mCRP-Induced Focal Adhesion Kinase-Dependent Monocyte Aggregation and M1 Polarization, Which Was Partially Blocked by the C10M Inhibitor.

Monomeric C-reactive protein (mCRP) has recently been implicated in the abnormal vascular activation associated with development of atherosclerosis, but it may act more specifically through mechanisms perpetuating damaged vessel inflammation and subsequent aggregation and internalization of resident macrophages. Whilst the direct effects of mCRP on endothelial cells have been characterized, the interaction with blood monocytes has, to our knowledge, not been fully defined. Here we showed that mCRP caused a strong aggregation of both U937 cell line and primary peripheral blood monocytes (PBMs) obtained from healthy donors. Moreover, this increase in clustering was dependent on focal adhesion kinase (FAK) activation (blocked by a specific inhibitor), as was the concomitant adhesive attachment to the plate, which was suggestive of macrophage differentiation. Confocal microscopy confirmed the increased expression and nuclear localization of p-FAK, and cell surface marker expression associated with M1 macrophage polarization (CD11b, CD14, and CD80, as well as iNOS) in the presence of mCRP. Inclusion of a specific CRP dissociation/mCRP inhibitor (C10M) effectively inhibited PBMs clustering, as well as abrogating p-FAK expression, and partially reduced the expression of markers associated with M1 macrophage differentiation. mCRP also increased the secretion of pro-inflammatory cytokines Interleukin-8 (IL-8) and Interleukin-1ß (IL-1ß), without notably affecting MAP kinase signaling pathways; inclusion of C10M did not perturb or modify these effects. In conclusion, mCRP modulates PBMs through a mechanism that involves FAK and results in cell clustering and adhesion concomitant with changes consistent with M1 phenotypical polarization. C10M has potential therapeutic utility in blocking the primary interaction of mCRP with the cells-for example, by protecting against monocyte accumulation and residence at damaged vessels that may be predisposed to plaque development and atherosclerosis.

The Influence of HLA Polymorphisms on the Severity of COVID-19 in the Romanian Population.

In this study, we aimed to investigate whether specific HLA alleles found in patients from Romania and the Republic of Moldova were associated with the severity of COVID-19 infection and its associated mortality. We analyzed the HLA alleles at the -A, -B, -C, -DRB1, and -DQB1 loci in a cohort of 130 individuals with severe and extremely severe forms of COVID-19, including 44 individuals who died. We compared these findings to a control group consisting of individuals who had either not been diagnosed with COVID-19 or had experienced mild forms of the disease. Using multivariate logistic regression models, we discovered that the B*27 and B*50 alleles were associated with an increased susceptibility to developing a severe form of COVID-19. The A*33 and C*15 alleles showed potential for offering protection against the disease. Furthermore, we identified two protective alleles (A*03 and DQB1*02) against the development of extremely severe forms of COVID-19. By utilizing score statistics, we established a statistically significant association between haplotypes and disease severity (p = 0.021). In summary, this study provides evidence that HLA genotype plays a role in influencing the clinical outcome of COVID-19 infection.

Genotoxic and Cytotoxic Activity of Fisetin on Glioblastoma Cells.

BACKGROUND/AIM: Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells. MATERIALS AND METHODS: We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis. RESULTS: Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 µM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells. CONCLUSION: Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.

A Mathematical Algorithm for Dried Blood Spot Quality Assessment and Results concerning Quality from a Newborn Screening Program.

BACKGROUND: In addition to newborn screening, dried blood spots (DBSs) are used for a wide variety of analytes for clinical, epidemiological, and research purposes. Guidelines on DBS collection, storage, and transport are available, but it is suggested that each laboratory should establish its own acceptance criteria. METHODS: An optical scanning device was developed to assess the quality of DBSs received in the newborn screening laboratory from 11 maternity wards between 2013 and 2018. The algorithm was adjusted to agree with the visual examination consensus of experienced laboratory personnel. Once validated, the algorithm was used to categorize DBS specimens as either proper or improper. Improper DBS specimens were further divided based on 4 types of specimen defects. RESULTS: In total, 27 301 DBSs were analyzed. Compared with an annual DBS rejection rate of about 1%, automated scanning rejected 26.96% of the specimens as having at least one defect. The most common specimen defect was multi-spotting (ragged DBS, 19.13%). Among maternity wards, improper specimen rates varied greatly between 5.70% and 49.92%. CONCLUSIONS: Improper specimen rates, as well as the dominant type of defect(s), are mainly institution-dependent, with various maternity wards consistently showing specific patterns of both parameters over time. Although validated in agreement with experienced laboratory personnel consensus, automated analysis rejects significantly more specimens. While continuous staff training, specimen quality monitoring, and problem-reporting to maternities is recommended, a thorough quality assessment strategy should also be implemented by every newborn screening laboratory. An important role in this regard may be played by automation in the form of optical scanning devices.