[Truncated abstract] A paradox exists in tumour immunology whereby progressive tumour growth exists in parallel with an anti-tumour T cell response. This defective T cell response is thought to result from the induction of T cell tolerance and/or tumour induced immunosuppression, which act to inhibit the activation, differentiation and function of tumour-specific CD8+ T cells. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are critical to the generation of effective CTL; however their function and phenotype is often defective or altered in tumour-bearing hosts, which may limit their capacity to mount an effective tumour specific T cell response. In this thesis, the role of DCs in the cross-presentation of tumour antigen was assessed in terms of their APC function, migration and location. In doing so the intention was to gain insight into the early processes that potentially contribute to the development of an ineffective anti-tumour immune response. This study examined cross-presentation of the nominal tumour antigen, influenza A hemagglutinin (HA) expressed by the murine malignant mesothelioma cell line, AB1-HA. Cross-presentation was predominantly restricted to the local draining lymph nodes throughout tumour growth and was mediated by CD8a+ and CD8a- DCs. This results in an ineffective CTL response due to the lack of DC activation and the presence of potentially immunosuppressive B7 molecules. However, the capacity of the CD8a- DC subset to cross-present antigen suggested a role for migratory tumour-resident DCs in this process. Analysis of tumour infiltrating DCs showed that they were paralysed in their capacity to cross-present tumour antigen and were immobilised at the tumour site. Conversely, cross-presentation of tumour antigen in the local draining lymph node was dependent on the continuous traffic of antigen from the tumour microenvironment. In this vein, small numbers of metastatic tumour cells were detected in the draining lymph nodes, however their isolation was dependent on the removal of DCs and T cells, suggesting immune control of metastatic spread. Thus, tumour cells may be the source of antigen for cross-presentation by DCs in the tumour draining lymph nodes. .... In conclusion, the results presented in this thesis support a role for DCs in the generation of tumour-specific T cell responses that fail to control tumour growth. In addition the results provide a basis for further investigation into the effects of chemotherapy on the source and form of tumour antigen for cross-presentation by specific DC subsets in the tumour bearing host. These findings may have important implications for the development of future anti-cancer immune therapies targeting DCs.

Hepatitis B virus (HBV) is a life-threatening pathogen with major economic significance. Acute infection in adults is common, albeit usually self-limiting. Importantly, infection in infants typically results in chronic infection and increased incidence of hepatocellular carcinoma (HCC). Furthermore, the infectious carrier state is perpetuated in chronically infected individuals. Successful immuno-therapeutic vaccination would reduce the incidence of chronic infection and of HCC as well as reduce transmission of the disease. Recovery from acute and chronic HBV infection typically occurs in the presence of robust antigen-specific humoral and cellular immune responses (CMI), whereas these responses are low or absent in chronically HBV-infected individuals. Therefore, it was hypothesised that effective stimulation of both humoral and CMI responses, in conjunction with currently available antiviral therapies, may contribute significantly to development of vaccines for treatment of chronic HBV infection. The duck hepatitis B virus (DHBV) model of HBV infection was used to test novel vaccine strategies that could complement existing antiviral therapeutic approaches to treat chronically HBV-infected humans. To this end, three separate vaccine studies were conducted to investigate potential therapeutic regimes. Methods to assess the efficacies of the vaccine strategies included immunoperoxidase detection of viral antigen and immune cell markers within the liver and development of sensitive assays to monitor levels of DHBV DNA, duck hepatitis B virus surface antigen (DHBsAg), antibodies to duck hepatitis B core (anti-DHBc) and surface antigens (anti-DHBs) in serum were developed and validated which allowed monitoring of the kinetics of the humoral immune response following vaccination and the course and outcome of