Background: This study aims to evaluate the role of parvovirus B19 (B19V) in the pathogenesis of myocarditis in a paediatric population, including post-mortem samples from two children. Methods: From 2004 to 2023, endomyocardial biopsies (EMBs) from children under 16 years of age were analyzed using histology, immunohistochemistry, and molecular pathology. A total of 306 children with acute and 1060 children with chronic lymphocytic myocarditis were identified. Results: B19V infection was more frequent in acute myocarditis than in chronic myocarditis (43% vs. 14%), with higher viral loads in acute cases regardless of age. The most prominent cardiac CD3+ T cell infiltration was noted in children < 2 years, correlating with high cardiac B19V loads. In two male infants who died from B19V infection, B19V DNA was localized in the endothelial cells of multiple organs using in situ hybridization. Virus replication was found in the endothelial cells of small cardiac arterioles and venules but not in capillaries. B19V DNA/mRNA was also detected in immune cells, especially in the spleen and lymph nodes, revealing virus replication in B lymphocytes. Conclusions: B19V can induce severe lymphocytic myocarditis, especially in young children. The simultaneous histopathological and molecular assessment of EMBs is important for early diagnosis of viral myocarditis, preventing severe disease, and ensuring appropriate therapy.

The pathophysiology of COVID-19 associated thrombosis seems to be multifactorial, involving interplay between cellular and plasmatic elements of the hemostasis. We hypothesized that COVID-19 is accompanied by platelet apoptosis with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca ²⁺ ) concentration, and phosphatidylserine (PS) externalization by flow cytometry. Platelets from intensive care unit (ICU) COVID-19 patients (n=21) showed higher ΔΨm depolarization, cytosolic Ca ²⁺ concentration and PS externalization, compared to healthy controls (n=18) and COVID-19 non-ICU patients (n=4). Moreover significant higher cytosolic Ca ²⁺ concentration and PS was observed compared to septic ICU control group (ICU control). In ICU control group (n=5; ICU non-COVID-19) cytosolic Ca ²⁺ concentration and PS externalization was comparable to healthy control, with an increase ΔΨm depolarization. Sera from ICU COVID-19 13 patients induced significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca ²⁺ concentration and PS externalization). compared to healthy volunteer and septic ICU control. Interestingly, immunoglobulin G (IgG) fractions from COVID-19 patients induced an Fc gamma receptor IIA dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca ²⁺ concentration and PS externalization). Enhanced PS externalization in platelets from ICU COVID-19 patients was associated with increased sequential organ failure assessment (SOFA) score (r=0.5635) and DDimer (r=0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared to those without. The strong correlations between apoptosis markers and increased D-Dimer levels as well as the incidence of thrombosis may indicate that antibody-mediated platelet apoptosis potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients. Key points: Severe COVID-19 is associated with increased antibody-mediated platelet apoptosis. Platelet apoptosis in severe COVID-19 is correlated with D-Dimer and higher incidence of thromboembolisms.

Introduction Transfusion of platelet concentrates (PCs) is an essential medical strategy to treat bleeding or to prevent it. However, the standard storage at room temperature enhances the risk of bacterial contamination. We reported that cold-stored PCs had better functionality but reduced survival due to cold-induced apoptosis. In this study, we investigated the impact of apoptosis inhibition on platelet functions and half-life during cold storage. Method PCs collected from healthy volunteers were stored for 1, 4, 7 and 10 days at 4°C with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). The apoptotic signal was analysed by flow cytometry detecting phosphatidylserine (PS) and mitochondrial membrane potential (MMP). The functionality was assessed: by flow cytometer, testing CD63 and CD62 upon TRAP; by aggregometry and performing an adhesion assay (on fibrinogen coated surfaces). The thrombus formation ability was analysed by thromboelastography. Platelet survival was investigated using the NOD/SCID mouse model. Results We found that inhibition of RhoA GTPase significantly reduced the percentage of apoptotic cells exposing PS, starting from day 7 in comparison to untreated cells (day 7, p=0.030; day 10, p=0.012). Accordingly, MMP was better conserved in cells stored with G04 (day 7, p=0.009; day 10, p=0.008). Interestingly, upon TRAP stimulation the CD63 levels were significantly higher in the presence of G04 (CD63: day 7, p=0.035; day 10, p=0.049) and CD62 expression was comparable to cells stored in buffer. Similarly, a significantly higher aggregation ability in response to both TRAP (p=0.038) and ristocetin (p=0.042) was measured after incubation with G04 on day 10, compared to untreated cells. Next, we observed that the presence of G04 better maintained the adhesion capability of cold-stored platelets after 4 days, in comparison to buffer (p=0.0493). Next, the thrombus formation ability was not affected by the apoptosis inhibitor. More importantly, a higher percentage of circulating human platelets, after 7 days of cold storage, was detected in the mouse bloodstream upon incubation with G04 compared to untreated cells (2h post injection, p=0.0387, 5h post injection, p=0.0355). Conclusion Our results show that the inhibition of cold-induced apoptosis significantly reduces the clearance of cold-stored platelets without affecting the haemostatic functionality of the cells. Therefore, the use of apoptosis inhibitor/s may be a promising strategy to prolong the storage time, improve the platelet survival and reduce the risk of bacterial infection post transfusion. Article published online: 20 February 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany