The host immune response contributes to Haemophilus influenzae virulence

SummaryBackgroundThere is compelling evidence that infections with non-typeable Haemophilus influenzae (NTHi) are associated with exacerbations in COPD patients. However, NTHi has also been isolated frequently during clinically stable disease. In this study we tested the hypothesis that genetically distinct NTHi isolates obtained from COPD patients differ in virulence which could account for dissimilarities in the final outcome of an infection (stable vs. exacerbation).ResultsNTHi isolates (n = 32) were obtained from stable COPD patients, or during exacerbations. Genetically divergent NTHi isolates were selected and induction of inflammation was assessed as an indicator of virulence using different in vitro models. Despite marked genomic differences among NTHi isolates, in vitro studies could not distinguish between NTHi isolates based on their inflammatory capacities. Alternatively, when using a whole blood assay results demonstrated marked inter-, but not intra-individual differences in cytokine release between healthy volunteers irrespective of the origin of the NTHi isolate used.ConclusionResults suggest that the individual immune reactivity might be an important predictor for the clinical outcome (exacerbation vs. no exacerbation) following NTHi infection

Interspecies discrimination of A. fumigatus and siblings A. lentulus and A. felis of the Aspergillus section Fumigati using the AsperGenius® assay

The AsperGenius® assay detects several Aspergillus species and the A. fumigatus Cyp51A mutations TR34/L98H/T289A/Y121F that are associated with azole resistance. We evaluated its contribution in identifying A. lentulus and A. felis, 2 rare but intrinsically azole-resistant sibling species within the Aspergillus section Fumigati. Identification of these species with conventional culture techniques is difficult and time-consuming. The assay was tested on (i) 2 A. lentulus and A. felis strains obtained from biopsy proven invasive aspergillosis and (ii) control A. fumigatus (n=3), A. lentulus (n=6) and A. felis species complex (n=12) strains. The AsperGenius® resistance PCR did not detect the TR34 target in A. lentulus and A. felis in contrast to A. fumigatus. Melting peaks for L98H and Y121F markers differed and those of the Y121F marker were particularly suitable to discriminate the 3 species. In conclusion, the assay can be used to rapidly discriminate A. fumigatus, A. lentulus and A. felis.

Efficacy of IFN-λ1 to protect human airway epithelial cells against human rhinovirus 1B infection.

Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-β. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-β (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-β treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-β induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-β. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-β

HRV1B induced interferon response in PBECs and its comparison with other stimuli.

<p>PBECs were infected with HRV1B for 4(n = 4). The mRNA expression of ISGs was determined with qPCR and fold changes were calculated with the 2^−ΔΔCt method (A). Cells were infected with RSV (MOI-1, 1 h) or HRV1B (4 h). After infection, virus-containing medium was removed and replaced with fresh medium. Additionally, cells were stimulated with poly(I:C)/LyoVec (500 ng/ml) for 24 h (n = 3). Then cells were collected and IFNβ/λ1 mRNA expression was determined by qPCR and relative amount of mRNA was calculated with the 2^−ΔCt method (B). *, p<0.05 control vs rest of the conditions.</p

Long-lasting antiviral state induced by IFNs.

<p>A549 were treated with IFNs for 18-treatment approaches, cells cultured for another 72 h and collected afterwards for further analyses (n = 4). mRNA expression of different genes was determined by qPCR and fold changes were calculated with the 2^−ΔΔCt method (A & B). Moreover, to evaluate the antiviral status of the cells after 72 h of culturing, cells were infected with HRV1B and incubated for another 24 h (n = 5). After that, viral copies were determined by qPCR and fold changes were calculated with the 2^−ΔΔCt method (C). *, p<0.05 HRV1B vs IFNs+HRV1B, control vs IFNs. #, p<0.05 IFN-λ1 vs IFN-β.</p

IFN-induced antiviral state protects against HRV1B infection in A549.

<p>A549 were treated with IFNs for 18(n = 6). Fold-changes were calculated with the 2^−ΔΔCt method (A). A549 were first treated with IFNs for 18 h. Next, according to the continuous and pre-treatment approaches, cells were infected with HRV1B and incubated for another 24 h (n = 6). After that, cells were collected for further analyses. Viral copies were determined by qPCR and fold-changes were calculated with the 2^−ΔΔCt method (B). *, p<0.05 Control vs IFNs, HRV1B vs IFNs+HRV1B. #p<0.05 IFN-λ1 vs IFN-β.</p

Priming effect of IFNs on HRV1B-induced interferon response.

<p>A549 were first treated with IFNs for 18-treatment approaches. After that cells were infected with HRV1B for 4 h in the same medium. After the infection period, either same medium was maintained on the cells (continuous) or replaced by fresh medium (pre-treated). Thereafter, cells were incubated for another 24 h and then collected for further analyses. mRNA levels of IFN-β (A) and IFN-λ1 (B) were determined by qPCR and fold changes were calculated with the 2^−ΔΔCt method. HRV1B induced average IFN-β expression was 1.8 (continuous) and 0.99 (pre-treated) folds and average IFN-λ1 expression was 1.92 (continuous) and 0.90 folds (pre-treated). *, p<0.05 control vs rest of conditions. #, p<0.05 IFN vs IFN+HRV1B. n = 4.</p

Validation of a new Aspergillus real-time PCR assay for direct detection of Aspergillus and azole resistance of Aspergillus fumigatus on bronchoalveolar lavage fluid

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