experimental DHBV infection. The first vaccine study tested the protective efficacy of DNA vaccines encoding either the small form of DHBsAg (DHBs) protein or the larger antigen (DHBpre-S/S), These were administered to ducks at day 4 and 14 of age. On the same day as the second vaccination, ducks were challenged intravenously with DHBV. Immunoperoxidase staining of biopsy tissue collected at day 4 p.i. showed significant decreases in the number of DHBV infected hepatocytes in ducks receiving the DNA vaccines compared to the mock-vaccinated control ducks. Significant protection against development of chronic DHBV infection was observed in ducks vaccinated with DNA vaccines expressing either pre-S/S or S protein. Although anti-DHBs antibodies were not detected prior to DHBV challenge, the decrease in the percentages of DHBV-infected hepatocytes at day 4 p.i is suggestive that neutralisation of the inoculum by low-level anti-DHBs antibodies in cohort with CMI responses induced by vaccination were the most probable mechanisms of action. The second vaccine study examined the protective efficacy of a novel whole-cell vaccine that expressed the DHBV core antigen (DHBcAg). Ducks were vaccinated on day 4 and 14 of age and DHBV challenge was administered 4 days later. Detectable anti-DHBc antibodies were generated as soon as 4 days after the initial vaccination suggesting that this regimen elicited increased immunogenicity than vaccination with DNA vaccines alone. In contrast to the first vaccine study with DNA vaccines expressing DHBsAg, no significant differences in the percentage of DHBV-infected hepatocytes were observed in biopsy tissue collected at day 4 p.i.This finding is confirmation that anti-DHBc antibodies were not neutralising to the initial DHBV inoculum. However, significant protection against development of chronic DHBV infection was observed in the whole-cell vaccinated ducks suggesting that the mechanism of protection was consistent immune-mediated killing of DHBV-infected hepatocytes following CMI responses to determinants of DHBcAg. The final vaccine study involved a combination strategy of antiviral drug Entecavir (ETV) and prime-boost vaccination with DNA vaccines and recombinant fowlpoxvirus (rFPV) expressing DHBV antigens. Immediately following DHBV infection, ducks were dosed by oral gavage with the antiviral drug Entecavir (ETV) and at the same time received the priming DNA vaccines encoding DHBV antigens. Seven days later the boosting vaccination consisting of recombinant fowlpox viruses (rFPV) also expressing DHBV antigens was administered. Extraordinary protection was observed, with 100% of ducks given combination therapy rapidly resolved their DHBV infection while 100% of non-treated ducks developed chronic infection. It was concluded that protection resulted from a combination of at least three factors. First, reduction and control of DHBV levels with the aid of ETV; secondly, stimulation of surface antigen-specific humoral immune responses resulting in neutralisation of newly produced virions; and finally, the combined up-regulation of CMI responses against DHBV core and surface antigens, resulting in elimination of infected hepatocytes. The four manuscripts that comprise this thesis provide insights into the viral kinetics and immune responses that follow DHBV infection and/or vaccination of ducks. The results provide new directions for future vaccine studies aimed at developing effective treatments for chronic HBV infection. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.

Orientador: Márcia Guimarães da Silva / Banca: Maria Terezinha Serrão Peraçoli / Banca: José Antonio Simões / Resumo: A Rotura Prematura de Membranas Pré-Termo (RPM-PT) é um dos principais problemas da Clínica Obstétrica, com etiologia relacionada à ascensão bacteriana do trato genital inferior para a decídua e membranas corioamnióticas. Objetivo: quantificar a expressão das citocinas inflamatórias interleucina (IL) -1b, IL-6, IL-8 e fator de necrose tumoral (TNF-a) pelas membranas corioamnióticas de gestantes com RPM-PT, e avaliar a correlação dessa expressão com a presença e intensidade do infiltrado inflamatório nas membranas e decídua. Material e Métodos: Foram incluídas no estudo, 25 gestantes com RPM-PT em trabalho de parto e 15 gestantes com RPM-PT fora do trabalho de parto. Como grupo controle foram avaliadas 25 gestantes em trabalho de parto prematuro (TPP) e bolsa íntegra. No momento da resolução da gestação, após a dequitação, foram retirados fragmentos das membranas