Infecções fúngicas : diagnóstico e susceptibilidade genética humana

Tese de Doutoramento Ciências da Saúde - Ciências Biológicas e BiomédicasInvasive fungal infections represent nowadays a major public health problem with associated high mortality rates. The lack of adequate diagnostic methods, together with the fact that many emerging fungal species are resistant to the currently available antifungal agents, contributes to the profound impact of these diseases in the health care systems, especially when dealing with immunocompromised patients. Taking this into consideration, the development of novel diagnostic applications has been considered a critical issue in recent years. We described a multiplex PCR-based strategy allowing the identification of eight of the most clinically relevant Candida species. The strategy, based on the amplification of fragments from the internal transcribed spacer regions of the ribosomal RNA genes, was shown to present both high specificity and sensitivity, in addition to other attractive features, including the individual discrimination of species present in mixture and the direct identification from clinical specimens, characteristics reinforcing the potential clinical application of the method. In addition to the need of more satisfactory diagnostic methods, the understanding of the host-fungi interaction, namely at the level of host genetic susceptibility, is critical to advance the knowledge regarding these infections and, in particular, the individual risk factors predisposing to them. Differences in human susceptibility to infectious diseases have been widely described with recent examples focusing on genetic variations within genes of the innate immune system, such as Toll-like receptors (TLRs), which may alter host-pathogen defence mechanisms, thus affecting susceptibility to infectious diseases, and in particular, fungal infections. Taking this into account, we developed a simple and rapid method based in the bi-directional PCR amplification of specific alleles (Bi-PASA) for genotyping known sequence variants in the TLR genes to be used in the forthcoming association studies regarding genetic susceptibility to fungal infections. The development of this methodology also allowed us to perform a characterization of the general Portuguese population regarding these polymorphisms, that can be used in future association studies, besides providing valuable information regarding stratification of patients most at risk of infection. Following the demonstration of the usefulness of Bi-PASA, we investigated the potential association between polymorphisms in the TLR genes and susceptibility to non-invasive forms of pulmonary aspergillosis. A significant association was observed between the presence of Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis. In the same way, this variation was also linked with fungal colonization in the haematopoietic stem cell transplantation (HSCT) setting, suggesting that an abnormal TLR4 extracellular domain may be impairing the recognition of the fungus, thus contributing to an increased predisposition to these diseases. However, the same polymorphism was previously shown to have a protective role against invasive aspergillosis in HSCT patients. Thus, as shown for hyper-inflammatory states, such as atherosclerosis, impairments in the production of inflammatory cytokines contributing to disease susceptibility may be compensating the effect of the defective TLR4. Furthermore, susceptibility to another form of pulmonary aspergillosis, allergic bronchopulmonary aspergillosis, as well as viral infection in the HSCT setting, were shown to be associated with T-1237C (TLR9), highlighting the divergent function of TLRs in the pathogenesis of these infections. A shared susceptibility mechanism involving the T-1237C polymorphism in the promoter region of TLR9 was also observed to predispose to the development of non-Hodgkin lymphoma (NHL). This disease includes a set of heterogeneous lymphoproliferative malignancies often associated with an altered immunological function of the host and chronic inflammatory type of infections, in which TLR9 was already shown to play a critical role. The T-1237C polymorphism introduces a regulatory site that is trans-activated by the IL-6-dependent transcription factor IL-6 response element binding protein (IL-6 RE-BP), thus resulting in increased expression of TLR9. TLR9 activation of B lymphocytes leads to yet increased gene expression levels and sequentially to augmented proliferation rates, as well as higher production of IL-6. This was shown to result in a TLR9 activation loop leading to B lymphocyte-specific uncontrolled proliferation, making these cells more prone to acquire transforming mutations associated with the development of NHL. Besides uncovering a major risk factor for the development of NHL, the presented information has important implications on the recent usage of CpG agonists on several therapeutic strategies in cancer and autoimmune diseases. In summary, we have contributed to show that management of fungal infections, both invasive and non-invasive, involves not only the development of more satisfactory diagnostic procedures, but also considerable attention that has to be given to individual genetic variants that, as we showed, are able to alter susceptibility to these infections. These findings have potential relevance for the stratification of patients most at risk, not only of fungal infections, but also to diseases of other aetiological natures, whose