A. fumigatus é um patógeno oportunista que causa infecções invasivas em hospedeiros imunocomprometidos. Estudos de respiração mitocondrial sugeriram a presença de componentes alternativos em sua cadeia respiratória envolvidos com processos de adaptação a ambientes adversos, como a proteína desacopladora (UCP). UCPs são proteínas mitocondriais cuja atividade dissipa o potencial de membrana gerado durante o transporte de elétrons. Um gene contendo características das três assinaturas moleculares das Proteínas Transferidoras de Energia foi clonado e sequenciado. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois íntrons que, após o splicing, codifica uma proteína contendo 341 aminoácidos, com uma massa molecular de 37 kDa e um pI de 10,02. A fim de se avaliar as propriedades bioenergéticas da UCP-like, essa sequência foi clonada no vetor pYES2 e leveduras S. cerevisiae foram transformadas. Esferoplastos foram preparados e o potencial elétrico transmembrana mitocondrial foi estimado. Os resultados mostraram que o potencial de membrana de esferoplastos de leveduras expressando a proteína UCP-like foi ligeiramente menor e que o decréscimo momentâneo do potencial associado com a fosforilação do ADP foi mais lento quando comparado com o controle, indicando desacoplamento da respiração. Além disso, esse comportamento dos esferoplastos recombinantes foi similar ao controle quando GDP foi adicionado ao meio de reação, sugerindo uma inibição da proteína por esse composto. Para sua caracterização funcional em sistemas reconstituídos, a sequência foi clonada no vetor pET SUMO. A expressão foi realizada em E. coli e a proteína recombinante, purificada por cromatografia em resina de níquel, foi analisada por Western blot com anticorpos anti-(His)6-tag e anti-UCP2 e por espectrometria de massas. A formação dos lipossomos foi confirmada através de medidas de distribuição de partícula por espalhamento de luz dinâmico, as quais sugeriram a formação de vesículas estáveis. Em adição, foi investigada a participação da UCP-like na proteção do A. fumigatus contra danos oxidativos. O nível de mRNA foi determinado por PCR em tempo real na presença de paraquat e menadiona. Em A. fumigatus, a presença dessas drogas pró-oxidantes resultou em um aumento no nível de mRNA desse gene, sugerindo que essa proteína possa também fazer parte de um sistema de defesa antioxidante do fungo. / A. fumigatus is an opportunistic pathogen that causes invasive infections in immunocompromised hosts. Mitochondrial respiration studies suggested the presence of alternative components on its respiration chain, which are involved with the adaptation to hostile environments, such as the uncoupling protein (UCP). UCPs are mitochondrial proteins whose activity dissipates the membrane potential generated during electron transport. A gene containing features of three molecular signatures of Energy Carrier Protein was cloned and sequenced. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 341 amino acids protein with a molecular mass of 37 kDa and a pI of 10.02. In order to study bioenergetics properties of UCP-like, the cDNA sequence was cloned into pYES2 vector and transformed in S. cerevisiae. Spheroplasts were prepared and the mitochondrial electrical transmembrane potential was estimated. The results showed that, compared with control cells, mitochondrial electrical transmembrane potential of transformant spheroplasts was slightly smaller and the transient potential decrease associated with ADP phosphorylation was longer, indicating uncoupling of respiration. Moreover, this behavior of recombinant spheroplasts was similar to control cells when GDP was added to the reaction medium, suggesting the inhibition of uncoupling protein. For its functional characterization in reconstituted systems, the cDNA sequence was cloned into pET SUMO vector. The expression was carried out in E. coli and the recombinant protein, purified by chromatography on a nickel-chelating resin, was analyzed by Western blot using anti- (His)6-tag or UCP2 antibodies and by mass spectrometry. Liposome formation was confirmed by light scattering, suggesting the formation of stable vesicles. In addition, the participation of UCP-like in A. fumigatus protection against oxidative damage was investigated. mRNA level was determined by real time PCR in the presence of paraquat and menadione. In A. fumigatus, the presence of these pro-oxidants drugs resulted in increased mRNA level of this gene, suggesting that this protein might also be part of an antioxidant defense system of this fungus.

