The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population.... Show moreThe increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population. Autophagy-related processes such as LC3-associated phagocytosis have been shown to be activated in the host response against fungal infection, but their overall effect on host resistance remains uncertain. To analyze the relevance of these processes in vivo, we used a zebrafish animal model of invasive Aspergillosis. To confirm the validity of this model to test potential treatments for this disease, we confirmed that immunosuppressive treatments or neutropenia rendered zebrafish embryos more susceptible to A. fumigatus. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of A. fumigatus conidia, and found that both wild-type and melanin-deficient conidia elicited Lc3 recruitment. In macrophages, we observed GFP-Lc3 accumulation in puncta after phagocytosis, as well as short, rapid events of GFP-Lc3 decoration of single and multiple conidia-containing vesicles, while neutrophils covered single conidia-containing vesicles with bright and long-lasting GFP-Lc3 signal. Next, using genetic and pharmacological stimulation of three independent autophagy-inducing pathways, we showed that the antifungal autophagy response improves the host survival against A. fumigatus infection, but only in the presence of phagocytes. Therefore, we provide proof-of-concept that stimulating the (auto)phagolysosomal pathways is a promising approach to develop host-directed therapies against invasive Aspergillosis, and should be explored further either as adjunctive or stand-alone therapy for drug-resistant Aspergillus infections. Show less
Outline of the Thesis Part I Pneumocystis in kidney transplant recipients: transmission, risk factors , new diagnostic and chemo-prophylactic strategies. Chapter 2 describes the characteristics of... Show moreOutline of the Thesis Part I Pneumocystis in kidney transplant recipients: transmission, risk factors , new diagnostic and chemo-prophylactic strategies. Chapter 2 describes the characteristics of a large outbreak of Pneumocystis pneumonia among kidney transplant recipients. By performing a classical outbreak investigation and by application of new molecular genotyping techniques, the potential of the __interhuman transmission hypothesis__ is addressed and discussed. In Chapter 3 all currently available data on reported outbreaks of Pneumocystis pneumonia is systematically reviewed with the emphasis on mortality data, clinical risk factors and transmission analyses. In the case-control study described in Chapter 4, we performed a detailed risk factor analysis for development of PCP in kidney transplant recipients and used the multivariate output data to estimate the effects of several chemoprophylactic strategies by modeling the expected incidence and number-needed-to-treat to provide efficient PCP chemoprophylaxis over a 2-year period post transplantation. Chapter 5 reports the data of a prospective study on the serum markers S-adenosylmethionine and (1-->3)-_-D-glucan serum levels and correlation with clinical parameters in HIVnegative immunocompromised patients __ the majority kidney transplant recipients - with Pneumocystis pneumonia. Potential applicability for treatment monitoring and assessment of P. jirovecii pulmonary load is also discussed. Part II Genetic predisposition for development of invasive aspergillosis in stem cell transplant recipients Chapter 6 describes a multicenter study on the impact of the Y238X stop mutation in the human Dectin-1 receptor (which senses and attaches to glucan moieties of the fungal cell wall) on the risk of development of invasive aspergillosis in stem cell transplant recipients. In Chapter 7 a retrospective study of the influence of genetic variation in the macrophage activation route with respect to the relative additional risk for development of invasive aspergillosis is presented. Part III Experimental markers for detection of fungal infection: scintigraphic imaging. In Chapter 8 the clinical applicability of radiolabeled antimicrobial peptides and antifungal drugs for the diagnosis of invasive fungal infections is reviewed, together with a concise discussion about how promising agents should be further developed. The results of the thesis are summarized and discussed in Chapter 9. Show less
