O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

Orientador: Rinaldo Poncio Mendes / Resumo: Introdução. A correlação entre gravidade da paracoccidioidomicose e patogenicidade e imunogenicidade dos fungos causadores tem sido pouco investigada e foi o objetivo deste estudo. Metodologia. As cincos cepas Pb192, Pb234, Pb326, Pb417 e Pb531 foram identificadas pelo seqüenciamento da região Exon 2 da gp43. A patogenicidade foi determinada pelo cálculo da dose letal 50% (DL50%) e pela contagem do número de unidades formadoras de colônias, realizada na sexta semana pós-infecção de camundongos BALB/c. A imunogenicidade foi determinada pela avaliação da resposta imune humoral específica, utilizando-se a reação de imunodifusão dupla em gel de ágar e da imunidade celular, determinada pela concentração das citocinas interleucina -2, interleucina-10, interferon-γ, fator de necrose tumoral – α e do fator de crescimento do endotélio vascular, em tecido pulmonar. Quatro amostras clínicas foram recém-isoladas de pacientes com paracoccidioidomicose, provenientes da Região de Botucatu - os isolados Pb234 e Pb417, de pacientes com a forma crônica moderada; o Pb326 de um caso com a forma aguda grave; e o Pb531, de um caso com a forma crônica grave. As demais cepas Pb192, Pb01 e 8334 foram cedidas pelo laboratório de Moléstias infecciosas. Resultados. As cepas Pb417 e Pb326 agruparam-se às cepas identificadas como P. brasiliensis S1a, a Pb531 às P. brasiliensis S1b e as cepas Pb234 e Pb192 às cepas depositadas como P. restrepiensis (PS3). Os resultados demonstraram correlação direta entre ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction. The investigation of paracoccidioidomycosis and pathogenicity and immunogenicity of the provoking fungi has been little investigated and was the objective of this study. Methodology. As strains, Pb192, Pb234, Pb326, Pb417 and Pb531 were included by sequencing the Exon 2 region of gp43. The pathogenicity was determined by calculating the 50% lethal dose (LD50%) and by counting the number of colony forming units performed in the sixth week post-infection of BALB / c mice. Immunogenicity was determined by the humoral immune response, using the agar gel immunodiffusion reaction and the cellular immunity, determined the concentration of cytokines interleukin-2, interleukin-10, interferonγ, tumor necrosis factor - and factor of vascular endothelial growth in lung tissue. Surgical has been associated with patients with paracoccidioidomycosis, derived from the Botucatu - the Pb234 and Pb417; the Pb326 of a case with a severe severe form; and Pb531, of a case with severe chronic form. The other strains Pb192, Pb01 and 8334 were transferred by the laboratory of Infectious Diseases. Results. Pb417 and Pb326 strains were grouped into the associated strains P. brasiliensis S1a, Pb531 to P. brasiliensis S1b and strains Pb234 and Pb192 to strains deposited as P. restrepiensis (PS3). The results demonstrate the pathogenicity of disease error and severity. The small LD 50 values were measured in the following cases: severe disease, Pb531 and Pb326. Pb531 strain was considered to... (Complete abstract click electronic access below) / Mestre

