Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-11T19:43:11Z No. of bitstreams: 2 Dissertação - Maysa Paula da Costa - 2012.pdf: 1943875 bytes, checksum: b80f4f02efcb888e3b6b59c7d58f6bc8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-12T09:34:11Z (GMT) No. of bitstreams: 2 Dissertação - Maysa Paula da Costa - 2012.pdf: 1943875 bytes, checksum: b80f4f02efcb888e3b6b59c7d58f6bc8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-12T09:34:11Z (GMT). No. of bitstreams: 2 Dissertação - Maysa Paula da Costa - 2012.pdf: 1943875 bytes, checksum: b80f4f02efcb888e3b6b59c7d58f6bc8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Secondary metabolites that have been discovered from the plant kingdom are major sources of innovative therapeutic agents for infectious diseases. Plants have been demonstrated to present important biological activities and are considered as potential clinical application when show compatibility with the human organism. Hymenaea courbaril Linnaeus var. stilbocarpa a tree known as jatoba, presents chemicals products obtained from their leaves, stem, fruit and seeds that are used in folk medicine. Antimicrobial and cytotoxic activity of this plant is little known. An initial screening was performed to evaluate the in vitro susceptibility to sap and compounds of H. courbaril against dermatophytes and Cryptococcus isolates. The sap and the compound fisetin were more active and thus they were used to determine the in vitro susceptibility against eighteen dermatophytes and twenty-six Cryptococcus isolates. The results of in vitro susceptibility assay showed antifungal activity of H. courbaril against all fungi studied. The sap of H. courbaril showed minimal inhibitory concentrations (MIC) ranging between 32-128 μg/mL for dermatophytes and 8-256 μg/mL for complex C. neoformans yeast. The compound of H. courbaril fisetin inhibited the growth of dermatophytes and of Cryptococcus in concentration of 4-128 μg/ml, and of 8 a 128 μg/ml, respectively. In vitro cytotoxicity assays of sap and compound were performed with fibroblast 3T3-A31 by using the technique of absorption of the dye neutral red. Cytotoxicity assays showed that sap and compound have an low toxicity on these cells. The sap and the compound showed IC50 values of 109 μg/mL of 158μg/mL, respectively. The results of this study are relevant and promising because they show that the “fisetin” present a good biological activity for fungi, and is a compound biocompatible with basal cells of mammals. Products with such concentrations of MIC and IC50 can be considered of great value for further pharmacological studies. / As plantas e seus metabólitos secundários são uma grande fonte de inovação de agentes terapêuticos para inúmeras enfermidades, incluindo doenças infecciosas. Plantas de uso popular apresentam atividade biológica em potencial, sendo que devem ser consideradas a importância da aplicação clínica e a compatibilidade com o organismo humano. Hymenaea courbaril Linnaeus var. stilbocarpa uma árvore conhecida como jatobá, possui produtos químicos obtidos de suas folhas, tronco, frutos e sementes que são usadas na medicina popular, no entanto atividade antifúngica e a citotoxicidade desta planta ainda é pouco conhecida. Uma triagem inicial foi realizada para avaliação da suscetibilidade in vitro com a seiva e compostos de H. courbaril sobre isolados de dermatófitos e Cryptococcus. A seiva e o composto fisetina apresentaram-se mais ativos e desta forma foram usados para determinar a suscetibilidade de dezoito isolados de dermatófitos e vinte e seis de Cryptococcus. A seiva de H. courbaril apresentou concentrações inibitórias mínima (CIMs) que variaram de 32 a 128 μg/mL para os dermatófitos e de 8 a 256 μg/mL para as leveduras do complexo C. neoformans. O composto fisetina foi capaz de inibir os dermatófitos em CIMs que variaram de 4 a 128μg/mL e as leveduras do complexo C. neoformans em CIMs que variaram de 8 a 128 μg/mL. A avaliação da citotoxicidade in vitro com fibroblastos 3T3-A31 foi realizada para a seiva e o composto fisetina usando a técnica de absorção do corante vermelho neutro. Por este método foi possível verificar que a seiva de H. courbaril apresentou um IC50 de 109 μg/mL e o composto de 158μg/mL Os resultados encontrados neste estudo são relevantes e promissores, pois mostram que esta planta apresenta uma boa atividade biológica para fungos, além de possuir um composto isolado biocompatível com celulas basais de mamíferos. Produtos com tais concentrações de CIM e IC50 podem ser considerados de grande valor para posteriores estudos farmacológicos.

