Helmut W. Ott

Aspergillus fumigatus Activates Thrombocytes by Secretion of Soluble Compounds

During invasive aspergillosis, platelets might be involved in immune defense, but they also might contribute to the pathology of the disease. We tested the hypothesis that Aspergillus secretes factors that influence the activity and functionality of thrombocytes. Platelets were incubated with medium wherein Aspergillus fumigatus was grown. This fungal culture supernatant potently stimulated thrombocytes in a time- and dose-dependent fashion, inducing release of alpha and dense granules, membrane alterations, aggregation, and formation of microparticles. Fungus-induced platelet activation could be confirmed in vivo: thrombocytes from mice infected with A. fumigatus showed a higher activation level than platelets from noninfected animals. Two stimulating components in the fungal culture supernatant were identified: a fungal serine protease and the mycotoxin gliotoxin. Activation of platelets by fungal factors stimulates antifungal functions: platelets gain the capacity to interact with foreign particles, and they become able to inhibit fungal growth, thus supporting the host immune network. However, some consequences of platelet activation might also be harmful, including excessive inflammation and induction of thrombosis. These findings imply that measuring platelet activation in patients might be an interesting diagnostic parameter.

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

Analysis of von Willebrand factor (vWF) multimers allows classification of the subtypes of von Willebrand disease (vWD) in human serum and platelet lysates. A novel method for multimer analysis of vWF by 2-chamber, vertical (sodium dodecyl sulfate), agarose gel electrophoresis, designed for comparing discontinuous high- and low-resolving gels for plasma and platelets, followed by Western blotting and high-sensitivity fluorescence detection (HSFD) of cyanine (Cy)5-labeled vWF multimers is presented. HSFD shows that this method has high discriminatory power for visualization and densitometric analysis of platelets and plasma vWF multimers in various types of vWD and allows rapid classification of vWD types, to separate types 2A and 2B. The described procedures of vWF multimer analysis with high-sensitivity Cy5 fluorescence detection and direct comparison of high- and low-resolving gels for screening and detection of the complete range of high- and low-molecular vWF multimers is efficient and useful for screening, detecting, and classifying vWD subtypes and makes this method diagnostically and clinically relevant.

Virulence and thrombocyte affectation of two Aspergillus terreus isolates differing in amphotericin B susceptibility

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.

A novel N-chlorotaurine-corticosteroid combination as a preservative-free local disinfectant: influence on the ciliary beat frequency in vitro.

Conclusions. The combination of N-chlorotaurine (NCT) and a corticosteroid seems to be a very promising substance for the local therapy of ENT infections. As it can be used without any preservatives, the effect on the ciliary beat frequency (CBF) is much less than that of products containing benzalkonium chloride (BAC). The in vitro results obtained in this study encourage us to perform clinical trials on this novel combination for intranasal application. Objective. To investigate the influence of a novel mixture of NCT and a corticosteroid [fluticasone propionate (FP)] on the CBF of human ciliated cells in vitro.Material and methods. The study was designed as an in vitro study. CBF was measured by means of a photometric technique involving the combination of a light microscope, a photometer, a photographic multiplier and a computerized analyzing unit. Results. The combination of 1% NCT + 0.5 mg/ml FP decreased the CBF to 42.17% of its original value after 20 min. Treatment with BAC lowered the CBF depending on the concentration to 96.61% of its original value with 0.04 mg/ml, to 91.90% with 0.1 mg/ml, to 63.46% with 0.2 mg/ml and to 0% with 0.5 mg/ml. After rinsing in saline, the CBF of samples treated with 1% NCT + 0.5 mg/ml FP recovered to 68.93% of its original value.

Interaction of Platelets and Anidulafungin against Aspergillus fumigatus

ABSTRACT The combination of platelets and anidulafungin at 0.03 μg/ml significantly (P < 0.05) reduced the germination rate and hyphal elongation in Aspergillus fumigatus compared to those with either anidulafungin only or an untreated control. Platelets decreased the expression of the fks gene, which plays an important role in cell wall synthesis. Our results suggest that human platelets plus anidulafungin might contribute to defense against A. fumigatus.

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: Implication for the interpretation of satellite bands

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS‐agarose gel electrophoresis is the lack of satellite bands in the high‐resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub‐unit from the first dimension dissociates into three distinct sub‐bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS‐agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrands disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS‐agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrands disease subtypes.