A Paracoccidioidomicose (PCM), causada pelo fungo dimórfico Paracoccidioides brasiliensis, é uma importante micose endêmica na América Latina. Afeta principalmente trabalhadores rurais em idade produtiva. A doença é associada com vários graus de disfunções da imunidade celular de acordo com a severidade da apresentação clínica. Neste trabalho analisamos a atividade fagocítica, apresentação antigênica e a capacidade linfoproliferativa de células dendríticas derivadas de monócitos (moDC) in vitro, frente a glicoproteína de 43 kDa (gp43) do P. brasiliensis, um exoantígeno secretado pela parede do fungo que possui atividade proteolítica e o antígeno somático de Candida albicans (CSA) usado como controle. A suspensão de células enriquecida de monócitos foi obtida através do gradiente de densidade Ficoll-Hypaque/Percoll, utilizando-se amostras de indivíduos saudáveis e pacientes com PCM. Os monócitos foram então cultivados na presença do fator de crescimento para granulócitos e monócitos (GM-CSF) e interleucina-4 (IL-4). Após a diferenciação em células dendríticas (DC), foram realizados ensaios de fagocitose, estimulação antigênica, linfoproliferação e análise da expressão das moléculas de superfície CD14, CD11c, CD86, HLA-DR e CD1a por citometria de fluxo. Sobrenadantes foram coletados para análise da produção de citocinas. Os resultados obtidos sugerem que a gp43 inibe a ativação das DCs dos pacientes por um mecanismo que envolve diminuição da expressão de moléculas de superfície, causando disfunção na resposta imune associada com altos níveis de secreção de IL-10. As DCs de indivíduos saudáveis fagocitam a gp43 e apresentam o antígeno de forma eficiente, enquanto que as DCs dos pacientes apresentaram diminuição da função fagocítica. Por outro lado, foram encontrados altos níveis de fator de necrose tumoral alfa (TNF-alfa) produzidos pelas DCs dos pacientes, havendo uma contribuição para a resposta inflamatória e imunidade inata de pacientes com PCM / Paracoccidioidomycosis (PCM), caused by the dimorfic fungus Paracoccidioides brasiliensis, is the most important endemic mycosis in Latin America. It affects rural workers at their productive period of live. PCM is associated with varying degrees of cellular immune dysfunction according to the severity of the clinical presentation. Here, we analyzed the phagocytic activity, antigen presentation and lymphoproliferation capacity of human monocyte-derived dendritic cells (moDC) in vitro challenged with the exoantigen 43 kDa of P. brasiliensis (gp43) and Candida albicans somatic antigen as control (CSA). The monocytes enriched suspension was obtained through gradient of Ficoll-Hypaque/Percoll density from health individuals and patients with PCM. The cells were cultivated in the presence of granulocytemonocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). After differentiation, DCs were stimulated with gp43 and CSA and analysed phagocytosis capacity. The following surfaces molecules such as CD14, CD11c, CD86, HLA-DR and CD1a were analyzed by flow cytometry. The supernatants were collected for cytokine analysis. In addition, we also verify the cellular morphology by microscopy examination. Taken together, these results suggest that P. brasiliensis inhibits activation of immature DC of patients by a mechanism that involves decreased expression of the surfaces molecules and this can result in dysfunction of the hostimmune response, associated with and high levels of interleukin-10 (IL-10) secretion. The results suggest that health individuals DC internalized gp43 and produced antigen presentation very efficient, while the patients DC inhibits activation by a mechanism involving lesser expression of the surfaces molecules. On the other hand, the high levels of interleukin-12 (IL-12) and tumor necrosis factoralpha (TNF-a) in patients DC could contribute to inflammatory response and innate immunity in human PCM

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

Antibiotic resistance is an ever-growing topic of concern within the medical field causing researchers to examine the mechanisms of resistance to develop new antimicrobials. Bacteria’s ability to form biofilms is one mechanism which aids in antimicrobial resistance. Staphylococcus aureus is of special interest as it is one of the most frequent biofilm-forming bacteria found on medical devices causing infections and posing dangerous threats in a clinical setting. A recently developed antimicrobial gel has been shown to have profound effects on treating bacterial infections and wound healing. This research is centered upon examining the antimicrobial effects of this gel on the three different stages of biofilm formation in clinical and laboratory strains of S. aureus. Through a series of experiments examining the effects this gel has on S. aureus at the stages of biofilm attachment, maturation, and dispersion, the gel has shown significant levels of inhibition. These findings indicate that the novel gel disrupts biofilm forming processes of S. aureus, which provides useful information for fighting infections in the medical field. Further research on the uses and effects of this new gel could lead possibility using the antimicrobial compound for a variety of clinical purposes.