Objetivando estudar o perfil fenotípico de leveduras mantidas em banco de microrganismos de Cryptococcus neoformans, 40 cepas de origem clínica e 44 de origem ambiental foram escolhidas aleatoriamente. As cepas passaram por tipagem bioquímica para diferenciação em C. neoformans e C. gattii, e dentre as cepas clínicas 90% foram tipadas como C. neoformans e 10% como C. gattii, já dentre as cepas ambientais 95,5% foram C. neoformans e 4,5% C. gattii. As cepas cepas que foram positivas no teste bioquímico para C. gattii passaram por tipagem molecular (PCR-RFLP) e verificou-se que apenas quatro cepas eram realmente C. gattii (VGII), e duas outras C. neoformans (VNI e VNIII). Quando estudado os fatores relacionados a virulência, todas as cepas tanto clínicas quanto ambientais foram produtoras de fosfolipase, sendo que cepas de origem clínica produziram essa enzima em maior quantidade. Todas as cepas tanto clínicas quanto ambientais foram produtoras da enzima protease e todas também apresentaram intensidade de cor da colônia ( melanização). Quando avaliado a espessura capsular in vitro todas apresentaram cápsula, das cepas clínicas, 67% apresentaram cápsula média e das ambientais 70%. Quando avaliado a sensibilidade aos antifúngicos pelo métodos E-test todas as cepas clínicas e ambientais foram sensíveis aos antifúngicos anfotericina B, cetoconazol, fluconazol, voriconazol e posoconazol. O itraconazol também foi testado e apresentou cepas sensíveis à droga, porém uma cepa clínica e duas ambientais tiveram classificação dose dependente. Destacamos que a fosfolipase poderia ser usada como um marcador fenotípico para o complexo Cryptococcus neoformans e uma correta identificação das culturas mantidas em banco de microrganismos é necessário. / Aiming to study the phenotypic profile of yeasts maintened in stock culture identified as Cryptococcus neoformans, 40 clinical and 44 environmental strains, were chosen ran domly. The strains had undergone biochemical typing for differentiation as C. neoformans and C. gattii. Among the clinical strains 90% were typed as C. neoformans and 10% as C. gattii, already among the environmental strains were 95,5% C. neoformans and 4,5% C. gattii. The strains thah were positive in biochemical test for C. gattii underwent molecular typing (PCR-RFLP) and found that only four strain were actually C. gattii (VGII) and two other C. neoformans (VNI and VNII). When the factors related to virulence were studied, both the clinical and environmental strains were phospholipase positive but the clinical strains produced a greater amount of this enzyme. All strains were both clinical and environmental production of protease and also showed an intensity colony color (melanization). When the thickness of the capsule was evaluated, all of it showed a capsule, from the clinical strains 67% had an average capsule and from environmental strains 70% had an average capsule. When assessed by sensitivity to antifungal by E-test method all clinical and environmental strains were sensitive to the anphotericine B, ketoconazole, fluconazole, voriconazole and posoconazole. Itraconazole was tested and the samples showed drug sensitive-strains. We point out that phospholipase production would be used as marked to C. neoformans complex and a correct identification of strains maintened in stock culture is necessary.

Criptococose é uma doença causada pelo fungo C. neoformans que têm grande importância atualmente, devido ao aumento da população imunocomprometida,. Além disso, existem poucas opções terapêuticas contra micoses profundas. Neste trabalho foi avaliado se a via de biossíntese do triptofano seria um bom alvo para o desenvolvimento de novos antifúngicos. Com o uso da tecnologia de RNA de interferência, concluiu-se que esta via de síntese é essencial para a sobrevivência desta levedura, sendo, portanto, um ótimo alvo. Ainda neste estudo, demonstrou-se que a letalidade decorre da baixa captação de triptofano pelas permeases de aminoácidos, as quais sofrem repressão catabólica pela fonte de nitrogênio e efeito negativo da temperatura. Foram testados dois inibidores específicos que atuam sobre a antranilato sintase e a triptofano sintase, duas enzimas cruciais para a conversão do corismato em triptofano. Ambos compostos causaram inibição do crescimento de C. neoformans e C. gattii. / Cryptococcosis is a disease caused by C. neoformans, currently of great importance due to the increase in immunocompromised population. Furthermore, there are few therapeutic options for treating this disease. This study evaluated the tryptophan biosynthetic pathway as a possible target for the antifungal development. By using RNA interference technology we concluded that this metabolic pathway is essential for the survival of this yeast, and, therefore, it is a good target. In the same study, it was demonstrated that lethality results from the low uptake of the tryptophan amino acid by permeases, which undergo nitrogen catabolite repression and negative effect of temperature. Two specific inhibitors acting on the anthranilate synthase and tryptophan synthase, two key enzymes for the conversion of chorismate into tryptophan were tested. Both compounds caused growth inhibition of C. neoformans and C. gattii.

