Persisting IL-18 levels after COVID-19 correlate with markers of cardiovascular inflammation reflecting potential risk of CVDs development.

COVID-19 manifestation is associated with a strong immune system activation leading to inflammation and subsequently affecting the cardiovascular system. The objective of the study was to reveal possible interconnection between prolongated inflammation and the development or exacerbation of long-term cardiovascular complications after COVID-19. We investigated correlations between humoral and cellular immune system markers together with markers of cardiovascular inflammation/dysfunction during COVID-19 onset and subsequent recovery. We analyzed 22 hospitalized patients with severe COVID-19 within three timepoints (acute, 1 and 6 months after COVID-19) in order to track the impact of COVID-19 on the long-term decline of the cardiovascular system fitness and eventual development of CVDs. Among the cytokines dysregulated during COVID-19 changes, we showed significant correlations of IL-18 as a key driver of several pathophysiological changes with markers of cardiovascular inflammation/dysfunction. Our findings established novel immune-related markers, which can be used for the stratification of patients at high risk of CVDs for further therapy.

Immunosuppression Affects Neutrophil Functions: Does Calcineurin-NFAT Signaling Matter?

Neutrophils are innate immune cells with important roles in antimicrobial defense. However, impaired or dysregulated neutrophil function can result in host tissue damage, loss of homeostasis, hyperinflammation or pathological immunosuppression. A central link between neutrophil activation and immune outcomes is emerging to be the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, which is activated by neutrophil detection of a microbial threat via pattern recognition receptors and results in inflammatory cytokine production. This potent pro-inflammatory pathway is also the target of several immunosuppressive drugs used for the treatment of autoimmune disorders, during solid organ and hematopoietic cell transplantations, and as a part of anti-cancer therapy: but what effects these drugs have on neutrophil function, and their broader consequences for immune homeostasis and microbial defense are not yet known. Here, we bring together the emerging literature describing pathology- and drug- induced neutrophil impairment, with particular focus on their effects on calcineurin-NFAT signaling in the innate immune compartment.

NANOG/NANOGP8 Localizes at the Centrosome and is Spatiotemporally Associated with Centriole Maturation.

NANOG is a transcription factor involved in the regulation of pluripotency and stemness. The functional paralog of NANOG, NANOGP8, differs from NANOG in only three amino acids and exhibits similar reprogramming activity. Given the transcriptional regulatory role played by NANOG, the nuclear localization of NANOG/NANOGP8 has primarily been considered to date. In this study, we investigated the intriguing extranuclear localization of NANOG and demonstrated that a substantial pool of NANOG/NANOGP8 is localized at the centrosome. Using double immunofluorescence, the colocalization of NANOG protein with pericentrin was identified by two independent anti-NANOG antibodies among 11 tumor and non-tumor cell lines. The validity of these observations was confirmed by transient expression of GFP-tagged NANOG, which also colocalized with pericentrin. Mass spectrometry of the anti-NANOG immunoprecipitated samples verified the antibody specificity and revealed the expression of both NANOG and NANOGP8, which was further confirmed by real-time PCR. Using cell fractionation, we show that a considerable amount of NANOG protein is present in the cytoplasm of RD and NTERA-2 cells. Importantly, cytoplasmic NANOG was unevenly distributed at the centrosome pair during the cell cycle and colocalized with the distal region of the mother centriole, and its presence was markedly associated with centriole maturation. Along with the finding that the centrosomal localization of NANOG/NANOGP8 was detected in various tumor and non-tumor cell types, these results provide the first evidence suggesting a common centrosome-specific role of NANOG.

Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 µg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 µg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 µg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 µg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 µg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.