Aspergillus fumigatus is increasingly recognised as a cystic fibrosis (CF) pathogen with reported infection rates up to 50% and associated with increased hospitalisations and lung deterioration. Research investigating immune responses of CF phagocytes against A. fumigatus has been scarce and the role of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in anti-Aspergillus immune responses is not known. The studies in this thesis aimed to explore innate immune responses by CF phagocytes against A. fumigatus. Peripheral blood mononuclear cells (PBMC), monocytes (MNC) and polymorphonuclear cells (PMN) were isolated from blood samples of CF patients with at least one copy of the F508del mutation. CF phagocytes efficiently cleared A. fumigatus similar to healthy controls. Microtubuleassociated proteins 1A/1B light chain 3B (LC3) expression was found to be attenuated in CF PMN and MNC, indicating a disbalance in the autophagy or LC3-associated phagocytosis pathway. Regarding inflammatory responses, it was found that upon phagocytosis a resting A. fumigatus conidia, CF phagocytes produce up to 4-fold more reactive oxygen species (ROS) compared to controls. The excessive ROS was then shown not to be necessary for adequate killing, suggesting the increased ROS to be redundant in the antifungal response. Patient metrics obtained from the clinic showed that the excessive ROS production correlated to exacerbations and lung function. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that CF PMN only express 20-25% of the 4 haemoglobin subunits compared to healthy controls. Combining the LC-MS data suggests that CF PMN are under hypoxic stress. In conclusion, the effective clearance of A. fumigatus by CF phagocytes comes at the cost of an excessive respiratory burst which correlates to disease severity. Our data indicate that CF PMN are under hypoxic stress while circulating in the blood stream, which is likely to contribute to the hyperinflammatory phenotype observed upon interaction with A. fumigatus.

Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. In healthy individuals, local pulmonary host defence mechanisms can efficiently eliminate the fungus without any overt symptoms. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. However, local host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control the invasive fungal disease. In different immunocompromised murine models, myeloid but not lymphoid cells were strongly recruited upon infection. Notably, neutrophils and macrophages were recruited to infected lungs in different immunosuppressed regimens. Other myeloid cells, particularly dendritic cells and monocytes were only recruited in the corticosteroid model after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and were not recruited after A. fumigatus infection. Importantly, adoptive CD11b+ myeloid cell transfer rescued immunosuppressed mice from lethal A. fumigatus infection. These findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defence under immunocompromised conditions. Despite improved antifungal agents, invasive A. fumigatus lung infections cause a high rate morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and the functional defence mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. We aimed to assess the efficacy of granulocyte transfusion and functional defence mechanisms of corticosteroid treated granulocytes using mouse models. In this thesis, we show that transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection, increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that have received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) in in vitro and in vivo conditions. Further, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections. / Der Mensch kommt über die Atemluft in regelmäßigem Kontakt mit Sporen des saprophyitschen Pilzes Aspergillus fumigatus. Glücklicherweise eliminieren die lokalen Abwehrmechanismen der Lunge den Pilz in gesunden Individuen sehr effektiv und ohne offenkundige Symptome. In immunkomprimierten Patienten hingegen verursacht A. fumigatus verheerende Infektionen. Allerdings ist die lokale Immunreaktion gegen A.fumigatus-vermittelte Infektionen der Lunge unter immunsuppressiven Bedingungen immer noch nicht ausreichend definiert. In Anbetracht der dynamischen Veränderungen an Immunzellunterpopulationen im Gewebe nach immunsuppressiver Therapie haben wir die zeitliche und örtliche pulmonale Immunreaktion nach A. fumigatus infektion untersucht, um die grundlegenden immunologischen Geschehnisse aufzudecken, die in dieser Situation zur unzureichenden Kontrolle des Pilzes führen. In anderen immunsupprimierten Mausmodellen fand eine starke Rekrutierung myeloider Zellen nach Infektion statt. In besonderem Maße wurden nach der Infektion Neutrophile und Makrophagen in die Lunge immunsupprimierter Mäuse rekrutiert. Andere myeloide Zellen, insbesondere dendritische Zellen und Monozyten, wurden nur im Corticosteroid-Modell nach Infektion rekrutiert. Lymphoide Zellen, insbesondere CD4+ oder CD8+ Zellen und NK Zellen, waren nach Immunsuppression stark reduziert und wurden nach Infektion mit A. fumigatus nicht rekrutiert. Adoptiver Zelltransfer von CD11b+ myeloiden Zellen stellte die Abwehr immunsupprimierter Mäuse gegen A. fumigatus wieder her, was die wesentliche Bedeutung dieser Zellen in der Immunabwehr unterstreicht. Diese Erkenntnisse verdeutlichen, dass CD11b+ myeloide Zellen unter immunkomprimierten Bedingungen entscheidend für die Abwehr gegen A-fumigatus sind. ...

