Type of Publication: | Dissertation |
URI (citable link): | http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-23677 |
Author: | Daneshian, Mardas |
Year of publication: | 2006 |
Title in another language: | Detektion und Charakterisierung der immunstimulatorischen Eigenschaften luftgetragener Pyrogene |
Summary: |
Human whole blood is an accessible source of primary immune cells and plasma components involved in host defense against infection. It can be used both to study the human inflammatory reaction to microbes and their pyrogenic components and to detect their presence in samples by measuring the production of a representative inflammatory factor upon challenge. The latter was exploited by developing a method to evaluate the pyrogenic burden in air samples collected on filters. As fungal spores represent a large portion of airborne microbia, their contribution to the pyrogenic burden in the air was evaluated by characterizing their inflammatory activity and studying the mechanisms and molecules involved. The strategy to collect pyrogens from the air on a solid phase was further developed to enable detection of pyrogens in toxic or immunomodulatory drugs and to improve sensitivity for testing of the pyrogenic load in dialysis fluids and large volume parenterals by collecting pyrogens on albumin-linked beads. 1. An air sampling method to collect pyrogens from the air on a filter and assess cytokine release by blood incubated with the filter was established and an adequate reference material was developed, enabling field studies. These showed a good correlation between the inflammatory activity in the air and the live germ count. Donor comparison revealed that the immune response of allergic people differed from that of non-allergic individuals. Standardized, cryopreserved blood proved a suitable substitute for fresh blood that could exclude such donor variances. Comparison of this new method with the Limulus amoebocyte lysate test, which is limited to the detection of endotoxins, proved as expected that the latter underestimates the total inflammatory burden in the air. 2. The immunostimulatory capacity of fungal spores was assessed by characterization of the cytokine pattern they induced in human whole blood. The 44 species tested induced a highly homogenous pattern of cytokine release with similar kinetics, indicating that fungal spores share a surface structure that is targeted by the innate immune system. Analysis of donor variance revealed that the cytokine amounts released in human blood, both in response to fungal spores and to other stimuli employed, is determined mainly by the individual monocyte count. The relative response to the different stimuli was highly consistent. Comparison of the cytokine response of human blood with an alveolar macrophage cell line revealed a high correlation, suggesting that the blood monocytes and lung macrophages respond similarly. 3. Analysis of the role of fungal surface glycans by chemical and enzymatic detachment of sugar chains strongly questioned their proposed role as the immunostimulatory principle of fungal spores. Examination of TLR dependency of immune recognition of fungal spores using cells from respective knock-out mice revealed that the presence of TLR-2 is necessary for full cytokine response to fungal spores. Interestingly, the presence of TLR-4 appeared to suppress cytokine release. 4. The role of TLR-2 in the recognition of fungal spores suggested that the immunostimulatory principle might be structurally related to that of other TLR-2 agonists. Conidia of Cladosporium cladosporioides were subjected to butanol extraction and separation by FPLC and HPLC. This resulted in the purification of an immunostimulatory, amphiphilic compound that consists mainly of alkyl chains probably bearing sugar moieties as revealed by 1HNMR spectroscopic analysis. This compound induces a cytokine pattern consistent with that induced by the whole spores and showed the same TLR dependency. Its activity was not affected by coincubation with the LPS inhibitors Polymyxin B and LAL-F, but the LTA inhibitor PPG 1200 reduced its inflammatory capacity. 5. Developing the idea of collecting pyrogens onto a solid phase, a method was developed to collect pyrogens from liquid onto albumin-linked beads. This allowed the separation of the pyrogens from immunomodulatory or toxic drugs. The beads were washed and then brought into contact with human whole blood. It could be shown that the beads bind LPS, LTA or zymosan spikes and the sensitivity of detection was comparable to that of the standard tests for parenterals. The ability of the test to detect pyrogens could be shown in drugs known to interfere with other pyrogen tests, i.e. paclitaxel, gentamicin, prednisolone, liposomal amphotericin B and liposomal daunorubicin. 6. Further modifications of the setting aimed to accumulate very low concentrations of pyrogens in large samples on the albumin-linked beads. This increased the sensitivity of the assay by a factor of 250, which is useful for measurement of pyrogens in dialysis fluids and large volume parenterals. The tests developed in this thesis may help to evaluate the health risk of working and living environments posed by the burden of air-borne pyrogens and to improve drug safety and chronic problems in dialysis patients caused by exposure to pyrogens. The studies on the immunostimulatory qualities of fungal spores revealed that, like Gram-negative and Gram-positive bacteria, fungi also share common surface structures that are targeted by the human innate immune system. In contrast to previous suggestions, these appear to be amphiphilic molecules. A better understanding of the interactions between fungi and the immune system may provide new therapeutic targets.
