BACKGROUND: Thrombosis is linked to neutrophil release of neutrophil extracellular traps (NETs). NETs are proposed as a mechanism of resistance to thrombolysis. This study intends to analyze the composition of thrombi retrieved after mechanical thrombectomy, estimate the age and organization of thrombi, and evaluate associations with the use of thrombolysis, antiplatelets, and heparin. METHODS: This retrospective observational study involved 72 samples (44 from cerebral and 28 coronary arteries), which were stained with hematoxylin and eosin, anti-NE (neutrophil elastase) antibody, and anti-histone H2B (histone H2B) antibody, representing different components in NET formation, all detectable during the later stages of NETosis, for histochemical and digital quantification of NET content. The histological and morphological evaluations of the specimens were correlated, through univariate and mediation analyses, with clinical information and therapy administered before intervention. RESULTS: The results demonstrated that the composition of cerebral and coronary thrombi differs, and there were significantly more lytic cerebral thrombi than coronary thrombi (66% versus 14%; P =0.005). There was a considerably higher expression of NETs in the cerebral thrombi as testified by the higher expression of H2B ( P =0.031). Thrombolysis was remarkably associated with higher NE positivity (average marginal effect, 6.461 [95% CI, 0.7901–12.13]; P =0.02555), regardless of the origin of thrombi. There was no notable association between the administration of antiaggregant therapy/heparin and H2B/NE amount when adjusted for the thrombus location. Importantly, the age of the thrombus was the only independent predictor of NET content without any mediation of the thrombolytic treatment ( P =0.014). CONCLUSIONS: The age of the thrombus is the driving force for NET content, which correlates with impaired clinical outcomes. The therapy that is currently administered does not modify NET content. This study supports the need to investigate new pharmacological approaches added to thrombolysis to prevent NET formation or enhance their disruption, such as recombinant human DNase I (deoxyribonuclease I).

Cardiac allograft rejection following heart transplantation is challenging to diagnose. Tissue biopsies are the gold standard in monitoring the different types of rejection. The last decade has seen an increased emphasis on identifying non-invasive methods to improve rejection diagnosis and overcome tissue biopsy invasiveness. Liquid biopsy, as an efficient non-invasive diagnostic and prognostic oncological monitoring tool, seems to be applicable in heart transplant follow-ups. Moreover, molecular techniques applied on blood can be translated to tissue samples to provide novel perspectives on tissue and reveal new diagnostic and prognostic biomarkers. This review aims to provide a comprehensive overview of the state-of-the-art of the new methodologies in cardiac allograft rejection monitoring and investigate the future perspectives on invasive and non-invasive rejection biomarkers identification. We reviewed literature from the most used scientific databases, such as PubMed, Google Scholar, and Scopus. We extracted 192 papers and, after a selection and exclusion process, we included in the review 81 papers. The described limitations notwithstanding, this review show how molecular biology techniques and omics science could be deployed complementarily to the histopathological rejection diagnosis on tissue biopsies, thus representing an integrated approach for heart transplant patients monitoring.

Background: Heart transplant rejection, particularly acute cellular rejection (ACR), remains a critical post-operative concern, despite declining incidence rates. Current diagnostic standards rely on invasive endomyocardial biopsy, which presents limitations in sensitivity and reproducibility. There is an unmet need for noninvasive, accurate biomarkers that can detect and monitor rejection. This study aims to evaluate whether extracellular vesicle (EV) surface antigens, analyzed through flow cytometry and interpreted with artificial intelligence (AI), can serve as reliable biomarkers for ACR detection and monitoring in heart transplant recipients. Methods: We conducted a prospective longitudinal cohort study involving 24 heart transplant recipients over a median follow-up of 303 days. A total of 285 blood samples were analyzed for EV surface antigens exploiting two flow cytometry-based protocols. An adaptive AI model (random forest regressor) was developed to interpret EV antigen profiles, dynamically calibrating thresholds per patient. Results: Here we show that 14 EV surface antigens progressively increase with ACR severity. These changes are evident even before histological diagnosis. The AI model achieves an accuracy of 93.3% at leave-one-out testing (AUC 0.968), and 78.9% at validation in an independent cohort (AUC 0.832), with high specificity and negative predictive value. EV profiling outperforms conventional biochemical markers and provides anticipatory insight into rejection dynamics. Conclusions: EV profiling, enhanced by patient-specific AI modeling, offers a powerful noninvasive method for early detection and monitoring of ACR. This approach holds the potential to reduce reliance on biopsies and tailor immunosuppressive strategies more precisely.

