris), amines (poli-L-lisina e aminohexil) and the aminoacid D-tryptophan, all of them immobilized on agarose. Among them, Lens culinaris-agarose showed the best performance for IgE selective adsorption therapy, and Sepharose¿concanavalin A was considered the most appropriate for the production of IgE enriched solutions. Chromatographic experiments were accomplished in order to determine operating conditions (superficial velocity, number of times the plasma passed through the column, temperature, and ratio of plasma volume to bed volume) more favorable to IgE adsorption on Lens culinaris-agarose. The selected conditions were utilized in in vitro simulation assays of extracorporeal circulation, in which the Lens culinaris-agarose removed from 40.7% to 42.8% of total and specific IgE. The production of IgE enriched solutions was carried out with two chromatographic steps, employing affinity (columns jacalin-agarose and Sepharose-concanavalin A) and size exclusion (gel permeation) principles. The IgE enriched final solution contained IgA and IgG as the major impurities. As a result of both steps, 36.6% of IgE was recovered and the IgE enrichement number concerning IgA, IgG, IgM, and albumin was 75.8. Despite Sepharose-anti-IgE has better performance in removal and purification of IgE, Lens culinaris-agarose and Sepharose-concanavalin A adsorbents have more attractive costs / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutora em Engenharia Quimica

Para elucidar os mecanismos envolvidos na inibição da hipersensibilidade do tipo I em proles murinas mediada pela imunização materna com Ovalbumina (OVA), as proles foram avaliadas quanto aos linfócitos Tγδ produtores de IL-17 (CD27-). A imunização materna com OVA reduziu a expressão de CD27, o que também se refletiu nos pulmões, e a produção de IL-17 por linfócitos Tγδ das proles. A redução da expressão de CD27 também foi evidenciada em linfócitos Tγδ intratímicos das proles após a transferência passiva de anticorpos IgG alérgeno específicos para fêmeas gestantes não imunizadas e in vitro em resposta a estes mesmos anticorpos, efeito que parece envolver a expressão de receptores para IgG expressos por outras células presentes no timo. As evidências obtidas indicam que a imunização materna influi na maturação tímica de linfócitos inibindo a população que colaborara com a inflamação alérgica. Aparentemente, os anticorpos IgG maternos são responsáveis por este fenômeno e a população estudada está envolvida na inibição da inflamação alérgica observada nas proles. / To elucidate the mechanisms involved in inhibition of type I hypersensitivity in murine offsprring mediated by maternal immunization with Ovalbumin (OVA), the offspring were evaluated as IL-17-producing γδT cells (CD27-). Maternal immunization with OVA reduced the expression of CD27, which was also reflected in the lungs, and the production of IL-17 by γδT cells of offspring. Reduction of CD27 expression was also evidenced in intrathymic γδT cells of offspring after passive transfer of allergen-specific IgG antibodies to non-immunized pregnant females and in vitro in response to these same antibodies, effect that seems to involve the expression of IgG receptors expressed by other cells present in the thymus. Evidence obtained indicates that maternal immunization influences the thymic maturation of lymphocytes by inhibiting the population that collaborate with allergic inflammation. Apparently, maternal IgG antibodies are responsible for this phenomenon and the population studied is involved in the inhibition of allergic inflammation observed in offspring.