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Summary in another language: |
Humanes Vollblut ist eine gut zugängliche Quelle für primäre Immunzellen und Plasmakomponenten, welche an Immunabwehrreaktionen beteiligt sind. Es kann sowohl genutzt werden, um die humane Abwehrreaktionen gegen Mikroben und ihre pyrogenen Komponenten zu studieren als auch um in einer Probe eine pyrogene Kontamination durch die Messung eines repräsentativen entzündlichen Parameters nachzuweisen. Letztere Möglichkeit wurde genutzt, um eine Methode zu entwickeln, durch die durch Beprobung von Filtern mit Luftproben deren pyrogene Belastung beurteilt werden kann. Da Pilzsporen einen großer Anteil an luftgetragenen Mikroben darstellen, wurde ihr Anteil an der pyrogenen Belastung der Luft beurteilt indem ihre entzündliche Aktivität charakterisiert und beteiligte Moleküle und Mechanismen untersucht wurden. Ferner wurde die Strategie, Pyrogene aus der Luft auf einer Festphase zu sammeln, weiterentwickelt, um es zu ermöglichen durch den Einsatz von Albumin-gekoppelten Mikrosphären, Pyrogene in toxischen oder immunmodulatorischen Medikamenten nachzuweisen und um die Nachweisgrenze des Tests so zu erniedrigen, dass auch geringe Pyrogene Belastungen in Dialyseflüssigkeiten und grossvolumigen Parenteralia nachgewiesen werden können. 1. Eine Luftsammelmethode wurde etablieret, um Pyrogene aus der Luft auf Filtermembranen zu sammeln und um die Zytokinausschüttung von Blutzellen als Reaktion auf diese Filter zu beurteilen. Dazu wurde auch ein entsprechendes Referenzmaterial für Feldstudien generiert. Diese zeigten eine gute Korrelation zwischen der Menge an lebenden Mikroben in der Luft und der entzündlichen Aktivität der Luft. Spenderanalysen zeigten, dass Allergiker eine abweichende Immunreaktion im Verglich zu Nichtallergikern haben. Standardisiertes, kryopräserviertes Blut stellte einen adäquaten Ersatz für Frischblut dar, was es ermöglicht, die Spendervarianz auszuschlielßen. Der direkte Vergleich dieser neuen Methode mit dem Limulus-Amoebzytenlysat-Test, welcher nur Endotoxine detektiert, zeigte dass letzterer wie zu erwarten die Belastung der Luft unterschätzt. 2. Die immunstimulatorische Kapazität von Pilzsporen wurde durch Charakterisierung der Zytokinmuster, die durch sie im humanen Blut induziert wurden, untersucht. Die 44 getesteten Spezies induzierten sehr homogene Zytokinmuster mit ähnlicher Kinetik, was nahe legte, dass Pilzsporen gemeinsame Oberflächenstrukturen besitzen, die vom Immunsystem erkannt werden. Eine Analyse der Spendervarianzen zeigte, dass sowohl die durch Pilze induzierten als auch die durch andere Stimuli induzierten Zytokinmengen hauptsächlich durch die individuelle Monozytenzahl bestimmt werden. Die relative Antwort auf verschiedene Stimuli zeigte sich durchwegs als vergleichbar. Der Vergleich zwischen der Zytokinantwort von humanen Blutzellen mit der von murinen Alveolarmakrophagen zeigte eine starke Korrelation, was darauf hinweist, dass Blutmonozyten und Alveolarmakrophagen ähnlich reagieren. 