pathogenesis share common signalling/activation pathways such as those presented by the TLRs.Actualmente, as infecções fúngicas invasivas representam um importante problema de saúde pública, sendo responsáveis por elevadas taxas de mortalidade. A falta de métodos de diagnóstico adequados, associada à resistência que muitas das espécies fúngicas emergentes apresentam aos antifúngicos disponíveis, contribui para um profundo impacto destas doenças, especialmente no caso dos doentes imunocomprometidos. Tendo em consideração estes aspectos, o desenvolvimento de novas metodologias de diagnóstico tem sido encarado como uma necessidade prioritária. Neste sentido, desenvolveu-se uma nova estratégia, baseada em PCR multiplex, que permite a identificação de oito das espécies de Candida mais relevantes em termos clínicos. Este método, baseado na amplificação de fragmentos dos genes de RNA ribossomal, apresenta uma elevada especificidade e sensibilidade. A sua potencial aplicação clínica é ainda reforçada por outras características, nomeadamente a discriminação individual de espécies presentes em co-infecção e a identificação directa a partir de espécimes clínicos. Juntamente com a necessidade de desenvolvimento de métodos de diagnóstico mais satisfatórios, a compreensão da interacção hospedeiro-fungo, nomeadamente ao nível da susceptibilidade genética humana, é essencial para o avanço do conhecimento destas infecções. Diferenças na susceptibilidade humana a doenças infecciosas têm sido descritas, associadas nomeadamente a variações em genes do sistema imunológico inato, como os receptores “Tolllike” (TLRs). Recentemente, verificou-se que estas variações podem alterar os mecanismos de defesa antimicrobianos, afectando a susceptibilidade a doenças infecciosas. Assim, tendo em conta estes aspectos, desenvolvemos um método simples e rápido para genotipar polimorfismos nos genes TLR, de forma a aplicá-lo na análise da susceptibilidade a infecções fúngicas nos estudos de associação subsequentes. O desenvolvimento desta metodologia permitiu ainda a caracterização da população Portuguesa em relação a estes polimorfismos, dados que poderão ser usados em estudos futuros, para além de fornecer informação pertinente acerca da estratificação de pacientes com maior risco de desenvolverem infecções. Após a demonstração da utilidade da técnica de Bi-PASA, investigámos a potencial associação entre polimorfismos nos genes TLR e a susceptibilidade a formas não-invasivas de aspergilose pulmonar. De acordo com os resultsdos obtidos, observou-se uma associação entre a presença de Asp299Gly (TLR4) e a aspergilose pulmonar crónica cavitária. De forma idêntica, verificou-se uma associação entre o mesmo polimorfismo e a colonização fúngica em doentes sujeitos a transplante de células estaminais hematopoiéticas (HSCT), sugerindo que um domínio extracelular anómalo de TLR4 pode limitar o reconhecimento do fungo, contribuindo para uma maior predisposição para estas doenças. Contudo, a mesma variação apresentou um papel protector em relação a aspergilose invasiva, sugerindo que uma redução na produção de citocinas pró-inflamatórias poderá equilibrar o defeito anterior, tal como foi anteriormente demonstrado para estados patológicos de hiper-inflamação, como a aterosclerose. Adicionalmente, demonstrámos que o polimorfismo T-1237C (TLR9) estava associado a uma maior susceptibilidade a aspergilose broncopulmonar alérgica, uma outra forma de aspergilose pulmonar, assim como a infecções virais em pacientes HSCT, realçando a função divergente dos TLRs na patogénese destas doenças. Um mecanismo de susceptibilidade partilhado envolvendo o polimorfismo T-1237C surgiu também como predispondo para o desenvolvimento de linfoma não-Hodgkin (NHL). Este inclui um conjunto de doenças linfoproliferativas, frequentemente associadas a alterações imunológicas do hospedeiro e infecções associadas a respostas inflamatórias crónicas, nas quais já foi descrito um papel importante de TLR9. O polimorfismo T-1237C introduz um local de regulação que é trans-activado por um factor de transcrição dependente de IL-6, resultando numa elevada expressão de TLR9. A activação deste receptor em linfócitos B leva a um aumento da sua expressão genética e, consequentemente, a taxas de proliferação mais elevadas, assim como à produção excessiva de IL-6. Estas alterações, juntamente com uma activação persistente de TLR9, culminam numa proliferação descontrolada dos linfócitos B, tornando estas células mais susceptíveis à aquisição de mutações transformantes associadas com o desenvolvimento de NHL. Para além de termos evidenciado o polimorfismo T-1237C como um factor de risco para o desenvolvimento de NHL, a informação resultante apresenta ainda importantes implicações no uso, recentemente preconizado, de agonistas de TLR9 em diversas estratégias terapêuticas, nomeadamente cancro e doenças autoimunes. Em resumo, demonstrámos que a abordagem das infecções fúngicas deve abranger não só um interesse particular no fungo, através do desenvolvimento de métodos de diagnóstico mais eficazes, mas também uma atenção considerável às variações genéticas individuais que, como demonstrámos, podem modular a susceptibilidade a estas infecções. Os resultados aqui apresentados têm uma potencial relevância na estratificação de pacientes com maior risco para infecções fúngicas, bem como doenças de outras etiologias, cuja patogénese partilha vias comuns de sinalização/activação, como as apresentadas pelos TLRs.Fundação para a Ciência e a Tecnologia (FCT) bolsa SFRH/BD/11837/200

