Development of combination products such as tissue engineered constructs which combine biomaterials with biologics has prompted the need to clarify the role of biomaterial in potentiating the immune response towards the biological component due to adjuvant effect. In tissue engineering applications, immune responses are to be minimized while vaccine strategies seek to enhance the protective immune response. Thesis project presented herein showed that adjuvant effect of poly(lactic-co-glycolic acid) (PLGA) is mediated in part by maturation of dendritic cells (DCs), immune cells that orchestrate adaptive immune response. Maturation of human peripheral blood monocyte-derived DCs in response to PLGA contact was demonstrated in vitro and in vivo by increased co-stimulatory and MHC molecule expression, mixed lymphocyte reaction, cytokine release, and delayed type hypersensitivity reaction. In contrast to PLGA, agarose did not induce DC maturation, in accordance with its low inflammatory effect. Roles of various receptors involved in DC maturation and recognition of biomaterials were assessed by in vitro receptor blocking studies. In particular, role of Toll-like receptors were further investigated using DCs derived from bone marrows of murine model of Toll-like receptor 4 deficiency (C3H/HeJ). While PLGA induced maturation of DCs from C57BL6 mice, maturation was not observed in DCs from C3H/HeJ strain or control strain, C3H/HeOuJ, perhaps due to particular haplotypes of these animals. Collectively, these results establish the differential adjuvant effects of agarose and PLGA on the level of DC maturation, and begin to elucidate the mechanisms of biomaterial adjuvant effect. In addition, assays developed herein provide methods to screen for biomaterials to be used in combination products, such that biomaterials with desired levels of adjuvanticity as measured by DC maturation effects may be selected for given application.

The host response toward biomaterial component of tissue-engineered devices has been extensively investigated. The objective of this research was to understand the response of dendritic cells (DCs) to different biomaterials upon contact and identify biomaterials suitable for use in tissue engineering constructs for rheumatoid arthritis (RA) applications. Differential levels of functional DC maturation were observed depending on the type of biomaterial in 2-dimensional films or 3-dimensional scaffolds used to treat immature DCs; Poly(lactic-co-glycolic acid) (PLGA) or chitosan supported higher levels of DC maturation, as compared to immature DCs. Alginate supported moderate levels of DC maturation. Agarose did not support DC maturation whereas hyaluronic acid inhibited DC maturation. Further, these DCs treated with different biomaterials induced differential phenotype and polarization of autologous T cells upon co-culture of DCs and T cells; DCs treated with PLGA induced T helper type I with immunogenic response while DCs treated with agarose did T helper type II with tolerogenic response. Effect of different biomaterials (PLGA and agarose) was assessed in vivo upon implantation of them into the knee joint of RA-induced rabbit. Total leukocyte concentrations in the peripheral blood or in the joint lavage of the left knees (untreated control) were observed in differential levels depending on the biomaterial implant, possibly due to the systemic circulation of the peripheral blood. Furthermore, cartilage and bone healing progression was differentially observed in the osteochondral defect of the knee joint of RA-induced rabbit, depending on type of biomaterial scaffold implanted into the defect. Collectively, these results demonstrate the multifunctional impacts of inherently different biomaterials on in vitro immunomodulation of phenotype and polarization of DCs and autologous T cells. Furthermore, taken together with these immunomodulatory impacts of biomaterials, in vivo effects of different biomaterial scaffolds on RA environment shown in this study can suggest the criteria of selection and design of biomaterials for orthopedic tissue engineering, which may ultimately be best integrated into the diseased cartilage and bone.