corioamnióticas e acondicionados em RNA later para posterior quantificação do RNA mensageiro (RNAm) das citocinas inflamatórias pela técnica de PCR em tempo real. Outros fragmentos das membranas foram submetidos à análise histopatológica para avaliação da presença e semi-quantificação do infiltrado inflamatório. Resultados: No período do estudo, a incidência de RPM-PT foi de 4,6%. A presença de infiltrado inflamatório nas membranas corioamnióticas e/ou decídua foi de 75% no grupo RPM-PT. A concentração relativa de RNAm de IL-1b, IL-6 e IL-8 não foi estatisticamente diferente nos grupos estudados. Para TNF-a, a concentração relativa de RNA foi estatisticamente superior nas membranas de gestantes com TPP em relação aos grupos com RPM-PT. Na presença de infiltrado inflamatório nas membranas corioamnióticas, a concentração de RNAm de IL-1b foi estatisticamente maior no grupo RPM-PT fora de trabalho de parto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The preterm premature rupture of membranes (PPROM) is one of the major problems of Clinical Obstetrics. Its etiology is related to the ascending pathway of bacteria from the lower genital tract to the decidua and chorioamniotic membranes. Objective: To quantify the expression of the inflammatory cytokines interleukin-1 (IL) -1b, IL-6, IL-8 and tumor necrosis factor alpha (TNF-a) in the chorioamniotic membranes of pregnant women with PPROM and evaluate the correlation of this expression with the presence and intensity of the inflammatory infiltrates present in the membranes and decidua. Material and Methods: Twenty-five PPROM women in labor and 15 PPROM without labor women were studied. As a control group of 25 pregnant women in premature labor (PTL) were studied. After delivery, samples of the chorioamniotic membranes were collected for histopathological analyses and others fragments were conditioned in RNA later for posterior quantification of cytokine mRNA expression by real time PCR. Results: In the study period, the incidence of PPROM was 4.6% and in 75% of these samples the presence of inflammatory infiltrates in the chorioamniotic membranes and/or decidua was observed. mRNA expression of IL-1b, IL-6 and IL-8 was not statistically different in the groups studied. For TNF-a, the expression of mRNA was statistically higher in the PTL group in relation to the groups with PPROM. In the presence of inflammatory infiltrates in the membranes, the concentration of mRNA of IL- 1b it was statistically greater in the PPROM without labor group. No difference occurred in the intensity of cytokine mRNA in relation to the intensity of the infiltrated inflammatory in the groups studied. In 87.2% of the chorioamniotic membranes included in the study, mRNA was expressed for all the cytokines studied... (Complete abstract, click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:29:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-16Bitstream added on 2014-06-13T19:59:17Z : No. of bitstreams: 1 polettini_j_me_botfm.pdf: 2194860 bytes, checksum: a731ea318ab122b9bf8331b8c84cfef9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Rotura Prematura de Membranas Pré-Termo (RPM-PT) é um dos principais problemas da Clínica Obstétrica, com etiologia relacionada à ascensão bacteriana do trato genital inferior para a decídua e membranas corioamnióticas. Objetivo: quantificar a expressão das citocinas inflamatórias interleucina (IL) -1b, IL-6, IL-8 e fator de necrose tumoral (TNF-a) pelas membranas corioamnióticas de gestantes com RPM-PT, e avaliar a correlação dessa expressão com a presença e intensidade do infiltrado inflamatório nas membranas e decídua. Material e Métodos: Foram incluídas no estudo, 25 gestantes com RPM-PT em trabalho de parto e 15 gestantes com RPM-PT fora do trabalho de parto. Como grupo controle foram avaliadas 25 gestantes em trabalho de parto prematuro (TPP) e bolsa íntegra. No momento da resolução da gestação, após a dequitação, foram retirados fragmentos das membranas corioamnióticas e acondicionados em RNA later para posterior quantificação do RNA mensageiro (RNAm) das citocinas inflamatórias pela técnica de PCR em tempo real. Outros fragmentos das membranas foram submetidos à análise histopatológica para avaliação da presença e semi-quantificação do infiltrado inflamatório. Resultados: No período do estudo, a incidência de RPM-PT foi de 4,6%. A presença de infiltrado inflamatório nas membranas corioamnióticas e/ou decídua foi de 75% no grupo RPM-PT. A concentração relativa de RNAm de IL-1b, IL-6 e IL-8 não foi estatisticamente