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-07-04T16:01:12Z No. of bitstreams: 1 rafaelvalenteveiga.pdf: 4234191 bytes, checksum: 215e887a2d4ac1ec9993886981d707eb (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-08T15:16:30Z (GMT) No. of bitstreams: 1 rafaelvalenteveiga.pdf: 4234191 bytes, checksum: 215e887a2d4ac1ec9993886981d707eb (MD5) / Made available in DSpace on 2017-08-08T15:16:30Z (GMT). No. of bitstreams: 1 rafaelvalenteveiga.pdf: 4234191 bytes, checksum: 215e887a2d4ac1ec9993886981d707eb (MD5) Previous issue date: 2017-03-08 / Nas últimas décadas os casos de asma e demais doenças alérgicas tiveram grande aumento em todo o mundo, sendo atualmente um grande problema de saúde pública. As causas do aumento da prevalência destas patologias são desconhecidas, porém a hipótese mais aceita é que seja oriunda da redução de infecções na infância como consequência da recente melhora nas condições de higiene. Esta redução das infecções pode levar ao desenvolvimento anômalo do sistema imune, aumentando assim a chance de desenvolver alergias e a asma. Tanto a asma como as demais alergias são patologias complexas, causadas por fatores genéticos e ambientais, de modo que o uso de ferramentas computacionais, tais como a programação genética podem contribuir para a compreensão destas doenças. Aplicou-se a técnica de Programação Genética Gramatical Multiobjetivo (MGGP) em dados obtidos de um coorte de 1445 crianças entre 4 e 11 anos para gerar modelos os quais possam representar como as relações entre infecções e ambiente podem explicar o desenvolvimento de atopia e asma. Para avaliar a presença de asma foi usado um questionário do ISAAC fase II e para avaliar atopia foram realizadas medições de anticorpos IgE contra alérgenos comuns e teste de reatividade cutânea. Os resultados obtidos mostram que os modelos gerados pela MGGP apresentam desempenho em acurácia competitivos aos obtidos pelo algoritmo C4.5 e regressão logística múltipla para os diferentes desfechos. Os resultados obtidos pela MGGP são de fácil interpretação, e capazes de encontrar relações complexas relevantes para o entendimento destas patologias, sendo assim, a MGGP é uma poderosa ferramenta para ajudar a compreender essas condições. / In the last decades cases of asthma and other allergic diseases have increased greatly throughout the world, being nowadays a major public health problem. The causes of this increased prevalence are unknown, however the most accepted hypothesis is that it comes from the reduction of childhood infections as a consequence of the recent improvement in hygiene conditions. This reduction of infections can lead to anomalous development of the immune system, thus increasing the chance of developing allergies, including asthma. Asthma and other allergies are complex pathologies caused by genetical and environmental factors, so the use of computational tools such as genetic programming can contribute to the understanding of these diseases. We applied the Multiobjective Grammatical Genetic Programming (MGGP) technique to data obtained from a cohort of 1445 children to generate models which may represent how the relationships between infections and environment may explain the development of allergies and asthma. To assess the presence of asthma, a questionnaire was used and allergy measurements were performed on IgE antibodies against common allergens and skin reactivity test. The results obtained show that the models generated by MGGP show a performance in accuracy that is competitive with those obtained by the algorithm C4.5 and multiple logistic regression for the different outcomes. The results obtained by MGGP are easy to interpret, and capable of finding complex relationship relevant to the understanding of these complex pathologies, therefore MGGP is a powerful tool to help understand these conditions.

