Thesis--Wisconsin. / Includes bibliographical references (leaves 72-79).

The aflatoxins, carcinogenic and toxic mold metabolites of Aspergillus flavus, were isolated and determined in cottonseed meal and other feedstuffs. This new quantitative method uses an acetone soxhlet extraction to remove the toxins from the defatted meal. The residual triglycerides, phospholipids and pigments were removed from the acetone by filtration of the cooled solution. The aflatoxin was further isolated by evaporating the acetone and redissolving the residue in hot methanol. After cooling, the insolubles were discarded and the methanol-soluble materials taken up in chloroform. The chloroform solution was spotted on silicic acid thin layer chromatographic plates along with suitable standards. After development with 9:1 (v/v) chloroform:acetone, the chromatographic plates were examined under ultraviolet light. Complete recovery of aflatoxin, within limits of visual discrimination, was obtained by this isolation procedure as indicated by extraction efficiency and internal standard data. Twenty-five cottonseed meals were analyzed by this method and nine meals contained aflatoxin B₁ at levels from 17 to 190 parts per billion. Fluorodensitometry, a new instrumental technique, was used to compare standards and samples containing aflatoxin directly from the thin-layer chromatographic plate. This procedure eliminates the errors inherent in the visual comparison method and permits greater sensitivity and accuracy. Amounts of aflatoxin B₁ as low as 8.0 x 10⁻⁵ micrograms can be detected by this technique. The results obtained by this procedure were substantiated by duckling assay and trout feeding trials. / Graduation date: 1966