A criptococose é infecção fúngica oportunista causada pelo Cryptococcus neoformans com alta incidência em indivíduos imunodebilitados, principalmente entre pacientes com Aids, sendo o número de casos registrados da doença no Brasil, 474.279, de 1980 a junho de 2007. Para melhor compreensão do agente etiológico, da criptococose e dos fatores associados à virulência são necessários estudos adicionais sobre a patogênese deste microrganismo. Dentre os fatores relacionados à virulência, a melanina é um dos principais e sua síntese ocorre por ação da lacase. Assim sendo, foram realizados estudos para avaliar a produção do pigmento entre amostras clínicas e ambientais de C. neoformans sob diferentes condições de pH (5, 6 e 7) e temperatura (25 °C, 30 °C e 35°C). A relação do pigmento com o perfil de sensibilidade aos antifúngicos fluconazol e anfotericina B também foi avaliada. Os resultados mostraram que não houve comportamento uniforme de melanização entre as amostras. As maiores alterações de pigmentação e crescimento das leveduras foram observadas nos meios de cultura com pH 7 incubados a 35 °C. A técnica de eletroforese em gel PAGE nãodesnaturante mostrou-se uma ferramenta útil na análise da lacase produzida pelas amostras de C. neoformans com posterior quantificação de melanina por densitometria das bandas após reação com o substrato L-dopa. As amostras ambientais apresentaram os maiores valores de intensidade de melanina e ampla faixa de variação. Mais da metade das amostras clínicas (56,2 %) apresentaram as menores intensidades de melanina. A amostra clínica ICB 88 revelou duas bandas de melanina no gel indicando a presença de duas isoformas da lacase. Os testes de sensibilidade aos antifúngicos fluconazol e anfotericina B, através das metodologias de difusão em agar por disco e fita (E-test®), mostraram que a leitura após 72 h de incubação pode levar a erro na classificação quanto ao perfil de sensibilidade, tanto nos testes com ou sem adição de melanina. Adição de melanina pareceu não interferir nos resultados frente ao fluconazol nas metodologias de difusão em agar a partir de disco e fita (E-test®). Para anfotericina B houve aumento de CIMs para 90,9 % das amostras, sendo 45,4 % superior a 2 diluições com adição de melanina nos testes de sensibilidade na metodologia de difusão em agar a partir de fita (E-test®). / Cryptococcosis is an opportunistic fungal infection caused by Cryptococcus neoformans that has presented increased incidence with the great number of immunocompromised patients, mainly AIDS ones, being 474.273, the number of cases registered in Brazil since 1980 to june 2007. Additional studies about microorganism’s pathogenesis is necessary for better comprehension about the aethyological agent, criptococosis and associated factors to virulence. Among virulence’s factors, melanin is one of the mainly and its synthesis occurs by laccases action. So, some studies were performed to evaluate the melanin pigment’s production between clinical and environmental strains of C. neoformans under different conditions of pH (5, 6 and 7) and temperatures (25 °C, 30 °C and 35 °C). The pigment and susceptibility to fluconazole and amphotericin B relation’s was also evaluated. The results showed that there wasn’t comparable behaviour of melanization among the strains. The higher alterations on yeast’s pigmentation and growth were noted under pH 7 and at 35 °C. The electrophoresis technique on non-denaturant PAGE gel showed to be an useful tool to the analysis of laccase produced by C. neoformans strains and posterior quantification of melanin by spot’s densitometry after reaction with L-dopa substrate. The environmental strains showed the higher values of melanin intensities and large variation range. Over than a half of clinical strains (56.2 %) showed the lower melanin’s intensities. The clinical strain, ICB 88 revealed two melanin spots on gel indicating the presence of two laccase isoforms. The susceptibility antifungal tests with fluconazole and amphotericin B through disk diffusion and E-test® methods showed that reading after 72 hours can induce to mistakes on the susceptibility profile of strains, either in presence or absence of melanin. Addition of melanin on medium surface appears not change the results of susceptibility to fluconazole in disk diffusion and E-test®, but to amphotericin B was observed increase of MICs to 90.9 % of strains, been 45.4 % upper to 2 dilutions after addition of melanin in Etest ® methodology.

