Vaccine associated benign headache and cutaneous hemorrhage after ChAdOx1 nCoV-19 vaccine: A cohort study

Objectives Fatal complications have occurred after vaccination with ChAdOx1 nCoV-19, a vaccine against Covid-19. Vaccine-induced immune thrombotic thrombocytopenia (VITT) with severe outcome is characterized by venous thrombosis, predominantly in cerebral veins, thrombocytopenia and anti-PF4/polyanion antibodies. Prolonged headaches and cutaneous hemorrhages, frequently observed after the ChAdOx1 nCoV-19 vaccine, have therefore caused anxiety among vaccinees. We investigated whether these symptoms represent a mild form of VITT, with a potential for aggravation, e.g. in case of a second vaccination dose, or a different entity of vaccine complications Materials and methods We included previously healthy individuals who had a combination of headache and spontaneous severe cutaneous hemorrhages emerging after the 1st dose of the ChAdOx1 nCoV-19 vaccine. Twelve individuals were found to meet the inclusion criteria, and a phone interview, cerebral MRI, assessment of platelet counts, anti PF4/polyanion antibodies and other laboratory tests were performed. Results None of the symptomatic vaccinees had cerebral vein thrombosis, hemorrhage or other pathology on MRI. Platelet counts were within normal range and no anti-PF4/polyanion platelet activating antibodies were found. Moreover, vasculitis markers, platelet activation markers and thrombin generation were normal. Furthermore, almost all symptoms resolved, and none had recurrence of symptoms after further vaccination with mRNA vaccines against Covid-19. Conclusions The combination of headaches and subcutaneous hemorrhage did not represent VITT and no other specific coagulation disorder or intracranial pathology was found. However, symptoms initially mimicking VITT demand vigilance and low threshold for a clinical evaluation combined with platelet counts and D–dimer

Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia

Aims - We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients. Methods and results - We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits. Conclusions - The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT

Carotid Plaque Neovascularization Detected With Superb Microvascular Imaging Ultrasound Without Using Contrast Media

Background and Purpose— A significant proportion of ischemic strokes are caused by emboli from unstable carotid artery plaques with intraplaque neovascularization (IPN) as a key feature of plaque instability. IPN is not detectable with conventional Doppler ultrasound. Contrast-enhanced ultrasound (CEUS) can visualize IPN, but its use is limited in clinical practice because it requires an intravenous injection of contrast. Superb microvascular imaging (SMI) without contrast uses an algorithm to remove clutter and motion wall artifacts while preserving low-velocity blood flow signals, enabling visualization of IPN. Our aim was to assess the feasibility of SMI for the detection of IPN. Methods— Thirty-one patients with >50% carotid stenosis were included: 22 patients were symptomatic and 9 asymptomatic. All patients underwent conventional carotid ultrasound, CEUS, SMI, and blood tests. CEUS and SMI findings were compared and correlated to histological plaque assessments after endarterectomy. Results— There was significant positive correlation between an IPN visual 5-level classification of SMI and a semiquantitative analysis of CEUS (P<0.001, r=0.911). Plaques with higher SMI grades had higher numbers of neovessels quantified at histology (P=0.041, r=0.460). Hypoechoic plaques had higher grades of IPN on both CEUS and SMI (P<0.001). Higher visual IPN counts on SMI were associated with (1) increased areas of inflammation (P=0.043, r=0.457), (2) combined rank scores of granulation tissue, inflammation and lipids (P=0.02, r=0.494) at histology, and (3) higher peak-intensity values on quantitative CEUS (P=0.042, r=0.514). Conclusions— SMI ultrasound can detect neovascularization with accuracy comparable to CEUS, suggesting SMI to be a promising noninvasive alternative to CEUS for the assessment of carotid plaque stability

Increased Endothelial Activation in Patients with Mixed Connective Tissue Disease

Objective: Juvenile-onset mixed connective tissue disease (JMCTD) is a chronic inflammatory disease. We have previously demonstrated preclinical atherosclerosis in these patients, now exploring this further by assessing markers of endothelial dysfunction. Methods: Thirty-three patients with JMCTD and 33 age-and sex-matched controls were included. Soluble intercellular adhesion molecule-1 (sICAM-1), Il-6 and, von Willenbrand factor (vWF) were assayed from blood taken at the time of carotid ultrasound. Results: Our major findings were: (1) Levels of sICAM-1 ( P < .001), IL-6 ( P = .004), and vWF ( P = .001) were higher, whereas (2) high density lipoprotein cholesterol (<.01) and apolipoprotein A1 ( P < .01) were lower in the patient group compared to controls. Conclusions: Patients with JMCTD had significantly increased levels of markers of endothelial dysfunction

N6-methyladenosine in RNA of atherosclerotic plaques: An epitranscriptomic signature of human carotid atherosclerosis

Background More than 170 post-transcriptional RNA modifications regulate the localization, processing and function of cellular RNAs, and aberrant RNA modifications have been linked to a range of human diseases. The RNA modification landscape in atherosclerosis, the main underlying cause of cardiovascular diseases, is still largely unknown. Methods We used mass spectrometry to analyse a selection of RNA-modifying enzymes and the N6-methyladenosine (m6A) in carotid atherosclerotic lesion samples representing early and advanced stages of atherosclerosis as compared to non-atherosclerotic arteries from healthy controls. Findings (i) the detection of different levels of several enzymes involved in methylations occurring in rRNA and mRNA; (ii) these findings included changes in the levels of methyltransferases (‘writers’), binding proteins (‘readers’) and demethylases (‘erasers’) during atherosclerosis as compared to non-atherosclerotic control arteries, with generally the most prominent differences in samples from early atherosclerotic lesions; and (iii) these changes were accompanied by a marked downregulation of m6A in rRNA, the most abundant and well-studied modification in mRNA with a wide range of effects on cell biology. Interpretation We show for the first time that RNA-modifying enzymes and the well-studied RNA modification m6A are differentially regulated in atherosclerotic lesions, which potentially could help creating new prognostic and treatment strategies