3. Die Analyse der Rolle von pilzlichen Glykanen durch chemische und enzymatische Spaltung stellte ihre vorgeschlagene Rolle als immunstimulatorisches Prinzip von Schimmelpilzen in Frage. Die Untersuchung der TLR-Abhängigkeit der Immunerkennung von Schimmelpilzen durch den Einsatz von Zellen aus entsprechenden knockout Mäusen zeigte, dass für deren Erkennung der TLR-2 Rezeptor notwendig ist um das volle Ausmaß an Zytokininduktion zu bewirken. Interessanterweise unterdrückt das Vorhandensein von TLR-4 die Zytokinproduktion. 4. Die Beteiligung des TLR-2 Rezeptors an der Erkennung von Pilzsporen deutet darauf hin, dass die pilzlichen TLR-2 Agonisten strukturelle Ähnlichkeit mit anderen TLR-2 Agonisten haben könnten. Konidien von Cladosporium cladosporioides wurden einer Butanolextraktion unterzogen und der Extrakt wurde über FPLC und HPLC aufgetrennt. Dadurch konnte ein immunstimulatorisches, amphiphiles Molekül aufgereinigt werden, welches der 1H-NMR Analyse nach aus Alkylketten besteht und möglicherweise Zuckerreste trägt. Diese Verbindung induzierte dieselben Zytokinmuster wie die ganzen Sporen und zeigte auch dieselbe TLR Abhängigkeit. Koinkubationen mit den bekannten LPS-Inhibitoren Polymyxin B und LAL-F beeinflussten die Aktivität der gereinigten Substanz nicht, aber der LTA Inhibitor PPG 1200 konnte die inflammatorische Kapazität dieser Verbindung dämpfen. 5. Im Zuge der Anpassung des Bluttestes an die Sammlung von Pyrogenen auf einer Festphase, wurde eine Methode entwickelt, mit der Pyrogene aus Flüssigkeiten an Albumin-gekoppelten Mikrosphären gebunden werden konnten. Dadurch konnten die Pyrogene von immunstimulatorischen oder toxischen Medikamenten getrennt werden. Nach einem Waschvorgang wurden sie erst mit Blut in Kontakt gebracht. Es konnte gezeigt werden, dass die Mikrosphären LPS, LTA und zymosan binden und dass die Sensitivität der Detektion mit der des Standardtests für injizierbare Arzneimittel vergleichbar war. Das Vermögen dieses Tests, Pyrogene in schwertestbaren Medikamenten nachzweisen wurde anhand von einigen Beispielen nachgewiesen: Paclitaxel, Gentamicin, Prednisolone, liposomales Amphotericin B und liposomales Daunorubicin. 6. Durch weitere Modifikationen des Versuchsaufbaus konnten geringe Mengen an Pyrogenen auf den Mikrosphären gesammt werden. Dadurch wurde die Sensitivität des Tests um den Faktor 250 erhöht, was den Nachweis von Pyrogenen in Dialyseflüssigkeiten und in großvolumigen Medikamenten ermöglicht. Die Tests, die in dieser These entwickelt wurden, können dazu beitragen, Risikoeinschätzungen für die pyrogene Belastung am Arbeitsplatz und in Innenräumen zu treffen, die Arzneimittelsicherheit in speziellen Fällen zu verbessern und um chronische Probleme pyrogenen Ursprungs bei Dialysepatienten zu vermeiden. Die Untersuchung der immunstimulatorischen Aktivitäten von Schimmepilzsporen zeigte, dass Pilze, ähnlich wie Gram-negative und Grampositive Bakterien, gemeinsame Oberflächenstrukturen tragen, welche vom angeborenen Immunsystem erkannt werden. Diese Strukturen scheinen entgegen früheren Vorschlägen, amphiphile Moleküle zu sein. Ein besseres Verständnis von der Interaktion zwischen den Pilzen und dem Immunsystem, könnte Ansatzpunkte für neue Therapeutika liefern.