Submitted by JÉSSICA CRISTINA BILIZARIO NOGUEROL ANDRADE null (jessica_belizario@hotmail.com) on 2017-03-03T13:45:50Z No. of bitstreams: 1 Dissertação Jéssica.pdf: 2269215 bytes, checksum: d9bf169b1817858a769738dc3d9266f6 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-08T17:36:05Z (GMT) No. of bitstreams: 1 andrade_jcbn_me_bot.pdf: 2269215 bytes, checksum: d9bf169b1817858a769738dc3d9266f6 (MD5) / Made available in DSpace on 2017-03-08T17:36:05Z (GMT). No. of bitstreams: 1 andrade_jcbn_me_bot.pdf: 2269215 bytes, checksum: d9bf169b1817858a769738dc3d9266f6 (MD5) Previous issue date: 2017-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Criptococose é uma micose sistêmica, cuja incidência tem aumentado nas últimas décadas. É causada pelos fungos Cryptococcus neoformans e Cryptococcus gattii que se apresentam sob a forma de levedura capsulada. Uma importante característica da infecção com as duas espécies do fungo é que enquanto o C.neoformans está associado à doença em pacientes imunossuprimidos, o C.gattii é capaz de induzir a micose em individuos imunocompetentes. O entendimento dos mecanismos envolvidos nestas diferenças tem sido um desafio para os pesquisadores da área. No que se refere à resposta imune do hospedeiro contra o Cryptococcus, está bem estabelecido que a resistência/suscetibilidade a infecção envolve a participação de diferentes subpopulações de células CD4. No entanto, os estudos sobre o papel das subpopulações TH17 e Treg são escassos. Além disso, são raros os trabalhos que de uma forma sistemática objetivaram comparar o perfil de células CD4 induzido por uma ou outra espécie do fungo. Diferencas nesse perfil poderiam explicar a indução da doença em diferentes populações. Assim, um primeiro objetivo do presente estudo foi avaliar o perfil de células CD4 efetoras induzido pelo C.neoformans e C.gatti . A abordagem experimental foi a incubação de células mononucleares do sangue periférico humano (PBMCs) in vitro com leveduras vivas de C. neoformans e C.gattii, seguida da avaliação da expressão intracitoplasmática das citocinas IFN-(TH1), IL-4 (TH2), IL-17 (TH17) e o fator de transcrição Foxp3 (células Tregs) , por citometria de fluxo, bem como a expressão de mRNA para Foxp3 e RORyt (fator de transcrição para TH17). A análise global dos resultados mostrou que o C.neoformans não é capaz de induzir a diferenciação de TH1 e TH2, mas sim de células Treg e TH17. Por outro lado, o C.gattii induz apenas a diferenciação de células Treg. Outro aspecto da relação hospedeiro/ Cryptococcus que merece ser avaliado, refere-se ao achado de que vários microrganismos podem causar genotoxicidade em células do hospedeiro como uma forma de evasão dos mecanismos de defesa. Assim, um segundo objetivo do presente estudo foi avaliar se as diferentes espécies do Cryptococcus são capazes de induzir danos no DNA de PBMCs e se este processo está relacionado aos níveis de óxido nitrico (NO) produzidos por essas células. Detectamos que ambas as espécies são igualmente capazes de induzir genotoxicidade em PBMCs. No entanto, uma associação entre danos no DNA e altos níveis de NO foi detectada apenas em relação a C. gattii. Os resultados apontam para a possibilidade de que os pacientes com criptococose sejam mais suscetíveis ao desenvolvimento de outras doenças. / Cryptococcosis is a systemic mycosis, whose incidence has increased in the last decades. It is caused by the fungi Cryptococcus neoformans and Cryptococcus gattii which present as capsulated yeasts. An important feature of infection with both fungus species is that while C.neoformans is associated with the disease in immunosuppressed patients, C.gattii is able to induce mycosis in immunocompetent individuals. To understanding the mechanisms involved in these differences has been a challenge for researchers. Regarding the host immune response against Cryptococcus, it is well established that resistance/ susceptibility to infection involves the participation of different subpopulations of CD4 cells. However, studies on the role of TH17 and Treg subpopulations are scarce. In addition, there are few studies that systematically aimed to compare the CD4 cell subpopulations