O resveratrol, um polifenol de origem natural, é descrito como uma substância antiinflamatória, antioxidante, cardioprotetora e anticancerígena. Diversos estudos comprovam a atividade imunomodulatória do resveratrol in vitro e in vivo, estimulando ou diminuindo a secreção de citocinas envolvidas na resposta Th1/Th2. Além do uso em vacinas como adjuvantes, a descoberta de novas substâncias imunomodulatórias pode ser aplicada na profilaxia e tratamento de doenças imunodegenerativas com perda da tolerância sistêmica ou periférica. O objetivo desse estudo é relacionar o efeito modulador do resveratrol em ensaios de endocitose em macrófagos e de secreção de citocinas IL-6(produção de IgA) e IL-10(resposta Th2 e tolerância em mucosas) com a produção de anticorpos anti-ova IgG e IgA após imunização via-oral. Os resultados obtidos demonstraram que in vitro, houve aumento da endocitose em macrófagos e diminuição na secreção de IL-6 pelas células isoladas de placas de peyer em concentrações abaixo de 50 µM de resveratrol. Após a administração oral de resveratrol de 5 mg e 10 mg/kg observou-se o aumento significativo da secreção de IL-10 em esplenócitos isolados de camundongos Balb/C. Nos grupos imunizados com 1 mg de ovalbumina/animal e resveratrol (5 mg e 10 mg/kg) via oral 2 vezes, com 14 dias de intervalos, houve aumento significativo da produção de IgG sérico em relação ao grupo imunizado somente com ovalbumina. Porém a produção de IgA sérico e em lavado intestinal diminuiu, indicando um possível aumento da tolerância oral. Esses resultados demonstram o efeito imunomodulador do resveratrol in vitro/in vivo e a necessidade de maiores estudos sobre o uso desta substãncia como adjuvante de vacinas ou uma droga imunossupressora de mucosa. / Resveratrol, a polyphenol of natural origin, is described as a substance-inflammatory, antioxidant, cardioprotective and anticancer. Several studies have demonstrated the immunomodulatory activity of resveratrol in vitro and in vivo, stimulating or decreasing the secretion of cytokines involved in Th1/Th2 response. Besides the use as adjuvants in vaccines, the discovery of new immunomodulatory substances can be applied for prophylaxis and treatment of diseases imunodegenerativas with loss of peripheral tolerance or systemic. The aim of this study is to relate the modulating effect of resveratrol on tests of endocytosis in macrophages and secretion of IL-6 (IgA production) and IL-10 (Th2 response and mucosal tolerance) with the production of anti-ova IgG and IgA after oral immunization route. The results of in vitro tests showed an increase of endocytosis in macrophages and decrease in IL-6 secretion by cells isolated from Peyer\'s patches at concentrations below 50 mM of resveratrol. After oral administration of resveratrol 10 mg / kg was observed to significantly increase the secretion of IL-10 in splenocytes isolated from Balb / C. In groups immunized with 1 mg ovalbumin / animal and resveratrol (5 mg and 10 mg / kg) orally two times with 14 days intervals, significant increase of IgG level in relation to the group immunized with ovalbumin only. But the production of IgA in serum and intestinal lavage decreased, indicating a possible increase in oral tolerance. These results demonstrate the immunomodulatory effect of resveratrol in vitro / in vivo and the need for more studies on substance use as a vaccine adjuvant or immunosuppressive drugs mucosa.

Advances in technology have lowered the limits of viability in premature births to 24 weeks of gestation. This brought forth a new population of children, who are born 3-4 months early and spent considerable amounts of time in neonatal intensive care unit (NICU), instead of sterile environment of mother’s womb. Besides, other problems associated with prematurity, these children often undergo invasive procedures resulting in mucosal inflammation and/ or injury by feeding tubes, endotracheal tubes, and prolonged IV catheter. To test whether “ex-preemie-infants” were different than “term-infants” with regard to their immunity, preterm infants (< 32 weeks) and term infants (control) at the corrected age of 9-12 months were analyzed for their resting and stimulated immune responses. Preterm infants had a significant Th1 skewed response, higher number of activated and functionally competent T cells compared to term infants. The critical role of neonatal environmental exposure on immune system development is imminent; nevertheless detailed mechanistic studies on pathways are warranted.