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί νόσημα με διαταραχή σε επίπεδο αρχέγονου αιμοποιητικού κυττάρου (stem cell) που χαρακτηρίζεται από μη αποδοτική αιμοποίηση και κυτταροπενίες του περιφερικού αίματος που περιλαμβάνουν μία ή περισσότερες αιμοποιητικές σειρές. Διάφορες ανοσολογικές διαταραχές των ασθενών με ΜΔΣ, όπως, αυξημένη ευαισθησία σε βακτηριακές λοιμώξεις, αυτοάνοσα φαινόμενα και υψηλή συχνότητα κακοηθειών του λεμφικού ιστού, υποδεικνύουν αδυναμία των ασθενών με ΜΔΣ για ανοσολογική απάντηση, οι αιτίες των οποίων παραμένουν άγνωστες μέχρι σήμερα. Τα Δενδριτικά Κύτταρα (ΔΚ) είναι κύτταρα του ανοσολογικού μηχανισμού που προέρχονται από το μυελό των οστών. Ως αντιγονοπαρουσιαστικά κύτταρα (APC), είναι εξειδικευμένα για τη πρόσληψη, επεξεργασία, μεταφορά και παρουσίαση του αντιγόνου στα Τ λεμφοκύτταρα. Στη παρούσα μελέτη πραγματοποιήθηκε ανάλυση διαφορετικών ποσοτικών και λειτουργικών παραμέτρων των ΔΚ από ασθενείς με Μυελοδυσπλαστικό Σύνδρομο, in vivo ή in vitro. Αρχικά διερευνήθηκε ο αριθμός, ο φαινότυπος, η ικανότητα ενδοκύττωσης και η αλλογενής διεγερτική δυνατότητα των ΔΚ, προερχόμενων από μονοκύτταρα του περιφερικού αίματος (ΜοΔΚ) ασθενών με ΜΔΣ και υγιών μαρτύρων, σε διαφορετικά στάδια διαφοροποίησης. Τα μονοκύτταρα των ασθενών με ΜΔΣ χαρακτηρίστηκαν από μειωμένη ικανότητα διαφοροποίησης σε ΔΚ, λόγω του μειωμένου αριθμού των διαφοροποιημένων κυττάρων και τη χαμηλή έκφραση του CD1a αντιγόνου επιφανείας. Τα ΜοΔΚ των ΜΔΣ ασθενών παρουσίασαν χαμηλή έκφραση του υποδοχέα της μανόζης και μειωμένη ικανότητα ενδοκύττωσης. ΜοΔΚ των ΜΔΣ ασθενών επέδειξαν μειωμένη απάντηση ύστερα από διέγερση με TNF-α, καθώς η έκφραση των CD83, CD80 και CD54 αντιγόνων και η αλλοδιεγερτική ικανότητα ήταν μειωμένη, ενώ η επίδραση με LPS είχε ως αποτέλεσμα να εμφανίσουν φαινοτυπικά χαρακτηριστικά και ικανότητα διέγερσης των Τ-κυττάρων, όμοια με τα ΜοΔΚ των φυσιολογικών μαρτύρων. Σε δύο από τους ασθενείς με σύνδρομο 5q-, σχεδόν όλα τα μονοκύτταρα και τα ΜοΔΚ περιείχαν τη χρωμοσωμική διαταραχή, υποδηλώνοντας την προέλευσή τους από τον παθολογικό κλώνο. Στη συνέχεια διερευνήθηκε το δυναμικό πολλαπλασιασμού και διαφοροποίησης των CD34+ προγονικών κυττάρων του μυελού ασθενών με ΜΔΣ σε δενδριτικά κύτταρα (CD34-ΔΚ) σε υγρή καλλιέργεια παρουσία κυτοκινών. Παράλληλα, έγινε ανάλυση των κυκλοφορούντων ΔΚ περιφερικού αίματος στους ίδιους ασθενείς. Τα CD34+ προγονικά κύτταρα παρουσίασαν χαμηλή δυνατότητα ανάπτυξης ΔΚ in vitro, καθώς ο αριθμός των παραγόμενων ΔΚ ανά CD34+ κύτταρο ήταν χαμηλότερος συγκριτικά με τα δείγματα των υγιών μαρτύρων. Παρά την αυξημένη απόπτωση των προγονικών κυττάρων του μυελού των ΜΔΣ ασθενών, η επιβίωση και ο πολλαπλασιασμός των CD34+ κυττάρων στην καλλιέργεια, δεν συσχετίστηκε με την απόπτωση και αποτελεί αξιοσημείωτη παρατήρηση. Φαινοτυπικά, τα CD34-ΔΚ των ΜΔΣ ασθενών δεν διέφεραν από τα ΔΚ που παρήχθησαν από τα CD34+ κύτταρα του μυελού των φυσιολογικών μαρτύρων καθώς επέδειξαν όμοια έκφραση των CD83, CD80, CD40, HLA-DR και CD54 αντιγόνων. Κυτταροεπιλεγμένα CD1a+ κύτταρα ασθενών είχαν όμοια διεγερτική ικανότητα αλλογενών Τ κυττάρων με τα CD34-ΔΚ των φυσιολογικών ατόμων. Το ποσοστό των κυκλοφορούντων μυελοειδών- και πλασματοκυτταροειδών- ΔΚ στους ασθενείς με ΜΔΣ ήταν σημαντικά μειωμένο συγκριτικά με τους υγιείς μάρτυρες. Στους ασθενείς με 5q έλλειψη, τόσο τα CD34-ΔΚ, όσο και τα ΔΚ του αίματος, είχαν τη χρωμοσωμική ανωμαλία. Τα παραπάνω αποτελέσματα υποδηλώνουν ότι η διαδικασία παραγωγής δενδριτικών κυττάρων από το μυελό (‘δενδριτοποίηση’) των ασθενών με ΜΔΣ, είναι μέρος της κλωνικής διαταραχής με αποτέλεσμα την μη αποδοτική παραγωγή ΔΚ από τα προγονικά κύτταρα του μυελού και το χαμηλό ποσοστό των κυκλοφορούντων πρόδρομων ΔΚ. Όλες οι ΔΚ υποομάδες προέρχονται από τον παθολογικό κλώνο και χαρακτηρίζονται από ποσοτικές και ποιοτικές ανωμαλίες. Το σύνολο αυτών των διαταραχών που παρατηρήθηκαν στα ΔΚ πολύ πιθανόν να συμβάλει στη διαταραγμένη ανοσολογική απάντηση έναντι παθογόνων οργανισμών, στην επιβίωση και στην επικράτηση του παθολογικού κλώνου, όπως επίσης και στην εμφάνιση αυτοάνοσων φαινομένων, που παρατηρούνται στους ασθενείς με ΜΔΣ. / Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis and blood cytopenias involving one or several myeloid lineages. Various immune disturbances in MDS such as increased susceptibility to bacterial infections, autoimmune phenomena and high incidence of lymphoid malignancies reveal an underlying defect of the immune response in MDS patients, the reasons for which still remain unclear. Dendritic cells (DCs) are bone marrow derived cells. As the most potent antigen presenting cells (APC), they are specialized for the uptake, processing, transport and presentation of Ag to T cells. In the present study different quantitative and functional parameters of DCs in patients with MDS were analyzed either in vivo or in vitro. The number, phenotype, endocytic ability, and allostimulatory capacity of DCs derived from peripheral blood monocytes (MoDCs) were investigated in patients with MDS and healthy controls at different stages of differentiation using the maturation stimuli-TNF-á and LPS. Monocytes in MDS showed low potential to differentiate into DCs, as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of Mannose receptor and reduced endocytic capacity. When stimulated with TNF-á, MoDCs obtained from MDS patients showed a diminished response with low CD83, CD80 and CD54 expression and allostimulatory capacity, whereas in the presence of LPS MDS-MoDCs acquired phenotypic characteristics and ability to stimulate T-cells similar to MoDCs derived from controls. In two patients with 5q- syndrome the vast majority of both monocytes and MoDCs were positive for the 5q deletion, suggesting that they originate from the malignant clone. Second, we investigated the potential of bone marrow CD34+ progenitors in patients with MDS to proliferate and differentiate into DCs in a liquid cytokine supplemented culture system and also analyzed the status of blood DC subsets in those patients. CD34+ progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34+ cell was significantly lower compared to controls. Interestingly, although the increased apoptotic level of bone marrow progenitors in MDS, the survival and proliferation of CD34+ cells in culture was not correlated to the degree of apoptosis. Phenotypically the MDS CD34-DCs did not differ from DCs obtained from normal BM CD34+ cells, exhibiting similar expression of CD83, CD80, CD40, HLA-DR, and CD54. FACsorted CD1a+ cells from MDS patients were as efficient stimulators of allogeneic T cells as normal CD34-DCs. The percentage of both circulating DC subsets, MDCs and PDCs in MDS patients was extremely diminished compared to controls. In cases with the 5q deletion both CD34-DCs and blood DCs harbor the cytogenetic abnormality. The results indicate that “dendritopoiesis” in MDS is affected by the transformation process resulting in ineffective production of DCs from bone marrow progenitors with low circulating blood precursors. All DC subsets were derived from the malignant clone and exhibited quantitative and qualitative abnormalities. This constellation of DCs defects probably contribute to the defective immune response against pathogens, escape and expansion of the malignant clone, as well as autoimmune phenomena, observed in MDS patients.