Host-derived biomarkers for risk assessment of invasive fungal diseases

Host-Derived Biomarkers for Risk Assessment of Invasive Fungal DiseasesInvasive fungal diseases are major complications associated with the treatment of hematologic malignancies. The integration of host-derived biomarkers into clinical processes to predict the risk and progression of fungal disease is a promising approach in immunocompromised patients. Recent insights into human antifungal immunity have highlighted the remarkable influence of host genetics in modulating susceptibility to infection. In this chapter, we describe protocols to examine human genetic variation and to assess its functional consequences using the pattern recognition receptor PTX3 as an example.European Society of Clinical Microbiology and Infectious Diseases (Research Grant 2012 to A.C.) and the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BPD/96176/2013 to C.C. and IF/00735/2014 to A.C.). Further support was provided by FCT, cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), the Quadro de Referência Estratégico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER), and the Projeto Estratégico PEst-C/SAU/LA0026/2013info:eu-repo/semantics/publishedVersio

Responses of antioxidant defenses to Cu and Zn stress in two aquatic fungi

Aquatic hyphomycetes are fungi that play a key role in plant litter decomposition in streams. Even though these fungi occur in metal-polluted streams, the mechanisms underlying their tolerance to metals are poorly documented. We addressed the effects of Zn and Cu in Varicosporium elodeae and Heliscus submersus by examining metal adsorption to cell walls, plasma membrane integrity and production of reactive oxygen species at metal concentrations inhibiting biomass production in 50% or 80%. The activity of the enzymes catalase, superoxide dismutase and glucose-6-phosphate dehyhdrogenase was measured to elucidate their role in coping with oxidative stress induced by metals at short- (14 h) and long- (8 days) term exposure. Results show that V elodeae was more susceptible to the toxic effects induced by Cu and Zn than H. submersus, as indicated by more extensive inhibition of biomass production. Both metals, particularly Cu, induced oxidative stress in the two fungal species, as shown by the noticeable recovery of biomass production in the presence of an antioxidant agent. In both fungi, Cu induced a more severe disruption of plasma membrane integrity than Zn. Our studies on antioxidant defenses showed that catalase had a greater role alleviating stress induced by Zn and Cu than superoxide dismutase. Chronic metal stress also stimulated the production of NADPH, via the pentose phosphate pathway by increasing the activity of glucose-6-phosphate dehydrogenase. Our results suggest that the tolerance of aquatic hyphomycetes to Cu and Zn is associated with the ability of these fungi to initiate an efficient antioxidant defense system. (c) 2007 Elsevier B.V. All rights reserved.info:eu-repo/semantics/publishedVersio

Polymorphisms in host immunity modulating genes and risk of invasive aspergillosis: results from the aspBIOmics consortium

Recent studies suggest that immune-modulating single nucleotide polymorphisms (SNPs) influence the risk of developing cancer-related infections. Here, we evaluated whether 36 SNPs within 14 immune-related genes are associated with the risk of Invasive Aspergillosis (IA) and whether genotyping of these variants might improve disease risk prediction. We conducted a case-control association study of 781 immunocompromised patients, 149 of whom were diagnosed with IA. Association analysis showed that the IL4Rrs2107356 and IL8rs2227307 SNPs were associated with an increased risk of IA (OR=1.92, 95%CI: 1.20-3.09 and OR=1.73, 1.06-2.81) whereas the IL12Brs3212227 and IFN?rs2069705 variants were significantly associated with a decreased risk of developing the infection (OR=0.60, 0.38-0.96 and OR=0.63, 0.41-0.97). An allogeneic hematopoietic stem cell transplantation (allo-HSCT)-stratified analysis revealed that the effect observed for the IL4Rrs2107356 and IFN?rs2069705 SNPs was stronger in allo-HSCT (OR=5.63, 1.20-3.09 and OR=0.24, 0.10-0.59) than in non-HSCT patients, suggesting that the presence of these SNPs may render patients more vulnerable to infection especially under severe and prolonged immunosuppressive conditions. Importantly, in vitro studies revealed that carriers of the IFN?rs2069705C allele showed a significantly increased macrophage-mediated neutralisation of fungal conidia (P=0.0003) and, under stimulation conditions, produced higher levels of IFN? mRNA (P=0.049) and IFN? and TNFa cytokines (PLPS-96h=0.057, PPHA-96h=0.036 and PLPS+PHA-96h=0.030 and PPHA -72h=0.045, PLPS+PHA-72h=0.018, PLPS-96h=0.058 and PLPS+PHA -96h=0.0058, respectively). Finally, we also observed that the addition of SNPs significantly associated with IA to a model including clinical variables led to a substantial improvement in the discriminatory ability to predict the disease (AUC=0.659 vs. AUC=0.564, PLR=5.2•10-4 and P50.000Perm=9.34•10-5). These findings suggest that the IFN?rs2069705 SNP influences the risk of IA and that predictive models built with IFN?, IL8, IL12p70 and VEGFa variants might be used to predict disease risk and to implement risk-adapted prophylaxis or diagnostic strategies.This study was supported by grants PI12/02688 from the Fondo de Investigaciones Sanitarias (Madrid, Spain), PIM2010EPA-00756 from the ERA-NET PathoGenoMics (0315900A), and the Collaborative Research Center/Transregio 124 FungiNet. C.C. is supported by the Fundação para a Ciência e Tecnologia, Portugal (SFRH/BPD/96176/2013). This study also was supported by a donation of Consuelo González Moreno, an acute myeloid leukemia survivor. We thank Astella Pharma Inc. for supporting this work.info:eu-repo/semantics/publishedVersio