profile induced by one or another species of the fungus. Differences in this profile could explain the induction of the disease in different populations. Thus, a first objective of the present study was to evaluate the effector CD4 cells subpopulations profile induced by C.neoformans and C.gattii. The experimental approach was the incubation of human peripheral blood mononuclear cells (PBMCs) in vitro with living yeasts of C. neoformans and C.gattii, followed by evaluation of the intracytoplasmic expression of the cytokines IFN-γ (TH1), IL-4 (TH2), IL-17(TH17) and Foxp3 transcription factor (Tregs cells) by flow cytometry, as well as mRNA expression for Foxp3 and RORyt (transcription factor for TH17). Overall, the analysis of the results showed that C.neoformans is not able to induce the differentiation of TH1 and TH2, but induces Treg and TH17 cells. On the other hand, C.gattii only induces Treg cells differentiation. Another aspect of the host / Cryptococcus interaction that deserves to be evaluated relates to the finding that various microorganisms may cause genotoxicity in host cells as a mechanism of evading defense mechanisms. Thus, a second objective of the present study was to evaluate if the different species of Cryptococcus are able to induce DNA damage of PBMCs and whether this process is related to the levels of nitric oxide (NO) produced by these cells. We have detected that both species are equally capable of inducing genotoxicity in PBMCs. However, an association between DNA damage and high NO levels was only detected in relation to C. gattii. The results point to the possibility that patients with cryptococcosis are more susceptible to the development of other diseases.

A incidência de micoses tem aumentado drasticamente, representando a maioria de infecções oportunistas. As leveduras vêm se destacando pelo modo ao qual infectam o hospedeiro, por suas defesas contra o sistema imune dos hospedeiros e por serem amplamente distribuídas no ambiente. A criptococose é a terceira mais freqüente infecção em indivíduos HIV-positivos e atualmente o número de casos em pessoas ditas “aparentemente imunocompetentes” vem aumentando drasticamente. Os agentes etiológicos são ambas as leveduras Cryptococcus neoformans e Cryptococcus gattii. A infecção humana se dá pela inalação de propágulos infectivos de um sítio ambiental. Nesse estudo, nós relatamos um caso de criptococose disseminada em um individuo de 66 anos, do sexo masculino, HIV-negativo, o qual residia e trabalhava em uma Madeireira na cidade de Cachoeira do Sul – RS. O diagnóstico inicial foi realizado por biópsia de um nódulo subcutâneo e posteriormente foi realizada a confirmação da disseminação da doença através de Raio-X pulmonar e Ressonância Magnética Cerebral. Com base nesse caso clinico, foi proposto isolar os agentes causais da criptococose e outras leveduras dessa Madeireira. As leveduras foram isoladas para caracterizar a microbiota leveduriforme desse local e avaliar a que outras micoses emergentes os trabalhadores dessa Madeireira estão expostos. Foram coletadas amostras de monitoramento do ar, excretas de pombos e galinhas, serragem, pó de madeira, poeira, cascas e folhas de árvores do gênero Eucaliptus. As amostras foram coletas das quatro vezes no período de um ano (2005), sendo realizadas uma coleta por estação do ano. As amostras coletadas totalizaram 40 sítios amostrais do ar interno da madeireira, 40 amostras de excretas (pombos e galinhas) e 63 amostras no interior da madeireira (serragem, pó de madeira, poeira, casca de eucalipto e folha de eucalipto). De alguns desses sítios amostrais foram obtidos três isolados de C. neoformans var. grubii, um isolado de C. gattii e 180 leveduras. Dentre as leveduras, foram identificados 7 diferentes gêneros do Filo Basidiomycota e 10 pertencentes ao Filo Ascomycota. Dentre as espécies de leveduras identificadas, algumas são patógenos, porém a maioria é saprofítica. Os gêneros prevalentes são: Candida, Cryptococcus, Debaryomyces e Pichia. Estudos no ambiente doméstico de indivíduos infectados podem sugerir medidas profiláticas pelo conhecimento dos patógenos aos quais eles estão expostos.