Biomarkers Associated with Atrial Fibrillation in Patients with Ischemic Stroke: A Pilot Study from the NOR-FIB Study

Background and Purpose: Cardioembolic stroke due to paroxysmal atrial fibrillation (AF) may account for 1 out of 4 cryptogenic strokes (CS) and transient ischemic attacks (TIAs). The purpose of this pilot study was to search for biomarkers potentially predicting incident AF in patients with ischemic stroke or TIA. Methods: Plasma samples were collected from patients aged 18 years and older with ischemic stroke or TIA due to AF (n = 9) and large artery atherosclerosis (LAA) with ipsilateral carotid stenosis (n = 8) and age- and sex-matched controls (n = 10). Analyses were performed with the Olink technology simultaneously measuring 184 biomarkers of cardiovascular disease. For bioinformatics, acquired data were analyzed using gene set enrichment analysis (GSEA). Selected proteins were validated using ELISA. Individual receiver operating characteristic (ROC) curves and odds ratios from logistic regression were calculated. A randomForest (RF) model with out-of-bag estimate was applied for predictive modeling. Results: GSEA indicated enrichment of proteins related to inflammatory response in the AF group. Interleukin (IL)-6, growth differentiation factor (GDF)-15, and pentraxin-related protein PTX3 were the top biomarkers on the ranked list for the AF group compared to the LAA group and the control group. ELISA validated increased expression of all tested proteins (GDF-15, PTX3, and urokinase plasminogen activator surface receptor [U-PAR]), except for IL-6. 19 proteins had the area under the ROC curve (AUC) over 0.85 including all of the proteins with significant evolution in the logistic regression. AUCs were very discriminant in distinguishing patients with and without AF (LAA and control group together). GDF-15 alone reached AUC of 0.95. Based on RF model, all selected participants in the tested group were classified correctly, and the most important protein in the model was GDF-15. Conclusions: Our results demonstrate an association between inflammation and AF and that multiple proteins alone and in combination may potentially be used as indicators of AF in CS and TIA patients. However, further studies including larger samples sizes are needed to support these findings. In the ongoing NOR-FIB study, we plan further biomarker assessments in patients with CS and TIA undergoing long-term cardiac rhythm monitoring with insertable cardiac monitors

N6-methyladenosine in RNA of atherosclerotic plaques: An epitranscriptomic signature of human carotid atherosclerosis

Background More than 170 post-transcriptional RNA modifications regulate the localization, processing and function of cellular RNAs, and aberrant RNA modifications have been linked to a range of human diseases. The RNA modification landscape in atherosclerosis, the main underlying cause of cardiovascular diseases, is still largely unknown. Methods We used mass spectrometry to analyse a selection of RNA-modifying enzymes and the N6-methyladenosine (m6A) in carotid atherosclerotic lesion samples representing early and advanced stages of atherosclerosis as compared to non-atherosclerotic arteries from healthy controls. Findings (i) the detection of different levels of several enzymes involved in methylations occurring in rRNA and mRNA; (ii) these findings included changes in the levels of methyltransferases (‘writers’), binding proteins (‘readers’) and demethylases (‘erasers’) during atherosclerosis as compared to non-atherosclerotic control arteries, with generally the most prominent differences in samples from early atherosclerotic lesions; and (iii) these changes were accompanied by a marked downregulation of m6A in rRNA, the most abundant and well-studied modification in mRNA with a wide range of effects on cell biology. Interpretation We show for the first time that RNA-modifying enzymes and the well-studied RNA modification m6A are differentially regulated in atherosclerotic lesions, which potentially could help creating new prognostic and treatment strategies

Increased Levels of lectin-like oxidized low-density lipoprotein receptor-1 in ischemic stroke and transient ischemic attack

Background: Soluble lectin‐like oxidized low‐density lipoprotein receptor‐1 (sLOX‐1) has been shown to be increased in patients with acute ischemic stroke. Here, we evaluated plasma sLOX‐1 levels and vascular carotid plaque LOX‐1 (ie, OLR1) gene expression in patients with ischemic stroke and transient ischemic attack (TIA) with particular focus on their relation to time since symptom onset. Methods and Results: Plasma sLOX‐1 (n=232) and carotid plaque OLR1 gene expression (n=146) were evaluated in patients who were referred to evaluation for carotid endarterectomy, as well as in healthy control plasma (n=81). Patients were categorized according to presence of acute ischemic stroke or transient ischemic attack (n=35) ≤7 days, >7 days ≤3 months (n=90), >3 months (n=40), or no reported symptoms before study inclusion (n=67). Our major findings were the following: (1) Patients with carotid atherosclerosis had increased plasma sLOX‐1 levels as compared with controls. (2) Plaque OLR1 mRNA levels were increased in carotid plaques (n=146) compared with nonatherosclerotic vessels (ie, common iliac arteries of organ donors, n=10). (3) There were no differences in sLOX plasma levels or OLR1 gene expression when analyzed according to the time since relevant cerebral ischemic symptoms. (4) Also patients with severe carotid atherosclerosis without any previous ischemic events had raised sLOX‐1 levels. (5) Immunostaining showed colocalization between LOX‐1 and macrophages within the carotid plaques. (6) Also patients with acute stroke (within 7 days) caused by atrial fibrillation (n=22) had comparable raised sLOX‐1 levels. Conclusions: sLOX‐1 levels are elevated in patients with ischemic stroke and transient ischemic attack independent of cause and time since the ischemic event