Aspergillus fumigatus ist ein ubiquitär vorkommender Schimmelpilz, dessen Sporen vom Menschen täglich zu hunderten mit der Atemluft aufgenommen werden. Aufgrund ihrer geringen Größe gelangen die Konidien leicht bis in die Alveolen der Lunge, wo sie normalerweise sofort vom angeborenen Immunsystem beseitigt werden. Immunsupprimierte Menschen leiden an einer qualitativen oder quantitativen Einschränkung dieses Teiles der Immunabwehr, weshalb die Inokulation der Sporen bei ihnen zur Auslösung der lebensgefährlichen Invasiven Aspergillose führen kann, deren Mortalität in der Hochrisikogruppe der Patienten nach hämatopoetischer Stammzelltransplantation über 90% beträgt. Bei diesen Patienten gewinnt die adaptive Immunabwehr an Bedeutung. Dendritische Zellen gehören dem angeborenen Immunsystem an und besitzen einzigartige Fähigkeiten zur Aktivierung und Steuerung der erworbenen Immunabwehr. Durch die Aktivierung naiver T-Zelen und die Sekretion bestimmter immunmodulatorischer Zytokine bestimmen sie die Art der konsekutiv asugelösten T-Helferzellantwort. Lediglich eine T-Helfer 1-Zellantwort wird hierbei mit einer erhöhten Resistenz gegenüber der invasiven Aspergillose in Verbindung gebracht. Die Zytokinsekretion der Dendritischen Zellen wird durch intrazelluläre Signalkaskaden reguliert, welche sich an ihre PRRs anschließen. Für die Erkennung des bakteriellen LPS durch den TLR wurde die Regulation der immunmodulatorischen Zytokine durch die Serin-Threonin-Kinase GSK3 festgestellt. In der vorliegenden Arbeit wurde untersucht, ob es bei der Erkennung von Aspergillus fumigatus durch humane dendritische Zellen ebenfalls einen Zusammenhang mit GSK3 und der Sekretion immunmodulatorischer Zytokine gibt. Dafür wurden aus humanen Monozyten generierte dendritische Zellen mit rekombinanten Antigenen von Aspergillus fumigatus sowie verschiedenen Morphologien stiumuliert und teilweise mit dem GSK3-Inhibitor LiCl gehemmt. Anschließend wurde die Expression bestimmter immunmodulatorischer Zytokine und der GSK3 mittels quantitativer real-time RT-PCR bestimmt. Hierbei konnte für eines der getesteten Antigene, Aspf 1, ein deutlicher Einfluss auf die GSK3-Expression der Zellen festgestellt werden. Ein paralleler Anstieg der Expression proinflammatorischer Zytokine erhärtete die Annahme einer immunregulierenden Rolle der GSK3 bei der Erkennung von Aspergillus fumigatus. Einen deutlicheren Hinweis auf den Zusammenhang zwischen der Aktivität der GSK3 und der Expression proinflammatorischer Zytokine erbrachte die Inhibierung mittels LiCl. Die mit Aspergillus fumigatus stimulierten Zellen reagierten hierauf im Vergleich zu den nicht-inhibierten Zellen mit einer Reduktion der IL12p35-Expression um 86,1% sowie mit einer Reduktion der IL23-Expression um 49,5%. Der letzte Teil der Experimente sollte ausgehend von der Vorstellung, dass verschiedene Aspergillus-Morphologien von unterschiedlichen Rezeptoren erkannt werden quantitative Unterschiede der GSK3-Beteiligung bei der Erkennung von Aspergillus fumigatus durch dendritische Zellen zeigen. Obwohl alle Morphologien des Schimmelpilzes einen Einfluss auf die GSK3-Expression der Zellen zeigten, konnten hierbei keine einheitlichen quantitativen Unterschiede festgestellt werden. Zusammengefasst konnte in der vorliegenden Arbeit die Beteiligung von GSK3 bei der Erkennung von Aspergillus fumigatus durch humane dendritische Zellen gezeigt werden. Außerdem konnten Hinweise auf eine immunregulierende Rolle der GSK3 bei der Abwehr des fakultativ pathogenen Schimmelpilzes erbracht werden, wobei die genaue Einbindung der Serin-Threonin-Kinase in dieser Situation noch unklar ist und weitere Experimente erforderlich macht. / Aspergillus fumigatus is a ubiquitously occurring mould. Human beings inoculate the spores each day by breathing air. Because of their small size, the conidia easily reach the alveoles, where under normal conditions, they are immediately deleted by innate immunity. Immunocompromised humans suffer from a reduction in this part of the immune system, regarding both the quality of its deletion ability and the quantity of immune cells. Therefore, the inoculation of the spores in immunocompromised cases leads to a severe disease called invasive aspergillosis. The mortality in the high-risk group of patients undergoing stem-cell transplantation is more than 90%. Thus, the adaptive immunity of these patients gains importance. Dendritic cells are a part of innate immunity, and they have unique skills to activate and control the adaptive part of the immune system. By activating naive T-cells and secreting certain immunomodulatory cytokines, they affect the consecutive T-cell response. At this time, only a T-helper 1 response is associated with an increased resistance to invasive aspergillosis. Intracellular signaling systems attached to the pathogen recognition receptors (PRRs) of the dendritic cells regulate the secretion of cytokines. The serine-threonine kinase GSK3 (glycogen synthase kinase 3) controls the expression of the immunomodulatory cytokines from the Toll-like receptor (TLR) and the bacterial Lipopolysaccharide (LPS). This research investigated whether a correlation exists between the recognition of aspergillus fumigatus by human dendritic cells and the GSK3-controlled secretion of immunomodulatory cytokines, and thereby, the control of adaptive immunity. Dendritic cells were generated from human monocytes, subsequently stimulated with recombinant antigenes or various morphologies of aspergillus fumigates, and partially inhibited with the GSK3-inhibitor lithium chloride (LiCl). The expression of certain GSK3-controlled immunomodulatory cytokines was determined using quantitative, real-time RT-PCR (reverse transcription polymerase chain reaction). For one of the tested antigenes, Aspf 1, we revealed a considerable influence on the cells GSK3-expression. A parallel increase of the proinflammatory cytokine expression confirmed the presumption of a immunoregulatory role of GSK3 in recognizing certain Aspergillus fumigatus antigenes. A further indication of the correlation between the activity of GSK3 and the expression of proinflammatory cytokines was provided by inhibiting the cells using LiCl. Hereby, the inhibited cells reacted according to the non-inhibited cells with a 86,1% diminution of the IL-12p35 expression and a 49,5% diminution of the IL-23 expression. In the last part of this work, we investigated whether there is a difference in the GSK3 involvement during the recognition of different aspergillus morphotypes by dendritic cells, as these are recognized by different PRRs. Although all morphotypes showed an influence on the GSK3-expression, no consistent differences in quantity were discovered. Summarizing our work, we revealed the involvement of GSK3 during the recognition of aspergillus fumigatus by human dendritic cells. Besides, we found some evidence of the immunoregulatory role of GSK3 in this context, however the exact integration of the serine-threonine kinase remains unclear and needs further research.