<p><italic>Cryptococcus neoformans </italic>is a human fungal pathogen that is also ubiquitous in the environment. To cause disease inside a human host, <italic>C. neoformans</italic> must be able to sense and respond to a multitude of stresses. One of the major responses to the host is the induction of a polysaccharide capsule, which allows the fungus to resist damage and evade the host immune response. This capsule is regulated by a number of signal transduction cascades, but a major contributor is the conserved cAMP/PKA pathway. </p><p> Using genetic and molecular biology techniques, I identified Gcn5 and Rim101 as key transcriptional regulators of capsule within the host. I determined that <italic>C. neoformans</italic> Rim101 is activated by a combination of the canonical pH sensing pathway and the cAMP/PKA pathway. This novel connection potentially gives the pathogen greater flexibility in responding to environmental stimuli, thus allowing for a greater capacity for disease. </p><p> I determined that the Rim101 transcription factor regulates cell wall remodeling in the context of the host by deep mRNA sequencing, electron microscopy, and biochemical assays. Using chromatin immunoprecipitation, I confirmed that these cell wall changes are under direct control of Rim101. I then confirmed the importance of cell wall changes in the host by nanoString profiling of fungal RNA in the context of a murine lung infection. I also examined the lungs of infected mice for cytokine and immune cell infiltrate and determined that <italic>C. neoformans</italic> cell wall changes are important in avoiding triggering an aberrant host response. I hypothesize that this cell wall remodeling via Rim101 activation is required for full capsule attachment and for masking immunogenic molecules from the host immune system.</p> / Dissertation

Background: Cryptococcal induced visual loss is common and increasingly becoming a debilitating consequence in survivors of cryptococcal meningitis (CM). Conflicting reports of the optic neuritis and papilloedema models of visual loss have delayed the introduction of effective interventional strategies for prevention and treatment of visual loss in CM. Qualitative and quantitative diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) of the optic nerves have proven useful in the examination of the microstructure of the optic nerve especially in optic neuritis. Its application has been extrapolated to other optic nerve disorders such as ischaemic optic neuropathy and glaucoma. The aim of this study is to elucidate the pathogenesis of cryptococcal-induced visual loss using diffusion imaging of the optic nerve as an investigational tool. Method: Full ethical approval was obtained from the Greys Hospital, Department of Health and University of KwaZulu Natal Ethics Committees. Reliable and reproducible optic nerve diffusion techniques were first developed and optimized on 29 healthy volunteers at Greys Hospital, Neurology and Radiology departments using a Philips 1.5 Tesla Gyroscan. Informed consent was also obtained from 95 patients suffering from CM (≥18 yrs. of age), 14 patients with papilloedema and 14 patients with optic neuritis from other causes, recruited from Greys and Edendale Hospitals. Patients underwent full neuro-ophthalmological assessments, CSF examination, haematological workup, CD4 count, (viral load for some), electrophysiological assessment of vision [Visual evoked potential (VEP) and Humphreys visual fields (HVF)], Magnetic Resonance Imaging (MRI) scan of the brain and orbits and DWI and DTI of the optic nerves. Results and Discussion: Visual loss is common in CM, occurring in 34.6-48%. Optic neuritis was uncommon as evidenced by a lack of signal change and lack of enhancement within the optic nerve in all patients scanned. The peri-optic CSF space was not dilated and the optic nerve diameter was not increased regardless of CSF pressure and visual status. Swollen optic discs occurred in only 25% of patients whereas raised intracranial pressure (> 20cmCSF) was demonstrated in 69-71% of patients. Therefore visual loss could not be explained by papilloedema alone. The VEP P100 latency was shown to be a useful screening test for subclinical optic nerve disease in CM, but HVF was not. The optic nerve diffusion imaging used was reliable and reproducible and produced diffusion parameters equivalent to other investigators in the field. Neither optic nerve movement nor the CSF signal was demonstrated to impact significantly on optic nerve diffusion parameters. Optic nerve diffusion imaging did not demonstrate similarities between CM and papilloedema or optic neuritis regardless of CSF pressure or vision. Conclusion: The rarity of optic neuritis in CM and the disparity between papilloedema and visual loss together with the lack of support from diffusion studies suggest a 3rd mechanism of visual loss viz. the optic nerve compartment syndrome. Good clinical support is provided by a case report for this hypothesis that shows re-opening of the peri-optic CSF space and return of the peri-optic CSF signal on MRI with lowering of intracranial pressure and antifungal treatment. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Um die Rolle von Interleukin-12p75 (IL-12p75) und Interleukin-12p40 (IL-12p40) in der Abwehr einer Kryptokokken-Infektion im Mausmodell zu untersuchen, wurden Mäuse auf 129Sv/Ev Stammhintergrund intraperitoneal und intranasal mit Cryptococcus neoformans (C. neoformans) infiziert. Dabei wurden die Unterschiede im Infektionsverlauf und in der Immunreaktion von Wildtyp-, IL-12p35-/- und IL-12p35/p40-/--Mäusen analysiert. Unterschiede zwischen den Wildtyp- und den IL-12p35-/--Mäusen lassen auf die Bedeutung von IL-12p75 schließen, wogegen Unterschiede zwischen IL-12p35-/-- und IL-12p35/p40-/--Mäusen auf die Rolle von IL-12p40 schließen lassen. Untersucht wurden sowohl die Erregerkonzentration in den Organen, Antigenspiegel im Blut, histologische Veränderungen und Serumantikörperkonzentrationen. Nach intraperitonealer Infektion war die Keimbelastung der Organe bei den Wildtyp-Mäusen geringer als bei beiden IL-12-/--Mausstämmen. Bei Wildtyp-Mäusen waren nicht nur weniger lebende Kryptokokken in den Organen zu finden, sondern auch weniger Kryptokokken Antigen im Serum als bei beiden IL-12-/--Mäusen nachweisbar. Das zeigt, dass IL-12p75 für die Kontrolle der intraperitonealen Infektion mit C. neoformans notwendig ist. IL-12p40 hatte ähnlich wie IL-12p75, wenn auch in etwas geringerem Masse, eine protektive Rolle bei der Erregerabwehr. Ohne IL-12p40 war eine Kontrolle der Infektion auf einem geringen Niveau der Keimbelastung nicht möglich. Besonders deutlich wurde dieses Phänomen beim Antigentiter bei den IL-12p35/p40-/--Mäusen. Durch das Fehlen von IL 12p40 wurde bei den IL-12p35/p40-/--Mäusen viel mehr Antigen über das Blut im Serum verteilt als bei den IL-12p35-/-- oder den Wildtyp-Mäusen. Die Wirtsreaktion bei einer Infektion mit C. neoformans geht mit der Bildung von Granulomen einher. Ohne IL-12p75 kam es zwar noch zur Bildung von Granulomen, diese zeigten aber eine veränderte zelluläre Zusammensetzung. Die IL-12p35/p40-/--Mäuse waren nicht zur Ausbildung von typischen Granulomen fähig. Bei ihnen kam es zu einer vermehrten Ansammlung von Kryptokokken fast ohne Entzündungszellen. IL-12p40 ist also für die Ausbildung einer zellulären Entzündungsreaktion notwendig. IL-12p40 ist auch für die Antikörperbildung gegen C. neoformans erforderlich. Die IL 12p35/p40-/--Mäuse waren kaum in der Lage, spezifische Antikörper gegen C. neoformans zu bilden. IL-12p75 ist für die Ausbildung einer Th1-Antwort notwendig. Infizierte Wildtyp-Mäuse produzierten doppelt soviel IgG2a, welches für ein Th1-Antwort typisch ist, wie die IL 12p35-/--Mäuse. Der intranasale Infektionsweg kommt der natürlichen aerogenen Infektion recht nahe. Deshalb wurde – zusätzlich zur intraperitonealen Infektion - dieser Infektionsweg zur Untersuchung der Immunantwort gegen C. neoformans berücksichtigt. Auch bei intranasaler Infektion ist IL-12p75 für die Kontrolle der Keimbelastung der Organe notwendig. Interessanterweise war die Keimbelastung der Lunge bei den IL-12p35-/--Mäusen etwas höher als bei den IL-12p35/p40-/--Mäusen. Bei den Wildtypmäusen war die Dissemination der Kryptokokken aus der Lunge in die Milz und ins Gehirn gering. Ein Fehlen von IL-12p75 bewirkte allerdings eine Besiedlung besonders des Gehirns. Nach intranasaler Infektion kam es in der Lunge von Wildtyp-Mäusen zu atypischen Granulomen mit zentraler Einschmelzung von Gewebe und Kryptokokken. Diese Reaktion war bei den IL-12p35-/--Mäusen noch stärker ausgeprägt als bei den Wildtyp-Mäusen. Bei den IL-12p35/p40-/--Mäusen blieb eine Gewebsreaktion größerer Areale aus. Es waren nur eine Aktivierung des BALT zu sehen. IL-12p40 ist demnach auch nach intranasaler Infektion für eine zelluläre Entzündungsreaktion notwendig. Möglicherweise kann sich diese Eigenschaft von IL-12p40 bei intranasaler Infektion in einer immunpathologischen Reaktion äußern, die bei IL-12p35-/--Mäusen für eine massive Infiltration der Lunge mit Entzündungszellen verantwortlich ist. Der Gehalt an Kryptokokken-spezifischen