Bacteria secrete and sense extracellular signals from neighboring members of a colony in a phenomenon called quorum sensing. These signals vary from species to species but allow for changes in the behavior of a colony based on changes to cell density, environment, or nutrient supply. Of particular interest to human health is the quorum sensing system of Streptococcus pneumoniae as this pathogen accounts for around one million infection-related deaths per year and is difficult to combat largely due to its ability to form biofilms. These polysaccharide coverings protect entire bacterial colonies from antimicrobial agents as well as allow them to adhere well to the nasopharynx passages of organisms, making them hard to remove. To gain a better understanding of quorum sensing in S. pneumoniae, we propose experiments to study its biofilm formation and its interactions with black carbon, a biochar shown previously to interact with the quorum sensing systems of related bacteria species. We hypothesize that inhalation of black carbon will aggravate a S. pneumoniae infection by promoting biofilm-forming quorum sensing systems making it easier for this bacteria to adhere to and remain on mammal lungs. We propose to first explore the competency and biofilm quorum sensing systems in S. pneumoniae to identify any shared signals between the two using RT-PCR and FITC-Dextran experiments. Further experiments will analyze black carbon particles’ effects on bacterial colonies grown on plates and present on the lung linings of mammals.

Lyme disease is one of the most common infectious diseases present in the United States today and it is clear that the changing climate will affect the geographical range of it across the country. Climate change may impact the range of the Lyme vector species, ticks, which will in turn expand the range of human risk. Although I could not successfully map the possible spread of tick populations or Lyme disease incidence as a result of climate change, my research shows a direct connection between infected tick geographic distribution and key climatic variables, such as temperature, humidity, and precipitation. It is expected that as the climate changes, particularly as it warms, the range of suitable habitat for ticks will expand into high latitudes and altitudes. The expansion of tick populations will put previously unaffected human populations at greater risk of Lyme disease. It is essential that further research be done to confirm the possible consequences of climate change on Lyme disease in the United States and to gain a more precise understanding of how and where effects will be seen. Health officials and policymakers must be informed so they can properly educate and prepare people preemptively for potential Lyme disease outbreaks.

Thesis (Ph.D.) - Carleton University, 2007. / Includes bibliographical references (p. 129-156). Also available in electronic format on the Internet.

Paracoccidioidomicose (PCM) é uma micose sistêmica causada pelo fungo Paracoccidioides brasiliensis e é considerada a 10º causa de morte dentre doenças infecciosas e parasitárias no Brasil. As células dendríticas (DCs) são as mais eficientes na apresentação de antígenos, e sendo de 100 a 1000 vezes mais eficientes que um adjuvante não específico. P10, motivo específico de 15 aminoácidos derivado da gp43 excretada pelo fungo, é reconhecido por linfócitos T, direciona para resposta Th1 e confere proteção no modelo experimental. Nesse sentido, analisamos a capacidade de DCs pulsadas com P10 em ativar uma resposta protetora. In vitro observamos que a proliferação de esplenócitos foi maior quando a reestimução era feira com P10 e DCs juntos. In vivo, camundongos infectados e tratados com DCs pulsadas com P10 apresentaram diminuição da carga fúngica e melhoria do tecido pulmonar, aumento de IFN-<font face=\"Symbol\">g/IL-12 e diminuição de IL-4/IL-10. Esses resultados evidenciam o potencial das DCs atuando como adjuvante para o P10 em uma vacina para o tratamento e cura da PCM. / Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis. In Brazil, it is ranked as the tenth cause of death among the chronic infectious and parasitic diseases. Dendritic cells (DCs) are the most efficient antigen presenting cells, and they are at least 100-1000 times more efficient than a non specific adjuvant. The P10, 15-amino acid peptide motif derived from gp43 secreted by the fungus, is recognized by T cells, induces Th1 response and protects mice in a murine model of the disease. In the present work we analyzed the ability of DCs pulsed with P10 to elicit a protective response. In vitro results showed that a stronger proliferation of the splenocytes was reached when induced by DCs with P10. In vivo, mice infected and treated with DCs pulsed with P10 showed reduce of the fungi burden with less inflamed areas, higher levels of IFN-<font face=\"Symbol\">g/IL-12 and lower levels of IL-10/IL-4. All that point out the importance of the adjuvant role of DCs when developing a vaccine using P10 for the treatment or cure of PCM.

