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Genome-Wide Expression Profiling Reveals S100B as Biomarker for Invasive Aspergillosis

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Genome-Wide Expression Profiling Reveals S100B as Biomarker for Invasive Aspergillosis

Andreas Dix et al. Front Microbiol.

Abstract

Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy toward this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of hematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time reverse transcription polymerase chain reaction (RT-PCR) assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis.

Keywords: allogeneic stem cell transplantation; fungal infection; gene expression data; human biomarker; invasive aspergillosis.

Figures

Figure 1
Figure 1
The workflow of the feature selection process. We used random forest as classifier and performed leave-one-out cross-validation. The ranking of the genes was done once for each fold of the cross-validation for the unreduced input gene set.
Figure 2
Figure 2
The Venn diagram shows that 123 DEGs were identified for both IA and non-IA patients. Additionally, 379 DEGs and 8 DEGs were specific for IA and non-IA patients, respectively.
Figure 3
Figure 3
The average error rates and standard deviations across the decreasing number of genes in the feature selection process. The smallest error rate was calculated for using one gene, S100B.
Figure 4
Figure 4
Comparison of the expression intensities of S100B between the different conditions. The distribution of the values of the non-IA samples covers a broad range and is similar to the healthy controls. The IA samples show low S100B expressions.
Figure 5
Figure 5
In vitro analysis of S100B regulation in DCs. S100B was down-regulated in A. fumigatus stimulated DCs. DCs were either stimulated with A. fumigatus (MOI 1) or left untreated. mRNA level were quantified after 6 h by real-time PCR relative to reference gene ALAS1. Data of three independent experiments is illustrated as mean plus SEM (**p < 0.05 Student's paired t-test).
Figure 6
Figure 6
Influence of S100B knockdown on gene regulation. DCs were transfected by electroporation with either non-silencing siRNA (white bars) or with siRNA targeting siS100B (black bars). Twenty-four hours after electroporation, DCs were stimulated with zymosan depleted (100 μg/ml), Pam3CSK4 (100 ng/ml) (Invivogen), or left untreated. mRNA levels of S100B (A), IL1B (B), CXCL1 (C), and IL6 (D) were quantified after 6 h by real-time PCR relative to non-silencing control. ALAS1 served as reference gene. Data of four independent experiments is illustrated as mean plus SEM (*p < 0.01, ***p < 0.001 Student's paired t-test).

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