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Examination date (for dissertations): | Jun 9, 2006 |
Dissertation note: | Doctoral dissertation, University of Konstanz |
Subject (DDC): | 570 Biosciences, Biology |
Controlled Keywords (GND): | Pyrogen, Schimmelpilze, Atemluft, Immunbiologie, In vitro |
Keywords: | pyrogens, mould, air, immunobilogy, in-vitro |
Link to License: | In Copyright |
Bibliography of Konstanz: | Yes |
DANESHIAN, Mardas, 2006. Detection and characterization of the detection and characterization of the immunostimulatory properties of airborne pyrogens [Dissertation]. Konstanz: University of Konstanz
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It can be used both to study the human inflammatory reaction to microbes and their pyrogenic components and to detect their presence in samples by measuring the production of a representative inflammatory factor upon challenge. The latter was exploited by developing a method to evaluate the pyrogenic burden in air samples collected on filters. As fungal spores represent a large portion of airborne microbia, their contribution to the pyrogenic burden in the air was evaluated by characterizing their inflammatory activity and studying the mechanisms and molecules involved. The strategy to collect pyrogens from the air on a solid phase was further developed to enable detection of pyrogens in toxic or immunomodulatory drugs and to improve sensitivity for testing of the pyrogenic load in dialysis fluids and large volume parenterals by collecting pyrogens on albumin-linked beads. 1. An air sampling method to collect pyrogens from the air on a filter and assess cytokine release by blood incubated with the filter was established and an adequate reference material was developed, enabling field studies. These showed a good correlation between the inflammatory activity in the air and the live germ count. Donor comparison revealed that the immune response of allergic people differed from that of non-allergic individuals. Standardized, cryopreserved blood proved a suitable substitute for fresh blood that could exclude such donor variances. Comparison of this new method with the Limulus amoebocyte lysate test, which is limited to the detection of endotoxins, proved as expected that the latter underestimates the total inflammatory burden in the air. 2. The immunostimulatory capacity of fungal spores was assessed by characterization of the cytokine pattern they induced in human whole blood. The 44 species tested induced a highly homogenous pattern of cytokine release with similar kinetics, indicating that fungal spores share a surface structure that is targeted by the innate immune system. Analysis of donor variance revealed that the cytokine amounts released in human blood, both in response to fungal spores and to other stimuli employed, is determined mainly by the individual monocyte count. The relative response to the different stimuli was highly consistent. Comparison of the cytokine response of human blood with an alveolar macrophage cell line revealed a high correlation, suggesting that the blood monocytes and lung macrophages respond similarly. 3. Analysis of the role of fungal surface glycans by chemical and enzymatic detachment of sugar chains strongly questioned their proposed role as the immunostimulatory principle of fungal spores. Examination of TLR dependency of immune recognition of fungal spores using cells from respective knock-out mice revealed that the presence of TLR-2 is necessary for full cytokine response to fungal spores. Interestingly, the presence of TLR-4 appeared to suppress cytokine release. 4. The role of TLR-2 in the recognition of fungal spores suggested that the immunostimulatory principle might be structurally related to that of other TLR-2 agonists. Conidia of Cladosporium cladosporioides were subjected to butanol extraction and separation by FPLC and HPLC. This resulted in the purification of an immunostimulatory, amphiphilic compound that consists mainly of alkyl chains probably bearing sugar moieties as revealed by 1HNMR spectroscopic analysis. This compound induces a cytokine pattern consistent with that induced by the whole spores and showed the same TLR dependency. Its activity was not affected by coincubation with the LPS inhibitors Polymyxin B and LAL-F, but the LTA inhibitor PPG 1200 reduced its inflammatory capacity. 5. Developing the idea of collecting pyrogens onto a solid phase, a method was developed to collect pyrogens from liquid onto albumin-linked beads. This allowed the separation of the pyrogens from immunomodulatory or toxic drugs. The beads were washed and then brought into contact with human whole blood. It could be shown that the beads bind LPS, LTA or zymosan spikes and the sensitivity of detection was comparable to that of the standard tests for parenterals. The ability of the test to detect pyrogens could be shown in drugs known to interfere with other pyrogen tests, i.e. paclitaxel, gentamicin, prednisolone, liposomal amphotericin B and liposomal daunorubicin. 6. Further modifications of the setting aimed to accumulate very low concentrations of pyrogens in large samples on the albumin-linked beads. This increased the sensitivity of the assay by a factor of 250, which is useful for measurement of pyrogens in dialysis fluids and large volume parenterals. The tests developed in this thesis may help to evaluate the health risk of working and living environments posed by the burden of air-borne pyrogens and to improve drug safety and chronic problems in dialysis patients caused by exposure to pyrogens. The studies on the immunostimulatory qualities of fungal spores revealed that, like Gram-negative and Gram-positive bacteria, fungi also share common surface structures that are targeted by the human innate immune system. In contrast to previous suggestions, these appear to be amphiphilic molecules. A better understanding of the interactions between fungi and the immune system may provide new therapeutic targets.</dcterms:abstract> <dc:rights>terms-of-use</dc:rights> </rdf:Description> </rdf:RDF>
Daneshian_Diss.pdf | 1760 |