MP1, a gene previously identified in P. marneffei by cDNA library screening, encodes a secreted cell wall mannoprotein Mp1p. Thirteen MP1 homologues named MPLP1 to 13 were previously identified in P. marneffei by BLAST analysis. Two MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p were previously identified by expressed sequence tag library screening in Aspergillus fumigatus – an important fungal pathogen closely related to P. marneffei. Mp1p, Afmp1p and Afmp2p have previously been reported to be immunogenic. Mp1p was also reported to bind fatty acid and was suggested to contribute to virulence in a MP1 knockout P. marneffei strain in a mouse model and a cell line model although the Koch’s postulates has yet been met to establish MP1 as a novel virulence factor. With reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were speculated to have functions similar to Mp1p. BLAST searches against the A. fumigatus genome identified two novel AFMPs namely AFMP3 and AFMP4. Sequence analysis of Afmp3p and Afmp4p revealed the presence of putative N-terminal signal peptide and substantial sequence identity to Mp1p, Afmp1p and Afmp2p. Two MP1 knockdown P. marneffei mutants were constructed to demonstrate suppression of MP1 expression alone can result in loss of virulence and also the dosage effect of MP1 expression on P. marneffei virulence towards mice. Subsequent mice challenge experiments using MP1 like protein (MPLP) knockdown strains suggested MP1 to be the most important virulence factor among all its homologues in P. marneffei. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for P. marneffei mutants with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice. Mice challenge experiments using AFMP1 to 4 knockdown A. fumgiatus mutants suggested significant decrease in virulence of A. fumigatus upon AFMP4 knockdown and complete protection of challenged mice upon knockdown of AFMP1 to 4. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for A. fumigatus mutants with knockdown of AFMPs and effect of AFMPs on granuloma formation in infected mice. Mice experiments using Pichia pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on virulence are not caused by factors specific to P. marneffei or A. fumigatus. It was shown using a human peripheral blood mononuclear cells model that Mp1p and Afmp4p confer intracellular survival advantage to P. marneffei and A. fumigatus upon infection. Expression of Mp1p or Afmp4p in P. pastoris also confers survival advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells. Reduction in proinflammatory prostaglandin E2 production were noticed in human peripheral blood mononuclear cells infected by P. marneffei, A. fumigatus or P. pastoris strains that expressed Mp1p or Afmp4p. Such reduction in eicosanoids production also coincides with the inhibition of apoptosis as shown by enzyme activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood mononuclear cells. These findings suggest two novel virulence factors – Mp1p and Afmp4p, which confer survival advantages to P. marneffei and A. fumigates respectively. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Stuttgart, Univ., Diss., 2008.

Tübingen, Univ., Diss., 2006.

Techn. Univ., Diss., 2010--München.