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer. / The idea that a cell could kill transformed cells, like a Natural Killer (NK) cell, all the while exhibiting also the capacity to present antigens to T cells, like a Dendritic Cell (DC), may seem farfetched. However, in mice, a cell presenting these specific properties was identified in 2006. These cells were named Interferon-producing Killer Dendritic Cells (IKDC) and extensive studies revealed that they were endowed with an important immunological potential. Indeed, the fact that IKDCs exhibit properties of both DC and NK cells conferred them with an exceptional anti-tumor potential. Notably, on a per cell basis, the in vivo anti-tumor activity of IKDCs is more efficient than NK cells. Therefore, following their identification, IKDCs showed great therapeutic promise. However, a debate on the cell lineage origin of IKDCs emerged. Several independent groups could not replicate the finding that IKDCs showed functional antigen-presentation properties similar to DCs. Also, additional studies revealed that IKDCs are very similar to NK cells. These and other observations led the scientific community to believe that IKDCs were activated NK cells. Despite this controversy, IKDCs clearly exhibit a unique and outstanding anti-tumor potential, highlighting the relevance to further explore these cells. We must first close the debate regarding the lineage origin of IKDCs. We subsequently need to identify a means to specifically target IKDCs to facilitate their use in novel anti-tumor therapies. Thus, the objective of my thesis is first, to define the identity of IKDCs and second, to determine the genetic factors implicated in the regulation of these cells. For the first objective, we demonstrated that IKDCs do not represent activated NK cells, as previously suggested. We show that IKDCs are highly proliferative and exhibit a unique phenotype associated with very immature NK cells. In an attempt to verify if IKDCs could acquire a mature phenotype, we conducted an adoptive transfer experiment. We found that, after adoptive transfer, IKDCs adopt a mature phenotype, but also surprisingly differentiate into NK cells. These findings indicate that IKDCs represent an intermediate in NK-cell differentiation. For the second objective, we demonstrated that the IKDC proportion was highly variable between strains of different background origins, indicating that these cells are regulated by genetic factors. A genetic study revealed that genetic factors in distal arm of chromosome 7 associate with the proportion of IKDCs. The results presented in this thesis represent an important breakthrough for the research on IKDCs. They allowed to define the cell lineage origin of IKDCs and to identify a genetic region involved in the regulation of this cell type. These discoveries are valuable knowledge for the identification of human IKDCs and the development of novel anti-tumor therapies.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Habib Zaghouani. "May 2008" Includes bibliographical references.

Orientador: Amauri Garcia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-08-09T07:59:55Z (GMT). No. of bitstreams: 1 Rosa_DanielMonteiro_D.pdf: 8184541 bytes, checksum: 0b8d932ad9a1091a507ea6b10e4185f8 (MD5) Previous issue date: 2007 / Resumo: As morfologias das estruturas de solidificação, caracterizadas principalmente por arranjos celulares e dendríticos, e suas grandezas representadas por espaçamentos celulares e dendríticos primários, secundários e terciários, controlam os perfis de segregação e a formação de segundas fases dentro das regiões intercelulares ou interdendríticas, que determinam as propriedades finais das estruturas fundidas. O presente trabalho pretende contribuir para o entendimento do desenvolvimento microestrutural de ligas binárias através da análise de dois sistemas binários que possuem elevada importância para a indústria na fabricação de peças fundidas automotivas e grades de baterias: Al-Si e Pb-Sb, respectivamente. Os experimentos realizados utilizaram dois diferentes dispositivos em que o calor é extraído somente pelo sistema de resfriamento a água, localizado no fundo (solidificação ascendente) e no topo (solidificação descendente) da lingoteira. As variáveis térmicas de solidificação foram determinadas pela leitura de temperaturas a partir de termopares posicionados dentro da lingoteira em diferentes posições em relação à superfície refrigerada. Estas variáveis térmicas foram confrontadas com as previsões teóricas de um modelo numérico de solidificação. Os aspectos macroestruturais e microestruturais foram caracterizados ao longo dos lingotes através de microscopia óptica. Para as ligas Al-Si foi realizada uma análise complementar do efeito da convecção térmica e constitucional nos espaçamentos dendríticos terciários na solidificação unidirecional transitória descendente. Ligas hipoeutéticas Pb-Sb foram utilizadas para analisar as influências das variáveis térmicas de solidificação e da concentração de soluto nas estruturas celulares, na transição celular/dendrítica e nas estruturas dendríticas. Os espaçamentos celulares e dendríticos foram comparados com as previsões teóricas fornecidas pelos principais modelos de crescimento estacionário