Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Cumin Seed Oil Induces Oxidative Stress-Based Antifungal Activities on Fusarium graminearum.

In this study, the antifungal activity of cumin seed oil (CSO) was tested on Fusarium graminearum. (i) Minimum inhibitory concentrations (MICs) and related concentrations (IC75, IC50, and IC25) were detected; (ii) toxicity was evaluated by a water-soluble tetrazolium salt-1 (WST-1) assay; (iii) genomic/epigenomic alterations were evaluated by the coupled restriction enzyme digestion-random amplification (CRED-RA) method; (iv) oxidative stress was investigated by CAT expression, catalase activity, and DCF-DA staining; (v) deoxynivalenol biosynthesis was evaluated by tri6 expression; (vi) and potential effects of CSO on wheat were tested by a water loss rate (WLR) assay. MIC, IC75, IC50 and IC25 values were detected at 0.5, 0.375, 0.25, and 0.125 mg mL-1. In WST-1 assays, significant decreases (p < 0.001) were detected. Genomic template stability (GTS) related to methylation differences ranged from 94.60% to 96.30%. Percentage polymorphism for HapII/MspI values were as 9.1%/15.8%. CAT (oxidative stress-related catalase) and tri6 (zinc finger motif transcription factor) gene expressions were recorded between 5.29 ± 0.74 and 0.46 ± 0.10 (p < 0.05). Increased catalase activity was detected (p < 0.05) by spectrophotometric assays. DCF-DA-stained (oxidative stressed) cells were increased in response to increased concentrations, and there were no significant changes in WLR values. It was concluded that CSO showed strong antifungal activity on F. graminearum via different physiological levels.

Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.

Introduction: Chronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients' quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology. Methods: Twenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed. Results: In both MGG (p<0.0001) and ICC analysis (p<0.001) a significant decrease of eosinophils was seen under Dupilumab treatment. When patients were divided into a Eo-low- (<21%) and Eo-high- (≥21%) group according to the percentage eosinophils in nasal swab catology in the first study visit, the Eo-high-group showed a greater change of eosinophils over time (Δ17.82) compared to the Eo-low-group (Δ10.67) but, however, no better response to therapy. The polyp score, SNOT20 questionnaire, and total IgE concentration in peripheral blood showed a significant decrease during the observation period (p<0.0001). Discussion: Nasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.