Advances in technology have lowered the limits of viability in premature births to 24 weeks of gestation. This brought forth a new population of children, who are born 3-4 months early and spent considerable amounts of time in neonatal intensive care unit (NICU), instead of sterile environment of mother’s womb. Besides, other problems associated with prematurity, these children often undergo invasive procedures resulting in mucosal inflammation and/ or injury by feeding tubes, endotracheal tubes, and prolonged IV catheter. To test whether “ex-preemie-infants” were different than “term-infants” with regard to their immunity, preterm infants (< 32 weeks) and term infants (control) at the corrected age of 9-12 months were analyzed for their resting and stimulated immune responses. Preterm infants had a significant Th1 skewed response, higher number of activated and functionally competent T cells compared to term infants. The critical role of neonatal environmental exposure on immune system development is imminent; nevertheless detailed mechanistic studies on pathways are warranted.

No description available.

Thirty five million people are currently living with HIV-1 today with women accounting for half of infected individuals globally. Sexual transmission is the main route of HIV transmission with approximately 40% of HIV infections occurring when the mucosal lining of the female genital tract (FGT) is exposed to HIV in semen from an infected male partner. Seminal plasma (SP), the fluid portion of semen, is a complex fluid which plays an immunomodulatory role in the FGT for successful conception, largely due to its high concentrations of TGF-β1. Several factors in SP from HIV-uninfected men have been shown to either inhibit or enhance HIV infection in target cells, however it is not clear how SP from HIV infected men would modulate genital epithelial cells (GECs), the first cells that encounter HIV in the FGT. The overall goals of this thesis were to compare inflammatory and regulatory cytokine concentrations in SP from HIV-uninfected and infected men, and subsequently compare GEC cytokine responses following exposure to SP from HIV-uninfected and HIV-infected men. I also investigated how SP and TGF-β regulated cytokine production and barrier function in GECs in the presence of HIV. The results summarized in this thesis demonstrated that HIV infection leads to different cytokine profiles in SP, based on stage of HIV-1 infection. HIV-infected men in acute stage contained higher levels of proinflammatory cytokines in their SP compared to HIV-uninfected and chronically infected men (CI men) which subsequently lead to higher levels of proinflammatory cytokines from GECs compared to CI men. In the follow up to this study we found that active TGF-β, which was found in higher concentrations in SP from CI men and led to decreased inflammatory response from GECs, was compartmentalized between blood plasma and seminal plasma. Higher levels of active TGF-β in SP correlated with decreased semen viral load and the immune activation marker sCD14 leading us to believe that ART-naive CI men in our cohort were naturally controlling their immune activation status, as active TGF-β levels were lower in ART-treated men. Short-term exposure of GECs to SP from CI men or TGF-β at comparable concentrations to SP protected the GEC barrier against HIV by decreasing inflammatory cytokines and preventing tight junction breakage. However, long-term exposure to TGF-β in the presence of HIV further increased inflammation in GECs suggesting a biphasic role for TGF-β in the FGT. This body of work summarized in this thesis demonstrates for the first time how semen from HIV-infected men modulates FGT epithelial cell cytokine responses and barrier function, an important consideration in the design of local therapeutic strategies to protect the FGT against HIV infection. / Thesis / Doctor of Philosophy (Medical Science)

No description available.