Abstract The aim of the South Karelian Allergy Research Project (SKARP) was to quantify the occurrence of children’s food allergy and food-associated hypersensitivity symptoms and their associated factors. The study population comprised all children born between April 2001 and March 2006 and living in the province of South Karelia, in the south-eastern part of Finland. The questionnaire survey was conducted in co-operation with the child health clinics in the area in 2005–2006. Concurrently with but independently of the questionnaire study, the results of allergy tests regarding the same child population were collected from patient records. The participation rates in the questionnaire study were 54% (644/1194) among the newborn infants and 69% (3308/4779) among the children aged 1 to 4 years. The lifetime prevalence of parent-reported food allergies was 9% and that of parent-perceived food-associated hypersensitivity symptoms 21% by the age of 4 years. In addition, another 19% of children adhered to an elimination diet without previous symptoms associated with any food items. The prevalence of children with such diets decreased by age. Up to the age of 4 years, 19% of the participants had undergone a food allergy test and 8% of the participants had obtained a positive result in these tests. Physician-diagnosed food allergies and food allergies based on the tests were more common for milk, egg and cereals than for other food items. The tested children and those with a positive test result were only slightly overrepresented among the participants. Allergic manifestations in either biological parent doubled and in both biological parents tripled the incidence of a positive food allergy test. The spring season coinciding with the end of the first trimester of pregnancy predicted sensitisation to food items in the children. In early childhood, food allergies and food hypersensitivities were found to be common in a child population. New population-based knowledge regarding the inheritance of these conditions was obtained. Additionally, an association was observed between the timing of the 11th gestational week in spring and the sensitisation to food items, the detailed reasons and immunological mechanisms of which must be confirmed in further studies. / Tiivistelmä Etelä-Karjalan allergiatutkimus (EKAT) käynnistettiin selvittämään lasten ruoka-allergioiden ja ruokayliherkkyysoireiden esiintyvyyttä ja niihin liittyviä tekijöitä. Tutkimusväestöön kuuluivat kaikki ne Etelä-Karjalan maakunnan alueella asuvat lapset, jotka olivat syntyneet huhtikuun 2001 ja maaliskuun 2006 välisenä aikana. Kyselyaineisto kerättiin yhteistyössä alueen neuvoloiden kanssa vuosina 2005–2006. Kyselytutkimuksen kanssa samanaikaisesti mutta siitä riippumattomasti kerättiin samaa lapsiväestöä koskevat allergiatestitulokset alueen potilasrekistereistä. Kyselytutkimuksen osallistumisaste oli vastasyntyneiden ikäluokassa 54 % (644/1194) ja isommilla lapsilla 69 % (3308/4779). Kolmannen ikävuoden loppuun mennessä vanhemmat ilmoittivat 9 %:lla lapsista todetun ruoka-allergiaa ja lisäksi 21 %:lla ruokayliherkkyys perustui ainoastaan vanhempien havaintoihin. Näiden lisäksi 19 % lapsista vältti yhtä tai useampaa ruoka-ainetta ilman, että minkään ruoka-aineen olisi todettu aiheuttaneen oireita. Dieettien noudattajien osuus väheni selvästi tarkasteltaessa ikäluokkia yksivuotiaista neljävuotiaisiin. Kolmannen ikävuoden loppuun mennessä ruoka-allergiatestattuja oli 19 % ja positiivisen testituloksen saaneita 8 % tutkimukseen osallistuneista. Lääkärin toteamat ja testeihin perustuneet ruoka-allergiat olivat tavallisempia maidolle, kananmunalle ja viljoille kuin muille ruoka-aineille. Allergiatestatuilla ja positiivisen testituloksen saaneilla oli vain lievä yliedustus kyselytutkimukseen osallistujien joukossa verrattuna kyselyyn vastaamattomiin. Allergiset sairaudet toisella biologisella vanhemmalla kaksinkertaistivat ja molemmilla vanhemmilla kolminkertaistivat positiivisen ruoka-allergiatestin ilmaantuvuuden. Raskauden ensimmäisen kolmanneksen lopun ajoittuminen kevääseen ennakoi syntyvän lapsen herkistymistä ruoka-aineille. Varhaislapsuuden ruoka-allergioiden ja -yliherkkyyksien todettiin olevan tavallisia väestössä. Näiden sairauksien periytyvyydestä saatiin uutta väestötietoa. Lisäksi havaittiin yhteys 11. raskausviikon ajoittumisen kevääseen ja ruoka-aineille herkistymisen välillä. Yhteyden yksityiskohtaiset syytekijät ja immunologiset mekanismit täytyy ehdottomasti vahvistaa jatkotutkimuksin.