e transitório da literatura. Foram também examinados os efeitos da taxa de resfriamento no crescimento celular da liga Pb 0,85%Sb e a influência do tamanho celular e do perfil de macrossegregação correspondente na resistência à corrosão / Abstract: The morphologies of as-cast microstructures, characterized mainly by cellular and dendritic patterns, and their scales represented by primary, secondary and tertiary arm spacings, control the segregation profiles and the formation of secondary phases within intercellular and interdendritic regions, which determine the final properties of castings. The present work aims to contribute to the understanding of microstructural development of binary alloys by analyzing two binary systems, which possess high industrial importance in the manufacture of as-cast automotive components and battery grids: Al-Si and Pb-Sb, respectively. Experiments have been carried out by using two castings assemblies, which were designed in such way that heat was extracted only through the water-cooled system, located at the bottom (upward solidification) and at the top (downward solidification) of the casting. The solidification thermal variables have been determined from thermal readings acquired by thermocouples located inside of the casting in different positions from the cooled surface. Such experimental thermal variables have been compared with theoretical predictions of a numerical model of solidification. Macrostructural and microstructural aspects along the casting were characterized by optical microscopy. For Al-Si alloys a complementary analysis of the influence of thermosolutal convection on the tertiary dendrite arm spacing during the downward unsteady-state directional solidification has been carried out. Hypoeutectic Pb-Sb alloys have been used to analyze the influences of solute concentrations and solidification thermal variables in the development of cellular structures, the cellular/dendritic transition and dendritic structures. Experimental cellular and dendritic spacings have been compared with the theoretical predictions furnished by the main steady-state and unsteady-state growth models from the literature. The effect of cooling rate on the cellular growth of a Pb 0.85wt%Sb alloy and the influences of cell size and of the corresponding macrosegregation profile on the resultant corrosion behavior have also been examined. / Doutorado / Materiais e Processos de Fabricação / Doutor em Engenharia Mecânica

<p>The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Orientador: Iracilda Zeppone Carlos / Resumo: A esporotricose é uma micose de distribuição universal causada por fungos termodimórficos do complexo de espécies Sporothrix schenckii (S. schenckii). No Brasil, a esporotricose é considerada endêmica, sendo normalmente adquirida pela inoculação acidental do seu agente causal através da pele ou através da transmissão zoonótica por gatos infectados. As formas clínicas podem variar entre cutânea, linfocutânea e sistêmica, esta última sendo mais comumente observada em pacientes imunodeprimidos. A ineficácia do tratamento antifúngico contra esta micose, especialmente em pacientes imunocomprometidos, tem levado à busca de terapias mais eficazes e seguras. Com base em vários estudos que mostram a eficiente utilização de células dendríticas como ferramenta para o desenvolvimento de vacinas contra diferentes fungos, o objetivo deste trabalho foi avaliar a capacidade protetora de células dendríticas derivadas da medula óssea (BMDCs) ativadas com as proteínas da superfície celular de S. schenckii (PSCs) em camundongos infectados com S. schenckii strictu sensu. As BMDCs foram estimuladas com PSCs e analisadas quanto à expressão superficial de moléculas co-estimulatórias, bem como à secreção de citocinas pró-inflamatórias. Posteriormente, camundongos sádios foram vacinados com uma ou duas doses de BMDCs para avaliar a sua imunogenicidade e, por último, foi avaliado o efeito das BMDCs em camundongos infectados por S. schenckii. Nossos resultados mostram que as PSCs foram capazes de ativar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sporotrichosis is a universally distributed mycosis caused by thermodimorphic fungi of the Sporothrix schenckii (S. schenckii) species complex. In Brazil, sporotrichosis is considered endemic and is usually acquired by accidental inoculation of its causative agent through the skin or through zoonotic transmission by infected cats. Clinical forms may vary between cutaneous, lymphocutaneous and systemic, the latter being more commonly observed in immunosuppressed patients. The ineffectiveness of antifungal treatment against this mycosis, especially in immunocompromised patients, has led to the search for more effective and safe therapies. Based on several studies showing the efficient use of dendritic cells as a tool for the development of different fungal vaccines, the aim of this work was to evaluate the protective capacity of bone marrow derived dendritic cells (BMDCs) activated with cell surface proteins of S. schenckii (ScCWP) in mice infected with S. schenckii strictu sensu. The BMDCs were stimulated with PSCs and analyzed for surface expression of costimulatory molecules and the secretion of proinflammatory cytokines. Subsequently, healthy mice were vaccinated with one or two doses of BMDCs to assess their immunogenicity, and finally the effect of BMDCs on S. schenckii infected mice was evaluated. Our results show that the ScCWPs were able to activate BMDCs. Immunization of healthy mice with ScCWPs-stimulated BMDCs induced a Th17 profile immune response, with increased T... (Complete abstract click electronic access below) / Doutor