A Paracoccidioidomicose (PCM), causada pelo fungo dimórfico Paracoccidioides brasiliensis, é uma importante micose endêmica na América Latina. Afeta principalmente trabalhadores rurais em idade produtiva. A doença é associada com vários graus de disfunções da imunidade celular de acordo com a severidade da apresentação clínica. Neste trabalho analisamos a atividade fagocítica, apresentação antigênica e a capacidade linfoproliferativa de células dendríticas derivadas de monócitos (moDC) in vitro, frente a glicoproteína de 43 kDa (gp43) do P. brasiliensis, um exoantígeno secretado pela parede do fungo que possui atividade proteolítica e o antígeno somático de Candida albicans (CSA) usado como controle. A suspensão de células enriquecida de monócitos foi obtida através do gradiente de densidade Ficoll-Hypaque/Percoll, utilizando-se amostras de indivíduos saudáveis e pacientes com PCM. Os monócitos foram então cultivados na presença do fator de crescimento para granulócitos e monócitos (GM-CSF) e interleucina-4 (IL-4). Após a diferenciação em células dendríticas (DC), foram realizados ensaios de fagocitose, estimulação antigênica, linfoproliferação e análise da expressão das moléculas de superfície CD14, CD11c, CD86, HLA-DR e CD1a por citometria de fluxo. Sobrenadantes foram coletados para análise da produção de citocinas. Os resultados obtidos sugerem que a gp43 inibe a ativação das DCs dos pacientes por um mecanismo que envolve diminuição da expressão de moléculas de superfície, causando disfunção na resposta imune associada com altos níveis de secreção de IL-10. As DCs de indivíduos saudáveis fagocitam a gp43 e apresentam o antígeno de forma eficiente, enquanto que as DCs dos pacientes apresentaram diminuição da função fagocítica. Por outro lado, foram encontrados altos níveis de fator de necrose tumoral alfa (TNF-alfa) produzidos pelas DCs dos pacientes, havendo uma contribuição para a resposta inflamatória e imunidade inata de pacientes com PCM / Paracoccidioidomycosis (PCM), caused by the dimorfic fungus Paracoccidioides brasiliensis, is the most important endemic mycosis in Latin America. It affects rural workers at their productive period of live. PCM is associated with varying degrees of cellular immune dysfunction according to the severity of the clinical presentation. Here, we analyzed the phagocytic activity, antigen presentation and lymphoproliferation capacity of human monocyte-derived dendritic cells (moDC) in vitro challenged with the exoantigen 43 kDa of P. brasiliensis (gp43) and Candida albicans somatic antigen as control (CSA). The monocytes enriched suspension was obtained through gradient of Ficoll-Hypaque/Percoll density from health individuals and patients with PCM. The cells were cultivated in the presence of granulocytemonocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). After differentiation, DCs were stimulated with gp43 and CSA and analysed phagocytosis capacity. The following surfaces molecules such as CD14, CD11c, CD86, HLA-DR and CD1a were analyzed by flow cytometry. The supernatants were collected for cytokine analysis. In addition, we also verify the cellular morphology by microscopy examination. Taken together, these results suggest that P. brasiliensis inhibits activation of immature DC of patients by a mechanism that involves decreased expression of the surfaces molecules and this can result in dysfunction of the hostimmune response, associated with and high levels of interleukin-10 (IL-10) secretion. The results suggest that health individuals DC internalized gp43 and produced antigen presentation very efficient, while the patients DC inhibits activation by a mechanism involving lesser expression of the surfaces molecules. On the other hand, the high levels of interleukin-12 (IL-12) and tumor necrosis factoralpha (TNF-a) in patients DC could contribute to inflammatory response and innate immunity in human PCM