diferente nos grupos estudados. Para TNF-a, a concentração relativa de RNA foi estatisticamente superior nas membranas de gestantes com TPP em relação aos grupos com RPM-PT. Na presença de infiltrado inflamatório nas membranas corioamnióticas, a concentração de RNAm de IL-1b foi estatisticamente maior no grupo RPM-PT fora de trabalho de parto... / The preterm premature rupture of membranes (PPROM) is one of the major problems of Clinical Obstetrics. Its etiology is related to the ascending pathway of bacteria from the lower genital tract to the decidua and chorioamniotic membranes. Objective: To quantify the expression of the inflammatory cytokines interleukin-1 (IL) -1b, IL-6, IL-8 and tumor necrosis factor alpha (TNF-a) in the chorioamniotic membranes of pregnant women with PPROM and evaluate the correlation of this expression with the presence and intensity of the inflammatory infiltrates present in the membranes and decidua. Material and Methods: Twenty-five PPROM women in labor and 15 PPROM without labor women were studied. As a control group of 25 pregnant women in premature labor (PTL) were studied. After delivery, samples of the chorioamniotic membranes were collected for histopathological analyses and others fragments were conditioned in RNA later for posterior quantification of cytokine mRNA expression by real time PCR. Results: In the study period, the incidence of PPROM was 4.6% and in 75% of these samples the presence of inflammatory infiltrates in the chorioamniotic membranes and/or decidua was observed. mRNA expression of IL-1b, IL-6 and IL-8 was not statistically different in the groups studied. For TNF-a, the expression of mRNA was statistically higher in the PTL group in relation to the groups with PPROM. In the presence of inflammatory infiltrates in the membranes, the concentration of mRNA of IL- 1b it was statistically greater in the PPROM without labor group. No difference occurred in the intensity of cytokine mRNA in relation to the intensity of the infiltrated inflammatory in the groups studied. In 87.2% of the chorioamniotic membranes included in the study, mRNA was expressed for all the cytokines studied... (Complete abstract, click electronic access below)

Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T17:18:35Z (GMT). No. of bitstreams: 1 Bianco_JuaresEdnaldoRomero_M.pdf: 23225852 bytes, checksum: 523781fa325c40e61fa0fe33c59406a1 (MD5) Previous issue date: 2006 / Resumo: Durante o período peri-implantacional na gestação de humanos e roedores, ocorre no estroma da mucosa uterina um conjunto de fenômenos que envolvem modificações dos componentes celulares e da matriz extracelular. Este conjunto de modificações é conhecido como decidualização ou reação decidual. Concomitante a este processo ocorre a migração de leucócitos provenientes de órgão hematopoéticos para este estroma. Dentre estes leucócitos predominam as subpopulações de linfócitos denominados de células ¿uterine Natural Killer¿ (uNK), que atuam na gestação reconhecendo o embrião alogeneico, modulando a reação decidual e o crescimento placentário. Porém, a sua participação nos casos de aborto e o possível desencadeamento do potencial lítico e citotóxico relacionado com a resposta imune inata, não foi ainda totalmente elucidado. O presente trabalho buscou desenvolver estratégias para o estudo in situ das células uNK, sem a influência de fatores oriundos do embrião, empregando o modelo da pseudogestação em camundongos. Foram utilizados fêmeas pseudogestantes obtidos através do acasalamento com machos vasectomizados e inoculação intraluminal do óleo vegetal (arachis) ou mineral (nujol) no útero de 4° dia de pseudogestação (dpg). Estes animais pseudogestantes foram subdivididos em grupos submetidos ou não a ovarectomia e com suplementação hormonal de estrógeno e/ou progesterona ou hormônio gonadotrófico coriônico (hCG). Foram coletados o útero e ovário e processados para análise em microscopia de luz e eletrônica de transmissão no 9°, 12° e 14° dpg. Fêmeas em gestação normal foram utilizados como grupo controle. As análises histológicas, citoquímicas e ultra-estruturais efetuadas demonstraram que a indução da pseudogestação com emprego tanto de óleo vegetal (arachis) quanto de óleo mineral resultam na reação decidual e presença de células uNK lectina DBA (Dolichos biflorus) positivas. A reação decidual resultante e mais exuberante com o óleo de arachis, porém, o período de manutenção do deciduoma é menor, mesmo com a suplementação hormonal de progesterona, se comparado ao grupo de