Allergi förekommer hos cirka 20 % av befolkningen i Sverige. Av de 20 % bedöms 40 % vara allergiska mot pollen. Allergi uppstår då en individ upprepade gånger exponeras för ett allergiframkallande ämne sk allergen. IgE-antikroppar bildas och binder till basofila granulocyter och mastceller. Den allergiska reaktionen eller överkänslighetsreaktionen av typ I uppkommer när ett allergen exempelvis pollen, kommer i direkt kontakt med IgE-antikroppar på mastceller eller basofila granulocyter. Allergiska individer har högre koncentration av IgE-antikroppar i blodet än icke allergiska. Normal koncentration av IgE i serum hos icke allergiker är <0.35 kU/L. Syftet med studien var att genom analys av serumprover från 50 patienter jämföra två immunokemiska metoder på analysinstrumenten, Siemens immulite® 2000 XPi och Thermo Fisher Phadia® 250, för detektion av IgE-antikroppar riktade mot en inhalationspanel respektive björkpollen. Immulite® 2000 XPi användes som referensmetod. Jämförelsen gjordes för att bedöma om metoderna gav överensstämmande resultat och om Phadia® 250 skulle kunna användas som ett alternativ till rutinmetoden. Studiematerialet bestod av 50 patienter i åldrarna 18–80 år, 17  män (34 %) och 33 kvinnor (66 %). Resultatet visade att metoderna överlag gav överrensstämmande resultat med avseende på positiva och negativa IgE-värden, men att de uppmätta värdena för metoderna saknade överrensstämmelse. Analysresultaten från de båda metoderna gav överlag god korrelation vid analys av specifikt IgE mot björk (R=0,977) men en svagare korrelation vid analys av inhalationspanel (R=0,609). Med hjälp av Man-Whitney u-test sågs en signifikant skillnad vid IgE-analys mot björk (p= 0,0006) däremot sågs inte en signifikant skillnad mellan metodernas IgE-värden mot inhalationspanelerna (p=0,2398). För att kunna utvärdera om Phadia® 250 skulle kunna användas som ett alternativ till rutinmetoden, krävs en större population av prover samt fler analystillfällen. / Allergy is nowadays a public health problem that’s affecting 20 % of the Swedish population. A very common allergen that is related to 40% of all the allergic cases is pollen. Allergic reactions occur when an  individual is repeatedly exposed to an allergenic substance called allergene. IgE antibodies then form and bind to basophilic granulocytes and mast cells. The allergic reaction or type I hypersensitivity reaction, occurs when an allergen such as pollen comes into direct contact with IgE antibodies on mast cells or basophilic granulocytes. IgE is produced by plasma cells under the influence of CD4+ T lymphocytes. The IgE antibodies synthesized then sensitize the mast cells and basophilic granulocytes which are the most active cell types involved in allergic reactions. Allergic individuals have a higher concentration of IgE antibodies in the blood than non-allergic. Normal serum IgE concentration in non-allergic patients is <0.35 kU / L. The aim of the study was to compare two immunochemical instruments, Siemens Immulite® 2000 XPi and Thermo Fisher Phadia® 250, by analyzing 50 serum samples to detect possible presence of IgE antibodies directed against an inhalation panel and a single allergen (IgE against birch). The study material consisted of 50 patients between the ages of 18-80, 17 men (34%) and 33 women (66%). The results showed that overall the two methods yielded consistent results due to positive and negative IgE values, but the measured values of the methods lacked concordance. The analysis results from the two methods generally yielded good correlation in the analysis of specific IgE against birch (R = 0.977) but a weaker correlation in the inhalation panel analysis (R = 0.609). Man-Whitney u-test showed a significant difference in IgE analysis against birch (p = 0.0006). However, a significant difference between the IgE values of the methods against the inhalation panels (p = 0.22398) was not observed. To be able to evaluate whether Phadia® 250 could be used as an alternative to the routine method, a larger population of samples and more analytical occasions is required.