The main aim of this study was to find a plant extract or isolated compound that could be used to combat aspergillosis in animals. Aspergillus fumigatus is one of the most common pathogenic fungal species in humans and animals. A. fumigatus is also an economically important fungus in the poultry industry. Current treatment of the disease is hampered by drug resistance of the organism to conventional antifungals and also its widespread toxicity to the animals. Seven tree species that had good antifungal activity against Cryptococcus neoformans in the Phytomedicine Programme database were selected for further work. These tree species were: Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae). The antimicrobial activity of leaf extracts of the selected plant species were determined against four important nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) and five important animal fungi (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Microsporum canis and Sporothrix schenckii) using a serial microplate dilution method. The minimal inhibitory concentrations (MIC), of an acetone extract of Loxostylis alata was the lowest against Aspergillus fumigatus with an MIC value of 0.05 mg/ml. The number of antifungal compounds in extracts was determined by bioautography. The acetone extract of L. alata had the most active zones (10). The antioxidant, antiplatelet and cytotoxic effects of the seven plant species were evaluated using established in vitro assays. All the extracts had comparably low toxicity except for the extract of C. harveyi that had high haemagluttination assay titre value, which indicates toxicity. The extracts of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi and C. vendae contained antioxidant compounds in the qualitative assay using DPPH. In the quantification of antioxidation using ABTS, only the extracts of P. longifolia, L. alata, and C. vendae had substantial antioxidant activity with respective TEAC value of 1.39, 1.94 and 2.08. Similarly, in the quantitative DPPH assay, L. alata. (EC50, 3.58 ± 0.23 μg/ml) and K. wilmsii (EC50, 3.57 ± 0.41 μg/ml) did not differ significantly (p ≤ 0.05) from the positive control (L-ascorbic acid). K. anthotheca had a much lower antioxidant activity (EC<su>50 176.40 ± 26.56 μg/ml), and differed significantly (p ≤ 0.05) from all the other extracts and control. In addition, the extract of C. vendae and C. harveyi had significant (p ≤ 0.05) antiplatelet activity and did not differ from the control (aspirin) with EC50 of 0.06 ± 0.01 μg/ml, 0.19 ± 0.00 μg/ml, respectively. Lower EC50 values in the antioxidant and antiplatelet studies are indicative of superior activity of the plant extract against oxidation and platelet aggregation. Based on the results obtained L. alata was selected for further examination. To simplify the isolation of the antifungal compounds from the L. alata fractions the acetone extract was first separated into six different fractions based on polarity in a mild solvent-solvent fractionation process. The fractions were aqueous methanol, butanol, carbon tetrachloride, chloroform, hexane and water fractions. The antimicrobial activities of the fractions as well as other relevant pharmacological tests on the different fractions were carried out. The number of antimicrobial compounds present in the aqueous methanol (AM), butanol (BT), carbon tetrachloride (CCl4), chloroform (CC), hexane and water fractions was determined by bioautography. The CCl4 extract was active against six out of the 9 microbial strains used and was particularly active against S. aureus, E. faecalis, A. fumigatus, C. albicans, C. neoformans and M. canis with MIC of 0.04, 0.04, 0.1, 0.1, 0.06 and 0.03 mg/ml, respectively. Microsporum canis was the most sensitive organism with the lowest average MIC of 0.16 mg/ml. Qualitative antioxidation using DPPH and quantitative assay using both ABTS and DPPH radicals revealed the presence of several antioxidant compounds in the AM, BT and water fractions of Loxostylis alata. This supported the usefulness of L. alata in treating fungal diseases, as aspergillosis and most fungal infections are associated with immune depression of the host. Antioxidants may reverse several conditions associated with immune deficiencies, resulting in increased levels of interleukin-2, elevated numbers of total lymphocytes and T-cell subsets. Loxostylis alata is used in southern African traditional medicine to control labour pain and to boost the immune system. Extracts and compounds isolated from leaves