Background and Aims: Tissular gene expression profiling applicable to formalin-fixed, paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) may refine the diagnosis of cardiac rejection while being easily implemented in clinical practice. This study aimed to develop and validate the first FFPE-based molecular diagnostic system dedicated to heart transplant rejection. Methods: An international study was conducted (NCT06436027), establishing a deeply phenotyped cohort of heart transplant recipients. EMBs were graded according to international classifications, and gene expression was analysed on FFPE-EMBs using the Banff Human Organ Transplant Panel. Molecular classifiers for antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were developed, with discrimination and calibration assessed in internal and external validation cohorts. Results: A total of 671 biopsies were included, with 591 in the main cohort (AMR: n = 188, ACR: n = 289, matched non-rejection: n = 114); this was split into a derivation set (n = 475) and an internal validation set (n = 116). The external validation cohort comprised 80 biopsies (AMR: n = 20, ACR: n = 32, non-rejection: n = 28). AMR was associated with significant transcripts related to the interferon-gamma pathway, endothelial activation, and monocyte-macrophage recruitment, while ACR was characterized by transcripts related to T-cell receptor signalling, CD3 receptor activation, and CD28 signalling. Molecular ACR and AMR models accurately identified rejection in the validation cohorts (internal: ROC-AUC: AMR = 0.812, ACR = 0.849; external: ROC-AUC: AMR = 0.822, ACR = 0.815) and were strongly associated with pathologic severity. Calibration was adequate. An automated report was developed to enhance the clinical applicability of these classifiers. Conclusions: A novel FFPE-based molecular diagnostic system accurately identified cardiac allograft rejection. This tool is readily applicable in clinical practice as a companion to pathology and has the potential to refine the diagnosis of rejection.

We report five cases of sudden intrauterine death due to premature closure of the ductus arteriosus. In four cases, this was caused by dissecting the hematoma of the ductus arteriosus with intimal flap and obliteration of the lumen. In one case, the ductus arteriosus was aneurysmatic, with lumen occlusion caused by thrombus stratification. No drug therapy or free medication consumption were reported during pregnancy. The time of stillbirth ranged between 26 and 33 gestational weeks. We performed TUNEL analysis for apoptosis quantification. The dissecting features were intimal tears with flap formation in four of the cases, just above the origin of the ductus arteriosus from the pulmonary artery. The dissecting hematoma of the ductus arteriosus extended downward to the descending aorta and backward to the aortic arch with involvement of the left carotid and left subclavian arteries. TUNEL analysis showed a high number of apoptotic smooth muscle cells in the media in two cases. Abnormal ductal remodeling with absence of subintimal cushions, lacunar spaces rich in glycosaminoglycans (cystic medial necrosis), and smooth muscle cell apoptosis were the pathological substrates accounting for failure of remodeling process and dissection.

Background: Heart failure remains a leading cause of morbidity and mortality, and current therapies rarely repair established myocardial damage. Nanoparticle-based interventions have been investigated across heterogeneous models of myocardial injury, remodeling, cardiomyopathy, and heart failure, but the distribution and translational maturity of this evidence remain unclear. Methods: A systematic search of PubMed, Embase, Scopus, and Web of Science was conducted from database inception to June 2024. Eligible reports were mapped according to disease model, experimental system, carrier-level nanoparticle platform, payload, route, comparator, outcomes, biodistribution, safety assessment, and translational characteristics. Reports of non-therapeutic nanoparticle exposure were retained in a separate contextual safety/toxicology stratum and were not included in the therapeutic evidence-density map. Risk of bias was evaluated using design-appropriate tools. Results: Of 2640 records screened, 157 independent studies met the criteria: 140 in the main therapeutic/platform evidence map and 17 in a separate contextual safety/toxicology stratum. Within the main corpus, polymeric systems were the largest platform class (n = 50), followed by inorganic/mineral (n = 35), biological/biomimetic (n = 24), lipid-based (n = 23), carbon-based (n = 5), and hybrid/multicomponent systems (n = 3). Evidence was concentrated in acute myocardial injury (n = 76), while direct same-agent comparisons, long-term safety assessment, repeated dosing, quantitative biodistribution, and clinically aligned heart-failure models remained limited. Conclusions: The field demonstrates substantial formulation diversity and biological activity, but translation is constrained by fragmented characterization, sparse comparative evidence, and incomplete assessment of biological fate and safety.