Chaque cadre doit contenir un résumé de 1700 caractères maximum, espaces compris. En cas de dépassement, lacoupure sera automatique.Le doctorant adresse son texte sous forme électronique selon les recommandations de la bibliothèqueLes dermocorticoïdes (DC) sont des anti-inflammatoires topiques largement prescrits pour traiter de nombreuses dermatoses. Actuellement, leur puissance, classée en quatre groupes, est déterminée par leur effet vasoconstricteur basé sur un test empirique obsolète, subjectif et réducteur vis-à-vis des nombreux autres effets des DC. L'objectif de ce travail était de comparer la puissance des DC dans trois de leurs indications et de la confronter à la classification actuelle:• Anti-inflammatoire. Une étude clinique randomisée contrôlée en double aveugle a été réalisée sur 21 patients allergiques aunickel traités par quatre DC. Les différents paramètres de l'inflammation ont été mesurés objectivement par des outils debiométrologie. La hiérarchisation de la puissance des DC variait en fonction du temps et des paramètres de l'inflammationétudiés. Sur ce modèle, la classification peut être simplifiée en trois groupes.• Antiprolifératif. L'efficacité de six DC sur la prolifération, le cycle cellulaire et l'apoptose de kératinocytes hyperproliféranten culture a été comparée. La classification de la puissance antiproliférative divergeait de la classification actuelle.• Antiprurigineux à médiation histaminique. L'effet anti-histaminolibérateur de huit DC a été comparé sur des explants depeau à l'aide d'un dosage par microdialyse sous-cutanée. La classification de la puissance anti-histaminolibératrice divergeaitde la classification actuelle.Nous avons démontré que la classification actuelle des DC n'est pas exactement corrélée à leur effet anti-inflammatoire dansl'allergie de contact, à leur effet antiprolifératif ainsi qu'à leur effet sur la libération d'histamine. Notre nouvelle approche est deparler des puissances DC au pluriel. Une classification multimodale adaptée à la situation clinique permettrait une prescriptionplus efficiente et limiterait les échecs et effets indésirables. / Topical corticosteroids (TCS) are topical anti-inflammatory widely prescribed to treat many skin diseases. Currently, their potency, ranged into four groups, is determined by their vasoconstrictor effect based on an empirical, outdated, subjective and too restrictive test. The aim of this work was to compare specifically the potency of TCS in three situations and to compare it to the current classification:• Anti-inflammatory. A randomized controlled double-blind clinical study was performed on 21 nickel-sensitive patientstreated by four TCS. Different inflammation parameters were objectively assessed with bioengineering tools. The ranking ofTCS potency varied according to time and parameters of the inflammation studied. According to this model, the classificationmay be simplified into three groups.• Antiproliferative. The efficacy of six TCS on immortal keratinocytes proliferation, cell cycle and apoptosis was compared. Theclassification of the antiproliferative potency differed from the current classification.• Antipruritic histamine-mediated. The histamine-release inhibition of eight TCS was compared on skin explants by asubcutaneous microdialysis dosage. The classification of the histamine-release inhibition potency differed from the currentclassification.We have demonstrated that the current classification of TCS potency is not strictly correlated to their anti-inflammatory effect in the context of allergie contact dermatitis, to their antiproliferative effect and to their histamine-release inhibitor effect. Our new approach is to talk about TCS potencies in the plural. A specifie classification adapted to the therapeutic target is needed to be more efficient and reduce side effects.

The prevalence of allergy is increasing and it is becoming a serious problem not on- ly in medicine, but also in social and economic terms. The most effective way to minimize the development of allergic diseases is preventive measures. In recent years, many studies have attempted to confirm or rebut the hypothesis that early administration of probiotic bacteria to newborns and pregnant women before birth could have preventive effects on the development of allergy. In the Czech Republic, the probiotic strain Escherichia coli O83:K24:H31 (EC O83), being registered with the State Health Institute for Drug Control under the name Colinfant Newborn, has long been used to prevent allergies and paediatri- cians have long been known and used it against various diarrhoea. The aim of this work was to elucidate the effect of EC O83 on CBMC (cord blood mononuclear cells) and to compare the ability of CBMC of healthy mothers (children with a relatively low risk of developing allergic disease) and allergic mothers (children at high risk of developing allergies) to form cytokines in response to EC O83 stimulation. Phytohemagglutinin was used as a positive control, Escherichia coli Nissle 1917 was used as a reference probiotic strain, which is much more known abroad than EC O83. Cytokine production was detected by...