of Loxostylis alata were therefore also evaluated for their in vitro antimicrobial, anti-inflammatory (cyclooxygenase-1 and -2) activities and evaluated for their potential toxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and Salmonella typhimurium tester strains TA98 and TA100. Antimicrobial activity was evaluated using a serial microdilution assay. The bacterial strains used were Staphylococcus aureus (ATCC29213), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922). The fungal strains used were Cryptococcus neoformans, Sporothrix schenckii, Aspergillus fumigatus, Microsporum canis and Candida albicans. A bioassay guided fractionation of the crude extract yielded two antimicrobial compounds namely, Lupeol and μ-sitosterol Lupeol had the most pronounced zone of inhibition against S. aureus and A. fumigatus., When MICs of the 2 compounds were determined, only lupeol had relatively good activity with MICs values ≤ 100 μg/ml against 8 out of 10 of the tested pathogens. However, β-sitosterol had activity against only S. aureus and E. coli with MICs values of 90 and 110 μg/ml, respectively. In addition β-sitosterol had selective inhibition of COX-1 (IC50 = 55.3 ± 2) None of the compounds isolated were toxic in the Salmonella typhimurium/microsome assay and MTT cytotoxicity test. The isolation of these two compounds is reported for the first time from Loxostylis alata. It was disappointing that the two antifungal compounds isolated from L. alata had such a low activity against Aspergillus fumigatus. This inhibits the development of a single compound that can be used therapeutically. Because the crude extract had very good activity we decided to investigate the safety and potential use of this extract in target animal species. At a dose of 300 mg/kg, the chicks had some signs of intoxication, but not at a dose of 200 mg/kg. Aspergillosis was induced experimentally, in broiler chicks. The degree of infection was assessed by comparing degree and severity of clinical signs, lesion scores and fungal re-isolation from treated chicks with those from infected chicks not treated with the extract. The extract at a dose of 100 and 200 mg/kg reduced significantly (p ≤ 0.05) the lesions due to aspergillosis and the amount of Aspergillus fumigatus isolated from infected chicks in an excellent dose related response.. The crude extract of L. alata leaves was as active as the commercially used ketoconazole against avian aspergillosis. It appears likely that the crude acetone extract could be produced at a much lower cost than ketoconazole or other chemical antimicrobial products. If these results can be confirmed in larger studies and if the crude extract does not have a negative effect on the production of the poultry the crude extract of L. alata may prove to be a viable and cost effective alternative to using current antimicrobial products. This study proves that it may be worthwhile to invest human and financial resources in searching for plant related products than can increase animal health and productivity. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Die invasive Aspergillose spielt in der modernen Medizin und speziell bei malignen Bluterkrankungen, die mit einer Immunsuppression des Patienten einhergehen, eine entscheidende Rolle. Die bisherigen Methoden erlauben eine Diagnose meist erst sehr spät oder liefern oft falsch-negative Ergebnisse. Bis zum heutigen Tag ist es nicht gelungen, ein standardisiertes Verfahren zur PCR-Diagnostik aus Blutproben zu etablieren. In den der Arbeit zugrunde liegenden Versuchsreihen wurden verschiedene Ansätze an Mastermix im Monoplex- und Duplexformat zur Aspergillus fumigatus-Detektion in Humanserum getestet und optimiert. Es wurde versucht, eine Extraktionskontrolle mit Bacillus subtilis in die Probe zu integrieren, um so die Aussagekraft der extrahierten Probe zu evaluieren. Dabei zeigte sich ein Elutionsvolumen von 35 µl am effizientesten. Der Cq-Wert war bei 35 µl Eluationsvolumen ca. einen Zyklus niedriger als bei 65 µl und 1,5-2 Zyklen niedriger als bei 100 µl. Bei den Versuchen im Monoplexformat konnte gezeigt werden, dass bei der Extraktion viel DNA in den Filtern zurückbleibt. Es wurden aus einer Serumextraktion zwei Eluate mit je 35 µl Elutionsvolumen erstellt und getestet. Hier zeigten sich in Abhängigkeit von der Menge der DNA teils hohe Cq-Werte im zweiten Eluat, bei einigen Proben aber sogar ein niedrigerer Cq-Wert im zweiten Eluat. Dies war sowohl für Aspergillus fumigatus als auch für Bacillus subtilis zu