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- ItemBiochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy(Basel : MDPI, 2021) Pistiki, Aikaterini; Ramoji, Anuradha; Ryabchykov, Oleg; Thomas-Rueddel, Daniel; Press, Adrian T.; Makarewicz, Oliwia; Giamarellos-Bourboulis, Evangelos J.; Bauer, Michael; Bocklitz, Thomas; Popp, Juergen; Neugebauer, UteBiochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.
- ItemEffect of Cold Atmospheric Plasma Jet Associated to Polyene Antifungals on Candida albicans Biofilms(Basel : MDPI, 2021) Leite, Lady Daiane Pereira; Oliveira, Maria Alcionéia Carvalho de; Vegian, Mariana Raquel da Cruz; Sampaio, Aline da Graça; Nishime, Thalita Mayumi Castaldelli; Kostov, Konstantin Georgiev; Koga-Ito, Cristiane YumiThe increasing incidence of antifungal resistance represents a great challenge in the medical area and, for this reason, new therapeutic alternatives for the treatment of fungal infections are urgently required. Cold atmospheric plasma (CAP) has been proposed as a promising alternative technique for the treatment of superficial candidiasis, with inhibitory effect both in vitro and in vivo. However, little is known on the association of CAP with conventional antifungals. The aim of this study was to evaluate the effects of the association between CAP and conventional polyene antifungals on Candida albicans biofilms. C. albicans SC 5314 and a clinical isolate were used to grow 24 or 48 h biofilms, under standardized conditions. After that, the biofilms were exposed to nystatin, amphotericin B and CAP, separately or in combination. Different concentrations of the antifungals and sequences of treatment were evaluated to establish the most effective protocol. Biofilms viability after the treatments was compared to negative control. Data were compared by One-way ANOVA and post hoc Tukey (5%). The results demonstrate that 5 min exposure to CAP showed more effective antifungal effect on biofilms when compared to nystatin and amphotericin B. Additionally, it was detected that CAP showed similar (but smaller in magnitude) effects when applied in association with nystatin and amphotericin B at 40 µg/mL and 60 µg/mL. Therefore, it can be concluded that the application of CAP alone was more effective against C. albicans biofilms than in combination with conventional polyene antifungal agents.