(EN) Reduced microbial stimulation of an immature neonatal immune system can lead to a poor balance adjustment of immune responses, thus contributing to the development of allergic diseases, whose incidence continues to rise. One of the promising precautionary measures seems to be an early preventive administration of probiotic bacteria to pregnant or nursing mothers, or to newborns. Previous works have described a beneficial effect of Escherichia coli O83:K24:H31 (E. coli O83) in the prevention of allergic diseases. In order to contribute to the clarification of E. coli O83 effects on the neonatal immune system, its immune- modulating properties were tested in vitro on umbilical cord blood cells. The ability of E. coli O83 to support the maturation of in vitro-derived dendritic cells from cord blood precursors (moDCs) of the children of healthy (children with a relatively low risk of allergy) and allergic (children at a relatively high risk of developing allergies) mothers was tracked by flow cytometry, qPCR and ELISA. Probiotic bacteria-stimulated moDCs were subsequently cultured with autologous naive CD4+ T lymphocytes and immune response polarization was also characterised by flow cytometry, qPCR, and ELISA. It was evident from the results that E. coli O83 promoted moDCs maturation. The presence of...

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-24T21:28:26Z No. of bitstreams: 1 Flavia Regina Greiffo.pdf: 886904 bytes, checksum: 4aa27a44bc94c9d06427df1b649e9b9e (MD5) / Made available in DSpace on 2016-05-24T21:28:26Z (GMT). No. of bitstreams: 1 Flavia Regina Greiffo.pdf: 886904 bytes, checksum: 4aa27a44bc94c9d06427df1b649e9b9e (MD5) Previous issue date: 2015-03-02 / In the last years, a growing number of studies have demonstrated that the aerobic physical training (APT), performed in the proper intensity, duration and frequency, display several benefits to asthmatic patients, including improvement in quality of life, reduction in the levels of anxiety and depression, improvements in minute ventilation, reduction in the number of crisis, reduction in the levels of exhaled nitric oxide, beyond reduced daily needing of corticosteroids. More recently, was demonstrated for the first time that APT presents a direct anti-inflammatory effect on the airways of asthmatic patient, since that APT reduced the number of eosinophils in the induced sputum of these patients. However, several questions and hypothesis were raised up about the possible mechanism involved in these anti-inflammatory effects of APT for asthma. In this way, a growing number of experimental studies using models of asthma have been published, revealing possible mechanism involved of action APT for asthma. On the other hand, until this moment no study evaluated the effects of APT on the via of purinergic receptors, a family of receptors that present a central role in the physiopathology of asthma. Therefore, the present study aim is to evaluate if the anti-inflammatory effects of APT for asthma is mediate, at least partially, by the inhibition of expression of purinergic receptor P2X7, as well as the extracellular adenosine triphosphate (ATP). Thus, using 40 C57Bl/6 mice, divided in 4 experimental groups (Control, n = 10), (Exercise, n = 10), (Asthma, n = 10) and (Asthma + Exercise, n = 10). The experimental model of asthma (chronic pulmonary allergic inflammation) was induced through the intra-peritoneal injection of ovalbumin solution on days 0, 14, 21 and 42 followed by inhalation of 1%, 3% and 5% ovalbumin, 3x/week, and beginning on 21st day until the 53rd day of experimental protocol. To evaluate the therapeutic effects of APT on the inflammation and on the via of purinergic receptors, the low intensity APT was performed 5x/week, 60 min/session, begining on 27th day of experimental protocol until the 53rd day, when the airways inflammation is already established. Twenty-four hours after the last inhalation and training session, mice were anesthetized, tracheotomized, canuled and the bronchoalveolar lavage was collected and analyzed for the ATP levels and also the total number of cells as well as the differential number of cells. The analysis of hyperresponsiveness was performed. The histological analysis was performed to evaluate and quantify the number of lymphocytes and eosinophils in the airways wall, and also the airway remodeling. The expression of purinergic receptor P2X7 in the lung tissue homogenate was performed using western blotting and also the immunohistochemistry technique. / Nos últimos