beobachten. Die Gründe für dieses Ergebnis könnten die längere Inkubationszeit und die zweite Zentrifugation des zweiten Eluats sein. Die Extraktionseffizienz hatte bei der Aufbereitung mit dem QIAamp Ultrasens Kit (Qiagen) einen Faktor von im Mittel 2,7 bei Aspergillus fumigatus und von 4,7 bei Bacillus subtilis beim GEX-Mastermix. Beim Sso-Mastermix lag der Faktor für Aspergillus fumigatus im Mittel bei 4,4 und der für Bacillus subtilis bei 5,4. Bei den Versuchen im Duplexformat wurden sowohl Aspergillus fumigatus als auch Bacillus subtilis in unterschiedlichen Konzentrationen in dieselbe Probe eingebracht. Hier zeigte sich, dass im Sso-Mastermix die Menge an Bacillus subtilis-DNA und an Aspergillus fumigatus-DNA einen deutlichen Einfluss auf die jeweiligen Cq-Werte und die Sensitivität haben. Zusätzlich zeigte der Sso-Mastermix immer ein positives Ergebnis für Bacillus subtilis zwischen 36-40 Zyklen. Beim GEX-Mastermix hatte die Menge an Bacillus subtilis-DNA ebenfalls einen deutlichen Einfluss auf die Cq-Werte und die Sensitivität für Aspergillus fumigatus. Die Cq-Werte für Aspergillus fumigatus erhöhten sich bei 10 Kopien mit 104 Kopien Bacillus subtilis um 6,1 Zyklen und bei 105 Kopien Bacillus subtilis um 10,6 Zyklen im GEX-Mastermix. Ein Einfluss auf die Cq-Werte für Bacillus subtilis konnte bei den Versuchen im Duplexformat mit GEX-Mastermix nicht verzeichnet werden. Zusammenfassend lässt sich sagen, dass eine Extraktionskontrolle mit Bacillus subtilis im Sso-Mastermix auf Grund der sich gegenseitig beeinflussenden Sonden und Primer sowie der DNA-Mengen an Aspergillus fumigatus und Bacillus subtilis nicht möglich ist, da keine Aussage über den Verlauf und die Effizienz der Extraktion getroffen werden könnte. Bei den Versuchen mit GEX-Mastermix konnte gezeigt werden, dass die Menge an eingegebener Bacillus subtilis-DNA einen entscheidenden Einfluss auf die Sensitivität für die Cq-Werte von Aspergillus fumigatus in niedriger Konzentration hat. Einen Einfluss auf die Cq-Werte für Bacillus subtilis wurde nicht detektiert. Eine Extraktionskontrolle mittels Bacillus subtilis wäre denkbar, wenn die Menge eingegebener Bacillus subtilis-DNA so reduziert werden könnte, dass stabile Cq-Werte für Bacillus subtilis erreicht würden, die dann aber keinen oder nur einen sehr geringen Einfluss auf die Sensitivität für Aspergillus fumigatus hätten. / In the test series on which the work is based, various approaches to mastermix in monoplex and duplex format for Aspergillus fumigatus detection in human serum were tested and optimized. An attempt was made to integrate an extraction control with Bacillus subtilis into the sample in order to evaluate the meaningfulness of the extracted sample.

Der ubiquitär vorkommende Schimmelpilz Aspergillus fumigatus (A. fumigatus) ist Haupterreger der invasiven Aspergillose (IA). Diese invasive Pilzinfektion tritt gehäuft bei Patienten mit immunsuppressiver Langzeit-Therapie z. B. nach allogener Stammzelltransplantation zur Prophylaxe und Therapie der Graft-versus-Host-Disease (GvHD) auf. Trotz der in-vitro-Suszeptibilität der Pilze gegenüber verschiedenen Antimykotika ist die Erkrankung in diesem Patientenkollektiv weiterhin mit einer hohen Letalität assoziiert. Neben ihren direkten, antifungalen Eigenschaften wurden durch Antimykotika auch indirekte, modulierende Wirkungen auf die Funktionalität von Immunzellen beschrieben, die damit möglicherweise das therapeutische Ansprechen dieser Erkrankung beeinflussen. Insbesondere neutrophile Granulozyten (PMN) spielen als Teil der angeborenen Immunität durch ihre Vielzahl an Abwehrmechanismen eine essentielle Rolle für die Bekämpfung von invasiven Pilzinfektionen und damit der IA. Von zusätzlicher Bedeutung und bislang kaum untersucht ist das kombinierte Einwirken von Antimykotika und Immunsuppressiva auf die Funktionalität dieser Zellen. In dieser Arbeit wurde daher in-vitro der Einfluss klinisch eingesetzter Antimykotika zur Therapie der IA (liposomales Amphotericin B bzw. Amphotericin B – Desoxycholat, Voriconazol) auf wichtige Effektorfunktionen humaner neutrophiler Granulozyten nach Exposition gegenüber A. fumigatus untersucht. Im Fokus stand hierbei die Analyse des oxidativen Burst, der Interleukin (IL)-8-Freisetzung, der Bildung von neutrophil extracellular traps (NETs) sowie der Phagozytose-Aktivität dieser Immunzellen. Außerdem wurde der Effekt einer zusätzlichen Gabe klinisch relevanter Immunsuppressiva (Ciclosporin A / Mycophenolat-Mofetil, Prednisolon), die zur Prophylaxe und Therapie