Natürliche Killer T-Zellen stellen ein verbindendes Element zwischen angeborener und adaptiver Immunität dar und sind durch die Expression von T- und NK-Zell-Markern, sowie eines invarianten T-Zell-Rezeptors charakterisiert. Damit erkennen sie Lipide, welche vorwiegend durch dendritische Zellen mittels des MHC-I-ähnlichen Moleküls CD1d präsentiert werden. Die protektive Rolle von iNKT-Zellen bei Autoimmun- und malignen Erkrankungen ist nachgewiesen, ebenso ihre wichtige Aufgabe bei der Abwehr bakterieller, viraler und parasitärer Pathogene. Inwiefern iNKT-Zellen mit Pilzen und im speziellen Aspergillus fumigatus (A. f.) interagieren ist jedoch unzureichend beschrieben. iNKT-Zellen gesunder Spender wurden ex vivo selektiv kultiviert und mit A. f. Konidien und Keimschläuchen kokultiviert. Microarray-Analyse, bzw. Multiplex-ELISA fanden Anwendung, um eine durch A. f. induzierte differentielle Genexpression bzw. Zytokinsekretion bei iNKT-Zellen zu detektieren. Zusätzlich wurde mittels Durchflusszytometrie gemessen, ob A. f. die Produktion des TH1-Zytokins IFN-γ beeinflusst. Schließlich wurde der fungizide Effekt von iNKT-Zellen auf A. f. via XTT-Assay quantifiziert. iNKT-Zellen zeigten einen ausgeprägten fungiziden Effekt auf A. f. Keimschläuche, welcher mit einem hohen Anteil IFN-γ+ iNKT-Zellen vergesellschaftet war. Hingegen zeigte sich kein Stimulus-spezifisches Zytokinsekretionsmuster. Besonders Keimschläuche induzierten eine differentielle Genexpression bei iNKT-Zellen. Diese war gekennzeichnet durch eine Hochregulierung von Genen des anaeroben Glukosemetabolismus. Als Zeichen einer möglicherweise lokalen Hypoxie waren VEGFA hoch-, bzw. CCL15 in ihrer Transkription herunterreguliert. Vor dem Hintergrund einer unbefriedigend hohen Mortalität der durch A. f. verursachten invasiven Aspergillose können diese ersten Ergebnisse der Interaktion des Schimmelpilzes mit iNKT-Zellen Grundlage für weitere Untersuchungen sein, um deren Wechselwirkung besser zu verstehen und möglicherweise zur Verbesserung der Therapie der Aspergillose beizutragen. / Objectives Invariant human Natural Killer T Cells (iNKT-Cells) are innate immune effector cells. They are characterized by their expression of both, T- and NK cell markers and therefore they are a connecting link between innate and adaptive immunity. These cells are further defined by their T-Cell receptor, consisting of a specific, invariant α-chain and a β-chain derived from a limited repertoire. They strongly react upon stimulation with lipid antigens presented in the context of the MHC-like molecule CD1d on dendritic cells (DCs) and thus they can be expanded in vitro by stimulation with their prototypic agonist α-galactosylceramide (α-GalCer). A protective role of iNKT cells in autoimmune diseases and cancer immunity, as well as their action against different pathogens is well documented. However, little is known about their direct interaction with fungi, especially with Aspergillus fumigatus. A. fumigatus, an omnipresent mold, is the most prevalent cause of a highly devastating opportunistic infection, affecting mainly immunocompromised patients. Methods In order to expand human iNKT cells (iNKT⁺CD3⁺), PBMCs were treated with α-GalCer and rhIL-2 for 15-20 days. After expansion, purity of the iNKT+CD3+ fraction was >90%. To further purify this fraction, a positive selection with CD3 microbeads was performed. After that the purity increased above 95%. iNKT cells were cocultured with different morphologies of A. fumigatus, resting conidia and germlings. Gene induction was evaluated by microarray analysis (Affymetrix U219) and the protein release by using multiplex ELISA assays (BioRad Bio-Plex). Additionally, the induction of IFN-γ, a major Th1 cytokine, was analyzed by flow cytometry. Finally, XTT assays were used to examine the toxic effects of iNKT cells on A. fumigatus. Results Stimulating iNKT cells with different A. fumigatus morphologies, we observed a time- and morphotype-dependent induction of INF-γ, with germlings to be shown more immunogenic than conidia. Furthermore, challenging iNKT cells with A. fumigatus germlings at an MOI=1 for 6h, we found that iNKT cells caused a significant fungal damage. We could also observe defined patterns of gene induction, especially regarding cytokine and chemokine gene expression profiles. By multiplex ELISA assays gene expression patterns could be confirmed. Conclusion We found that there is a specific interaction of iNKT cells with A. fumigatus, leading to defined cytokine induction and a fungicidal effect. INF-γ, a cytokine, which has a known protective role against IA, is produced by iNKT cells when confronted with A. fumigatus.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

Thèse d'exercice : Pharmacie : Université de Nantes : 2005. / Bibliogr. f. 158-169 [127 réf.].

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Georgia Davis. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 201 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Stephen A. Osmani, Dept. of Molecular Genetics. Includes bibliographical references (p. 184-201).

published_or_final_version / Microbiology / Master / Master of Medical Sciences