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Cholera is a waterborne disease caused by toxigenic strains of Vibrio cholerae. The spread of cholera in developing countries has largely been imputed to the unavailability of proper water treatment and sanitary infrastructure as well as poor hygiene. In order to prevent the contraction and spread of cholera the use of solar disinfection (SODIS) to treat water in waterborne endemic communities has been recommended by the World Health Organization (WHO). SODIS is a water sterilizing method that relies on natural sunlight to improve the microbiological quality of water. During SODIS the culturability of the water contaminating microorganisms is inactivated by the ultraviolet component of solar radiation. The success of SODIS treatment of water in alleviating the risks associated with the contraction of waterborne diseases such as cholera has been attributed to the effectiveness, with which the water is treated, simple application as well as low cost of materials required. Currently SODIS research has been dominated by studies geared towards understanding how the microbial inactivation occurs, enhancement of the disinfection process and health impact assessments. However, little to no research has been directed towards exploring the role played by the immune system following the consumption of the solar irradiated water pathogens such as V. cholerae. SODIS of microorganisms in water results in immunologically important microbial states and components that could induce an immune reaction or response. In view of the role of dendritic cells in shaping an immune response, the effect that solar irradiated V. cholerae in water has on the immunochemistry of the dendritic cells in vitro was investigated. Prior to the stimulation of the dendritic cells with the solar irradiated cultures of V. cholerae, the first objective required an evaluation on the impact that solar irradiation has on the production and secretion of the cholera toxin by V. cholerae in water. The results from this evaluation showed that solar ultraviolet radiation was incapable of inducing the secretion of the cholera toxin. Furthermore, there was extensive DNA degradation in the solar irradiated cultures of V. cholerae. The second objective was to investigate the ability for solar irradiated cultures of V. cholerae in water to induce the phenotypic maturation of immature dendritic cells in vitro. In order to achieve this objective, solar and non-solar irradiated, chemically/ heat inactivated and phosphate buffered saline (PBS) prepared cultures of V. cholerae as well as lipopolysaccharide (LPS) and cholera toxin-β (CTB) subunit were each used to stimulate immature dendritic cells. After 48 hours of stimulation the dendritic cells were assessed for the expression of CD54, CD80, CD83, CD86, MHC-I and MHC-II on their cell membrane. The results showed an increase in the expression of all the maturation phenotypic markers with CD54, CD86 and MHC-I being the most prominent ones on the surface of the dendritic cells stimulated with solar irradiated cultures of V. cholerae. The third objective was to assess the profile of the cytokines and chemokines secreted by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of dendritic cell stimulation the tissue culture media from each treatment was quantitatively and qualitatively analysed for the presence of interleukin (IL)-1α, IL-1β, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-23, IL-27, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and tumor necrosis factor (TNF)-α. The analysis revealed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore the profile of the cytokines and chemokines secreted by the dendritic cells in response to the solar irradiated cultures of V. cholerae in water was similar to that required to induce a T- helper (Th) Th2 immune response. The fourth objective was to assess the expression of the toll like receptor (tlr) genes by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of stimulation total RNA was extracted from the dendritic cells and subjected to real time quantitative polymerase chain reaction (RT qPCR) assay for tlr 1, 2, 3, 4, 5, 6, 9, 11, 12 and 13. The results showed no significant increase or decrease in the expression of most tlr genes in comparison to the unstimulated dendritic cells. This observation is synonyms with dendritic cell maturation. Taken together these findings show that solar irradiated cultures of V. cholerae were able to induce the maturation of immature dendritic cells in vitro. Furthermore dendritic cells stimulated with solar irradiated cultures of V. cholerae produced pro-inflammatory cytokines and chemokines. The results from this study suggests that the consumption of SODIS treated could provide immunological benefits.

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