der akuten GvHD eingesetzt werden, auf diese vier Funktionen evaluiert. Zusammenfassend ergaben unsere Analysen keinen Anhalt für eine relevante Beeinflussung des oxidativen Burst, der IL-8-Freisetzung und der Phagozytose-Aktivität neutrophiler Granulozyten durch die untersuchten Antimykotika, insbesondere gegenüber A. fumigatus. Weiterhin wurden in dieser Arbeit keine signifikanten Differenzen zwischen dem Einfluss von liposomalem Amphotericin B und Amphotericin B - Desoxycholat beobachtet. Durch Prednisolon konnten überwiegend bereits bekannte, immunsuppressive Wirkungen auf PMN bestätigt sowie zusätzlich Hinweise auf eine Modulation Antimykotika-vermittelter Immuneffekte durch Immunsuppressiva gewonnen werden. Die kurze Exposition der PMN gegenüber Ciclosporin A / Mycophenolat-Mofetil ergab keine signifikanten Veränderungen der Sekretion von reaktiven Sauerstoffspezies. Des Weiteren unterstützen unsere Daten Hinweise auf differente Aktivierungsmechanismen von verschiedenen Effektorfunktionen der PMN. Im Gegensatz zu den anderen untersuchten Funktionen konnte in dieser Arbeit eine signifikant verminderte Bildung von NETs durch liposomales Amphotericin B, Amphotericin B - Desoxycholat und auch Voriconazol beobachtet werden. Damit geben unsere Ergebnisse Anlass zu tiefergehenden Analysen des Zusammenspiels von Antimykotika insbesondere mit dem noch immer wenig verstandenen Mechanismus der Auslösung und Bildung von NETs. Zudem wären weitere Studien wünschenswert, um einen umfassenderen Überblick und ein besseres Verständnis auch hinsichtlich anderer invasiver Pilzinfektionen sowie weiterer Abwehrmechanismen zu erhalten. / Aspergillus fumigatus (A. fumigatus), an opportunistic pathogenic mould, is the most common pathogen to cause invasive aspergillosis (IA). This invasive fungal infection occurs primarily in patients with long-term immunosuppressive therapy, e.g. after haematopoetic stem cell transplantation during prophylaxis and treatment of graft-versus-host-disease (GvHD). Despite in vitro susceptibility to different antifungal drugs, IA is still associated with significant morbidity and mortality. Besides their well-known, direct antifungal properties, antifungal drugs have been reported to interfere with immune response mechanisms, thereby possibly affecting therapeutic outcome. In particular, neutrophils with their broad arsenal of antifungal mechanisms are essential in the innate immune response against invasive fungal infections, including IA. The combined influence of antifungal and immunosuppressive agents is of additional importance and has not been subject to much investigation. Therefore, this study aimed to characterize the in vitro influence of clinically relevant antifungal agents for therapy of IA (liposomal amphotericin B, amphotericin B deoxycholate, voriconazole) on major effector functions of human neutrophils in response to A. fumigatus, particularly oxidative burst, release of interleukin-8 (IL-8), formation of neutrophil extracellular traps (NETs) and phagocytic activity. Moreover, we assessed the combined effects of antifungals and clinically relevant immunosuppressive agents commonly used for prophylaxis and treatment of GvHD (ciclosporin A / mycophenolate-mofetil, prednisolone) on these four effector functions. In conclusion, our findings are not indicative of major impairment or enhancement of oxidative burst, IL-8-release or phagocytosis response of neutrophils to A. fumigatus in presence of the studied antifungal agents. Our results did not show significant differences between liposomal and conventional amphotericin B. We were able to confirm immunosuppressive effects of prednisolone on neutrophils, and observe a modulation of antifungal-mediated immunological effects through prednisolone. Short-time exposure of neutrophils to ciclosporin A / mycophenolate-mofetil did not cause significant alterations in oxidative burst response to A. fumigatus. However, our data provide indications of different activation patterns for distinct effector functions of neutrophils. In contrast to the other effector functions, this study reports a significantly lowered formation of NETs in presence of LAmB, d-AmB, or voriconazole. Therefore, these findings should give rise to a more in-depth analysis of the interference of these antifungal drugs with the still poorly understood mechanisms of NEToses induction and trap formation. Further research is necessary to acquire a deeper knowledge of other invasive fungal infections and further effector mechanisms.