anos um crescente número de estudos tem demonstrado que o treinamento físico aeróbio (TFA), quando realizado de maneira adequada em termos de intensidade, duração e freqüência, apresenta inúmeros benefícios para o paciente asmático, incluindo melhora na qualidade de vida, diminuição dos níveis de ansiedade e depressão, melhora da ventilação minuto, diminuição do número de crises, diminuição dos níveis de óxido nítrico exalado, além de diminuir a necessidade diária de corticoesteróides. Recentemente, foi demonstrado pela primeira vez que o TFA apresenta um efeito antiinflamatório direto sobre as vias aéreas do paciente asmático, uma vez que o TFA diminuiu o número de eosinófilos no escarro induzido desses pacientes. Entretanto, inúmeras perguntas e hipóteses foram levantadas sobre os possíveis mecanismos envolvidos nesse efeito antiinflamatório do TFA na asma. Nesse sentido, um crescente número de estudos com animais de experimentação em modelos de asma tem sido publicado, os quais têm revelado possíveis vias de atuação do TFA na asma. Entretanto, até o momento não existem publicações sobre os efeitos do TFA sobre a via dos receptores purinérgicos, os quais têm um papel central na fisiopatologia da asma. Portanto, o presente estudo teve com objetivo avaliar se os efeitos antiinflamatórios do TFA na asma é mediado, pelo menos em parte, pela inibição da expressão dos receptores purinérgicos, assim como pela diminuição dos níveis do principal ativador dos receptores purinérgicos, a adenosina trifosfato (ATP) extracelular. Para isso, foram utilizados 40 camundongos C57Bl/6 divididos em 4 grupos experimentais (n=10 / cada) Controle, Exercício, Asma e Asma + Exercício. O modelo de asma (inflamação pulmonar alérgica crônica) foi induzido por meio de injeção intra-peritoneal com ovalbumina nos dias 0, 14, 28 e 42 seguidos por inalação com solução de ovalbumina (1, 3 e 5%) três vezes por semana, a partir do dia 21 até o dia 53 do protocolo experimental. Com o intuito de avaliar o efeito terapêutico do TFA sobre a inflamação e sobre a via dos receptores purinérgicos, o TFA de baixa intensidade foi realizado 5x/semana, 60 minutos/sessão, iniciando no dia 27 do protocolo experimental até o dia 53, quando a inflamação das vias aéreas já estava estabelecida. Vinte e quatro horas após a última inalação e sessão de treinamento, os animais foram anestesiados, traqueostomizados, canulados e o lavado broncoalveolar foi coletado e analisado para os níveis de ATP e também para o número de células totais e contagem diferencial. A avaliação da expressão do receptor purinérgico P2X7 foi realizada no homogenato do tecido pulmonar por meio da técnica de western blotting, e também nos cortes histológicos por meio da técnica de imunohistoquímica. Através da técnica de histologia foi quantificado o número de linfócitos e eosinófilos nas vias aéreas, assim como o remodelamento brônquico. A hiperresponsividade brônquica das vias aéreas foi avaliada através do sistema Buxco. Os resultados obtidos neste trabalho corroboram com a literatura, uma vez que o TFA reduz a inflamação crônica das vias aéreas. Nossos resultados também sugerem que o TFA atua na via dos receptores purinégicos diminuindo a inflamação pulmonar.

This diploma thesis deals with the evaluation of clinical manifestations in infants with allergic colitis and their overall nutritional status, anthropometry, blood count and antibodies against cow's milk proteins. Allergic colitis is one of the most common manifestations of infant allergy to cow`s milk protein with gastrointestinal involvement. The theoretical part summarizes the anthropometric evaluation of growth and development, infant nutrition, the most important laboratory indicators of nutritional status. The part is generally devoted to food allergies, mainly allergy to cow's milk protein. The practical part deals with the evaluation of a sample of examined patients in gastroenterology outpatient clinics and nutritional outpatient clinic of the Department of Pediatrics and Adolescent Medicine of the 1st Faculty of Medicine, Charles University and General Hospital in Prague. The observed group of infants with allergic colitis includes infants exclusively breastfed, breastfed by a mother following a non-dairy diet, infants on an extensive hydrolyzate or on an amino acid formula. The main goal of the diploma thesis was to trace the most common clinical manifestations and whether there are any changes in their nutritional status during the disease and its treatment. A total of 45 randomly...