Detection and characterization of the detection and characterization of the immunostimulatory properties of airborne pyrogens

Abstract

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.