A maioria dos tratamentos antifúngicos são longos e não eficazes, portanto, há uma necessidade de pesquisa de novas alternativas. A fim de prospectar novos epitopos para uma vacina para Paracoccidioides lutzii e Cryptococcus spp.. Foi desenvolvido um sistema com o objetivo de purificar epitopos de macrófagos no contexto de MHCII. Além disso, houve pesquisa de epítopos na parede celular e sobrenadante de Paracoccidioides spp. A atividade antifúngica da miltefosina foi avaliada contra Paracoccicioides spp.. A miltefosina demonstrou atividade inibitória similar à anfotericina B. A atividade fungicida ocorreu em baixas concentrações, se observou alterações ultraestruturais e formação de melanina. A atividade do C7a in vitro e in vivo contra Candida spp. foi analisada. Os resultados mostraram atividade fungicida em valores baixos. Além disso, foi visto inibição da formação de hifas / pseudohifas e alterações morfológicas. Os ensaios in vivo demonstraram uma diminuição significativa na infecção em um modelo de candidíase vaginal e sistêmica. / Most of antifungal treatments are not completely effective or long, thus there is a need to search new alternatives. In order to prospect new epitopes for a vaccine to Paracoccidioides lutzii and Cryptococcus spp.. A system was developed with the purpose of purify epitopes from macrophages in the context of MHCII. Also, a search for epitopes in the cell wall and supernatant of Paracoccidioides spp. was done. The antifungal activity of miltefosina was evaluated against Paracoccicioides spp.. Miltefosine demonstrated inhibitory activity similar to amphotericin B. Fungicidal activity occurred at low concentrations, ultrastructural alterations and melanin production were observed. The activity of C7a in vitro and in vivo against Candida spp. was analyzed. The results showed fungicidal activity at low values. Also, it was revealed that inhibition of formation of hyphae/pseudohyphae and morphological alterations. In vivo assays demonstrated a significant decrease of fungal border of intravaginal and intravenous infected mice.

Adult immunizations effectively reduce morbidity, mortality, and transmission rates of multiple diseases; however, outpatient providers often a struggle to convince patients to accept vaccinations. This project’s aim is to address vaccination rates in our adult population, focusing first on the influenza vaccine in year one (2016), and then on pneumococcal vaccine in year two (2017), by 1) using a strong quality improvement strategy (known as a Champion Team) and 2) implementing a clinic program consisting of provider training, improved documentation, and informative posters targeted at patients. A quality improvement strategy known as a “Champion Team” provided a strong mechanism through which we developed and implemented the interventions across both years. Specifically, the Champion Team consisted of key stakeholders (nurses, residents, physician faculty, and informatics expert) who identified, developed, and evaluated the program. Programming included an annual health care professional training session for each vaccine (early fall of 2016 and 2017 for flu, spring 2017 for pneumococcal), improved documentation strategies and nursing uptake, and informative posters in the clinic. We assayed data from our patient electronic health record to evaluate: the percentage of our patient population for whom an immunization was documented relative to the number of unique patients seen in our clinic during that time frame. This approach in year one showed a marked increase in influenza vaccination rates in our clinic. During the 2014/2015 and 2015/2016 flu seasons our clinic vaccination rates were 39.98% and 42.05% respectively. After implementation of our champion team and clinic wide program to increase rates in 2016 our vaccination rates for the 2016/2017 flu seasons was 50.88%. Pneumonia data for a full year are under analyses and will be included in this presentation. We anticipate a similar increase in rates for our pneumococcal vaccinations. Our Champion Team and clinic wide program were perceived as relatively low-effort interventions yet appeared to increase vaccinations over the course of this study. The replication of these findings across pneumonia data (pending) and, in future work, with the herpes zoster vaccine (planned for Year 3), will increase our confidence that increases in rates were attributable to these very accessible interventions.