Master of Science / Periodontal disease is one of the most widespread diseases in humans and is characterised by chronic gingival inflammation and B cell accumulation and resorption of the crest of alveolar bone with subsequent loss of teeth. Porphyromonas gingivalis has been identified as a putative aetiological agent for periodontitis. The aim of the research presented in this thesis was to investigate, using in vitro systems, the responses of autoreactive B-1 and B-2 cells to enterobacterial and nonenterobacterial lipopolysaccharide (LPS) to shed light on the pathogenesis of chronic periodontitis and other diseases involving B cell accumulation and autoantibody production. The hypotheses tested were: (1) B cells respond differently to enterobacterial and non-enterobacterial LPS. (2) B-1 cells are activated by a lower concentration of LPS than B-2 cells. (3) LPS stimulation results in preferential accumulation of B-1 cells. Findings consistent with these hypotheses would provide new evidence for different roles for B-1 and B-2 cells in immune responses and that LPS stimulation could lead to B-1 cell accumulation in diseases thus characterised. Initial experiments investigated the responses of representative B-1 (CH12) and B-2 (WEHI-279) cell lines to preparations of P. gingivalis and Salmonella enteritidis LPS utilising flow cytometric and quantitative molecular methods. The cell lines responded differently to the two LPS preparations. There were significant but limited effects on viability and proliferation in the WEHI-279 cell line, but no significant changes in mRNA expression levels for genes including Toll-like receptors (TLR2, TLR4, RP105), immunoglobulin (IgM), cytokines (IL-6, IL-10), co-stimulatory molecules (CD80, CD86), and regulators of apoptosis (Bcl-2, Bax). In the CH12 cell line however, LPS stimulation had greater effect. Addition of S. enteritidis LPS from a threshold level of 100ng/mL was found to rescue the cells from death, reflected by the percentage viability and proliferation. Stimulation of CH12 cells with S. enteritidis LPS also led to a decrease in expression of RP105 mRNA, which may be part of a negative feedback loop. Interestingly, stimulation with low concentrations P. gingivalis LPS appeared to inhibit proliferation but high LPS concentrations stimulated proliferation of CH12 cells, although no further significant effects were noted in other analyses. Evidence was found that CH12 cells have a high basal level of activation. This suggests that this line is constitutively activated. Stimulation with P. gingivalis or S. enteritidis LPS did not affect the level of CD80 mRNA expression. It is possible that the CH12 line constitutively expresses a maximal level of CD80 (and possibly CD86) and further stimulation will not cause any increase. Since S. enteritidis LPS appeared to have more pronounced effects on both B cell populations, this LPS was used to further investigate B cell subset responses in a mixed splenocyte culture system. Experiments examining percentage viability and number of viable cells indicated that B-1 and B-2 B cells responded differently to LPS stimulation. A threshold level for B-2 cell response (significant increase in cell number) was found to be 100ng/mL LPS, in contrast to the B-1 B cell subset which were only significantly different to the unstimulated cells when stimulated with 50μg/mL LPS. By examining the expression of CD80, the majority of murine splenic B-1 cells were found to activated prior to any LPS stimulation in vitro. In contrast, the B-2 subset showed significant increase in CD80 expression only at high (≥10μg/mL) LPS concentrations. Studies of the division index of B-1 and B-2 cells showed a significant response in both subsets following stimulation with 1μg/mL and 10μg/mL LPS. However, overall, the results are inconsistent with LPS driving the preferential accumulation of B-1 cells in disease states. These experiments provided useful evidence that supported the idea that B-1 and B-2 cells respond differently to LPS. However, these studies were unable to directly address the role of P. gingivalis LPS in periodontitis. It may be that P. gingivalis LPS could have different effects to S. enteritidis LPS on primary B cells. It is still possible that B-1 cells may be more sensitive to P. gingivalis, as opposed to S. enteritidis LPS. Studies by other groups have suggested that the TH1/TH2 profile is skewed towards TH2 in chronic periodontitis and that P. gingivalis may drive this shift via its ability to signal through TLR2 (and modulate TLR4 signalling). Further, recent studies in our laboratories have found that P. gingivalis gingipains are able to polyclonally activate B cells and to break down both IFNγ and IL-12. Future studies should further examine the effects of B-1 and B-2 interactions in the mixed lymphocyte system together with subsequent studies utilising human periodontitis biopsies. The results presented in this thesis, together with work undertaken by other investigators, suggests that LPS could perturb the normal homeostatic mechanisms of the B-1 B cell-subset and increase polyclonal activation therefore contributing to the genesis of pathologies such as chronic periodontitis.

There is mounting evidence to support combined therapy with radiation (RT) and antiangiogenic agents (AA) for the treatment of brain tumors. However, the therapeutic benefit of this combined treatment hinges on the specific dose, schedule, and duration of each treatment. Early biomarkers that reflect tumor physiological responses provide key information that could guide these aspects of treatment. Pre-clinical tumor models are invaluable tools for identifying potential biomarkers, their optimal timing for measurement and their ability to guide therapy in clinical translation. This thesis demonstrates the feasibility and potential of serial MRI to guide the design, delivery and measure of early response to combined AA and RT in a murine intracranial glioma model. We identified promising biomarker changes reflecting early treatment response that may ultimately facilitate individualized spatio-temporal delivery of radiotherapy (RT) and anti-angiogenic agents (AA) for brain tumors.

There is mounting evidence to support combined therapy with radiation (RT) and antiangiogenic agents (AA) for the treatment of brain tumors. However, the therapeutic benefit of this combined treatment hinges on the specific dose, schedule, and duration of each treatment. Early biomarkers that reflect tumor physiological responses provide key information that could guide these aspects of treatment. Pre-clinical tumor models are invaluable tools for identifying potential biomarkers, their optimal timing for measurement and their ability to guide therapy in clinical translation. This thesis demonstrates the feasibility and potential of serial MRI to guide the design, delivery and measure of early response to combined AA and RT in a murine intracranial glioma model. We identified promising biomarker changes reflecting early treatment response that may ultimately facilitate individualized spatio-temporal delivery of radiotherapy (RT) and anti-angiogenic agents (AA) for brain tumors.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.