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

O objetivo deste trabalho foi a construção de linhagens geneticamente modificadas de B. subtilis capazes de expressar porções de intimina, principal componente envolvido na capacidade de colonização de linhagens enteropatogênicas de Escherichia coli (EPEC), como estratégia vacinal de administração oral contra diarréias infecciosas. As vacinas desenvolvidas empregaram cinco regiões da intimina de EPEC e linhagens de B. subtilis capazes de expressar e acumular proteínas recombinantes no citoplasma. Além disso, avaliamos o uso de esporos e células vegetativas como veículos vacinais para a entrega de antígenos recombinantes a partir de sistema de expressão epissomal. A eficácia do modelo vacinal foi demonstrada pela: (i) produção de anticorpos sistêmicos (IgG) e secretados (sIgA) contra intimina, (ii) capacidade de neutralização das intiminas expressas por diferentes linhagens de EPEC pelos anticorpos específicos gerados nos animais imunizados; e (iii) proteção a desafio com linhagens de EPEC a partir de modelo experimental que emprega camundongos recém-nascidos. Os resultados representam uma etapa importante na validação de uma nova estratégia vacinal para o controle de patógenos entéricos. Além disto, propomos a utilização de um modelo animal como uma nova ferramenta para se avaliar o potencial protetor de vacinas contra EPEC. / The objective of this work was the construction of genetically modified strains of B. subtilis able to express portions of intimin, the main component involved in colonization by enteropathogenic Escherichia coli strains (EPEC) as a strategy of oral vaccination against infectious diarrhea. The vaccines employed five regions of EPEC intimin and B. subtilis strains expressing recombinant proteins in the cytoplasm. Furthermore, we evaluated the use of spores and vegetative cells as vaccine vehicles for the delivery of recombinant antigens based on an epissomal expression system. The efficacy of the vaccines was demonstrated by: (i) production of systemic (IgG) and mucosal (sIgA) antibody responses to intimin, (ii) neutralizing of intimin expressed by different strains of EPEC by the antibodies generated in immunized animals, and (iii) protection to lethal challenges carried out with EPEC strains using an experimental model based in newborn mice. The results represent an important step in the validation of a new vaccine strategy for the control of enteric pathogens. Moreover, we propose the use of an animal model as a new tool to evaluate the protective potential of vaccines against EPEC.