Antikörpern war nach intranasaler Infektion fünf- bis zehnmal höher als nach intraperitonealer Infektion. Der intranasale Infektionsweg zeigte also eine wesentlich ausgeprägtere humorale Antwort. Der Typ der Immunantwort schien sich im Gegensatz zur intraperitonealen Infektion in Richtung Th2 (d. h. verstärkte Antikörperbildung) verschoben zu haben. Sowohl nach intraperitonealer wie auch nach intranasaler Infektion mit C. neoformans lassen sich die immunstimulatorischen Aktivitäten von IL-12p75 und von IL-12p40 nachweisen, auch wenn diese sich in Abhängigkeit vom Infektionsweg etwas unterschiedlich manifestieren. / To analyse the role of interleukin-12p75 (IL-12p75) and interleukin-12p40 (IL-12p40) in the defence against Cryptococcus neoformans (C. neoformans) a murine infection model was established and studied. Mice of wild-tpye 129Sv/Ev background as well as IL-12p35-/- and IL-12p35/p40-/- 129Sv/Ev mice were infected intraperitoneally or intranasally with C. neoformans. The differences between the immune response of these genotypes were analysed. Comparing wild-type and IL-12p35-/--mice allows for conclusions related to the importance of IL-12p75, comparing IL-12p40-producing IL-12p35-/- mice with IL-12p35/p40-/- mice shows the importance of IL-12p40. Fungal organ burden, serum antigen levels, inflammatory cell responses, and antibody production were examined. The fungal organ load in wild-type mice was smaller than in both mutant IL-12-/--mice. In wild-type mice fewer cryptococci were found in organs and less cryptococcal antigen in serum than in IL-12p35-/- and IL-12p35/p40-/- mice. This underlines the importance of IL 12p75 for the control of the infection with C. neoformans. In addition, IL-12p40 was found to have a similar but weaker role as IL-12p75 in protection against C. neoformans. In the absence of IL-12p40 IL-12p35/p40-/- mice developed higher antigen titers than IL-12p35-/- and wild-type mice. The host response against infection with C. neoformans is associated with granuloma formation. Recruitment of inflammatory cells to granulomas was altered in the absence of IL 12p75. In addition, IL-12p40 contributed significantly to granuloma formation since IL 12p35/p40-/- mice developed no or only very poor granulomatous responses. Therefore, IL 12p40 is required for inflammatory cell responses. IL-12p40 was also found to be required for antibody production against C. neoformans. Infected IL-12p35/p40-/--mice had only very low levels of specific antibodies against C. neoformans. IL-12p75 is known to be essential for protective Th1 response against intracellular microorganisms. Th1 responses are commonly associated with the production of IgG2a. Infected wild-type mice produced 2-fold higher IgG2a levels than IL-12p35-/--mice. To adapt the infection model more to the natural infection mode the intraperitoneal infection route was changed to an intranasal route. Following intranasal infection IL-12p75 also proved to be necessary for control of the fungal organ load. Interestingly the organ load was higher in IL-12p35-/--mice than in IL-12p35/p40-/-mice which suggest a role of IL-12p40 in cell recruitment. Following intranasal application of cryptococci fungal dissemination to spleen and brain was reduced as compared to the intraperitoneal infection route. Without IL-12p75 dissemination of C. neoformans to the brain occured. This shows that IL-12p75 is involved in control of dissemination from lung to brain. The inflammatory response of IL-12p35-/--mice was stronger than the tissue response of wild-type mice. The massive tissue reactions of IL-12p35-/--mice caused big areas of diffuse cellular infiltration in their lungs. In IL-12p35/p40-/--mice inflammatory responses could be observed only in the peribronchial tissue. This shows that IL-12p40 is not only needed for a cellular inflammatory response following intraperitoneal but also following intranasal infection. Following intranasal infection IL-12p40 can induce immunopathological effects. Intranasal infection of mice with C. neoformans resulted in five to ten times higher antibody responses than intraperitoneal infection. This suggests that intranasal infection of mice results in a more Th2-biased humoral response. In summary, these experiments show that besides IL-12p75 also IL-12p40 contributes to cellular immunity against C. neoformans. The immunostimulatory properties of both, IL 12p75 and IL-12p40, can be observed after intraperitoneal and intranasal infection routes with similar but also distinct manifestations.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.