animais induzidos com o óleo mineral e suplementada com HCG. As características morfológicas das células uNK foram melhor preservadas nos animais pseudogestantes induzidos com o óleo mineral, não ovarectomizados e suplementados com o HCG.Nestes, as células uNK foram encontradas aém do 12° dpg em meio ao endométrio que mantinha o deciduoma, enquanto nos demais grupos o deciduoma era destituído antes do 12º dpg. Porém, , à semelhança dos demais grupos experimentais, houve predominância de formas menos diferenciadas de células uNK (subtipos I e II), sendo raras as formas plenamente diferenciadas (subtipo III). Em nenhum dos grupos foi encontrado o subtipo IV, correspondentes à forma de degeneração destas células. Estes resultados demonstram que as características do endométrio e o comportamento das células uNK na pseudogestação, apresentam diferenças influenciadas pelos meios de indução utilizados e regimes hormonais adotados.Além disso, a ausência do concepto parece ser o fator limitante na continuidade da manutenção do utero seudogestante. Por conseguinte, a pseudogestação induzida com óleo mineral e mantida sob a regência ovariana supra-controlado pelo HCG poderia ser adotada como modelo experimental para estudo das células uNK sem a interferência do embrião. Este modelo deverá permitir novas estratégias para investigação dos diversos fatores de origem embrionário/fetal que possam afetar as atividades destas e/ou outras células de forma controlada in situ no ambiente uterino que simule a gestação / Abstract: During the period of pre-implantation in humans and rodents gestations, a set of phenomenon, involving, modifications of cellular components and extracellular matrix occur in the stroma of the uterus. These modifications, are known as decidualization or decidual reaction. During this processes a migration of leukocytes that came from the heamatopoetic organs occur into the uterine stroma. Among these leukocytes, there is a predominance of a subpopulation of leukocytes known as uterine Natural Killers (uNK) that play important roles during gestation by recognizing the allogeneic embryos and modulating the decidual reaction and the placental growth. However, the uNK participations in miss carriage and triggering of lytic and citotoxic potential related to innate immune response during pregnancy have not elucidated yet. The present work aimed to develop a strategy for uNK study without the influence of embryonaryfactors by using the pseudopregnancy model in mice. It was used the pseudopregnant females mice obtained by matting with vasectomized males and intra-luminal injection of arachis or mineral oil in the uterus on 4th day of pseudopregnancy (dpp). These animals were grouped according to treatment submitted of ovarectomy or not and with exogenous supplement of progesterone and/or estradiol or chorionic gonadotrophin hormone (HCG). The uteri and ovary were collected and processes for light and electron microscope on the 9th, 12thand 14thdpp. Normal pregnant females were used as controls. Histological analysis, cytochemistry and ultrastructure demonstrated that the induction of pseudopregnancy by using both arachis oil and mineral oil resulted in decidual reaction and presence of the DBA (Dolichos biflorus) lectin positive uNK cells. The decidual reactions induced by arachis oil were more intensive and exuberant, but the deciduomas were not sustained longer even under progesterone treatment, if compared to those, in the uterus induced with mineral oil, and supplemented with HCG. The morphological characteristics of the uNK cells were better preserved in the pseudopregnant animals induced with mineral oils, not ovarectomized and supplement with HCG. In these animals, the uNK cells were found over the 12th dpp in the endometrium preserving the deciduoma while it failed before 12th dpp in the other groups However, similarly to other experimental groups, there was predominance immature forms of uNK cells (subtypes I and II), being rare the fully differentiated forms (subtype III). No groups did not show the uNK sub type IV, corresponding to the degeneration form of this cell / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

This work is concerned with a study of the role of complement and antibodies in the serum of rabbits and of a non-susceptible strain of mice in the protection of Akr mice injected with active Akr tumor cells.

This thesis has been an attempt to observe the role antibodies play in extending the life span of tumor infected Akr mice.

No description available.