A maioria dos estudos da interação parasito-hospedeiro tem focado na interação de um único patógeno. Porém, o hospedeiro em um ambiente natural é comumente exposto a múltiplos patógenos sequencialmente ou mesmo simultaneamente. Diversos estudos têm utilizado o modelo de Ligadura e perfuração do Ceco (CLP) para estudar a sepse, mas nenhum deles apresentou modelo de coinfecção ou estudo avaliando o papel de infecções prévias no desfecho da sepse polimicrobiana experimental. Neste contexto, nossa hipótese é de que a infecção crônica por parasitos poderia alterar o curso da resposta durante a sepse polimicrobiana. Para testar essa hipótese, animais C57BL/6 ou BALB/c foram infectados com 5 ou 20 cistos da cepa ME 49 de Toxoplasma gondii e 40 dias após a infecção os animais foram induzidos à sepse polimicrobiana. Em nosso estudo, 100% dos animais cronicamente infectados por T. gondii morreram num período de 24 horas após CLP. O mesmo não foi observado quando animais foram infectados cronicamente com os parasitos Leishmania major e Trypanosoma cruzi ou com o fungo Paracoccidioides brasiliensis. Um dado interessante em nosso estudo foi que, nos animais previamente infectados com T. gondii, constatamos melhora na eliminação de bactérias liberadas pela CLP e aumento do recrutamento celular para o sítio da infecção. Apesar de esses animais apresentarem melhora na resposta contra as bactérias, verificamos a presença de lesão intestinal e maior infiltrado inflamatório neste órgão, associado a um aumento da produção de citocinas pró-inflamatórias (IFN-, TNF-, IL-6 e IL-1) e consequente aumento de óxido nítrico (NO), num período de 24 horas depois da CLP. Verificamos que as células TCD4+ e TCD8+ são responsáveis pela produção de IFN- e TNF- nesse modelo de coinfecção, e em modelo in vitro, que macrófagos podem ser responsáveis pela produção de IL-1 dependente de ativação do inflamassoma NLRP3/ASC/Caspase 1. Neste estudo, observamos que a rápida resposta contra a CLP acontece em função da presença de células de memória de padrão Th1, induzidas na infecção por T. gondii. Dessa forma, esse trabalho mostra que a infecção crônica por T. gondii agrava a sepse polimicrobiana subletal, por aumentar a produção de citocinas pró-inflamatórias IL-6, TNF- e IL-1, com a indução de hipotensão, predispondo ao choque séptico. / Most studies of parasite-host interaction have focused on the interaction of a single pathogen with cells or organism of the host. However, in a natural enviroment, the host is commonly exposed to multiple pathogens sequentially or even simultaneously. Several studies have used the model of cecal ligation and puncture (CLP) to study sepsis, but none of them evaluated the effect of the presence of previous infections to the outcome of polymicrobial sepsis. In this context, we hypothesized that chronic infection with Toxoplasma gondii could alter the course of host response against polymicrobial sepsis. To test this hypothesis, C57BL/6 or BALB/c mice were orally infected with 5 or 20 cysts of ME-49 strain of T. gondii and 40 days post infection, they were subjected to CLP. When mice were chronically infected with T. gondii, 100% of the animals died within 24 hours after CLP. The same phenomenons were not observed in animals previously infected with other parasites, such as Leishmania major and Trypanosoma cruzi or the fungus Paracoccidioides brasiliensis. Interestingly, when we evaluated the response against the CLP in animals that were infected with T. gondii, we found an improvement in the killing of bacteria released by CLP and an increase in recruitment of inflammatory cells to the site of infection. However, despite the fact that these animals have improved response against the bacterial infection, they presented intestinal damage and increased inflammatory infiltrate in this organ. The animals also had increased pro-inflammatory cytokines (IFN-, TNF-, IL-6 and IL-1), and nitric oxide (NO) detected within 24 hours after CLP. We also found that the TCD4+ and TCD8+ cells were responsible to produce IFN- and TNF-, and, using an in vitro model, we verified that macrophages are primarily responsible for the production of IL-1 in a pathway dependent on the activation of NLRP3/ASC/Caspase 1 inflamassoma. In this study, we found that early response against CLP happens due to the presence of mainly Th1 memory cells, induced by T. gondii infection. Finally, we found that chronic infection with T. gondii aggravates sublethal polymicrobial sepsis by increasing the cytokines IL-6, TNF- and IL-1, with induction of hypotension that predispose to septic shock.

In the present studies we have explored the link between food hypersensitivity and IgA nephropathy (IgAN) and evaluated treatment options in primary and recurrent disease. Approximately one third of our IgAN patients had a rectal mucosal sensitivity to gluten, as demonstrated by increased local mucosal nitric oxide production and/or myeloperoxidase release after gluten challenge. The gluten sensitivity seemed to be an innate immune reaction unrelated to the pathogenesis of celiac disease. Approximately half of the patients had a rectal mucosal sensitivity to soy or cow’s milk (CM). The levels of IgG antibodies to alfa-lactalbumin, beta-lactoglobulin and casein were significantly higher in CM sensitive as compared with non-sensitive IgAN patients, indicating that an adaptive immune response might be involved in addition to the innate immune reaction observed. With the knowledge of gastrointestinal reactivity enteric treatment was considered as a potential new treatment approach of IgAN. A 6-month prospective trial demonstrated proof-of-concept for the use of enteric budesonide targeted to the ileocaecal region of IgAN patients. We observed a modest, but significant reduction in urine albumin, a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation. eGFR calculated from the Cockcroft-Gault formula and cystatin C was not changed. In a retrospective study recurrence of IgAN and graft loss was evaluated in Norwegian and Swedish patients having received a primary renal transplant due to IgAN. Adjusting for relevant covariates, a multiple Cox-regression analysis on time to IgAN recurrence showed that use of statins was associated with reduced risk of recurrence and reduced risk of graft loss. The time lag from diagnosis to first transplantation and female gender were also associated with lower risk of recurrence. Improved graft survival was associated with related donor, low donor age and no or low number of acute rejection episodes.