Supplementary Material for: Molecular Imaging of Carotid Plaque Vulnerability

<p><b><i>Background:</i></b> Carotid endarterectomy (CEA) has been shown to be beneficial in patients with high-grade symptomatic carotid artery stenosis. Patients with high-grade asymptomatic stenosis may only exceptionally benefit from CEA during periods of increased plaque vulnerability. Imaging modalities to characterize unstable, vulnerable plaques are strongly needed for better risk stratification in these patients. <b><i>Summary:</i></b> Contrast-enhanced ultrasound (CEUS) is a novel and noninvasive technique capable to identify several surrogate markers of vulnerable carotid plaques. The use of specific ultrasound microbubbles allows a reliable detection of microulcerations due to an optimized visualization of the plaque-lumen border. As microbubbles are strictly intravascular tracers, the detection of individual microbubbles within the plaque corresponds to intraplaque neovessels. The accuracy of CEUS in the visualization of newly formed microvessels has been confirmed in histological studies on carotid endarterectomy specimens. Together with the formation of adventitial vasa vasorum, intraplaque neovascularization is a strong predictor for symptomatic disease. The phenomenon of late phase contrast enhancement is based on the adherence of microbubble-containing monocytes on inflamed endothelium. Recent studies suggest that late phase contrast enhancement may reflect endothelial inflammation or activation within carotid plaques. The development of conjugated microbubbles that bind to specific ligands such as thrombotic material or neovessels has led to the term ‘molecular imaging'. CEUS with microbubbles targeted to P-selectin and VCAM-1, key molecules in leukocyte trafficking, was used to detect an inflammatory plaque phenotype, whereas microbubbles coupled to the VEGF-receptor may allow for a detection of neovascularization. Even though imaging with targeted microbubbles is yet in an experimental stage, this technique can visualize active plaque reorganization with increased vulnerability leading to generation of arterio-arterial embolism. <b><i>Key Messages:</i></b> The use of contrast-enhanced ultrasound can be recommended to assess atherosclerotic carotid lesions at risk for rupture. Prospective clinical studies are needed to validate the use of CEUS in patients with high risks of recurrent large artery strokes. In particular, this applies to the detection of intraplaque neovascularization, a well-established marker in preclinical and observational studies, while the clinical significance of late phase contrast enhancement still needs to be determined.</p

Supplementary Material for: Etiologic and Clinical Characterization of Patients with Recurrent Spontaneous Intracerebral Hemorrhage

<p><b><i>Background:</i></b> The impact of recurrent stroke has been extensively addressed with regard to ischemic stroke, revealing potentially different etiologies of recurrent events in the individual patient. In contrast, data on recurrent intracerebral hemorrhage (ICH) are scarce, especially considering etiologic characterization. We aimed to determine the etiology of recurrent ICH at each event to identify potential etiologic changes. <b><i>Patients and Methods:</i></b> We analyzed the data of patients admitted to our stroke unit with recurrent ICH between 1998 and 2014 with regard to clinical characteristics and etiology. <b><i>Results:</i></b> Thirty-three patients (2.6%) with recurrent ICH were identified. Mean age (mean ± SD) at the initial event was 69 ± 9 and 72 ± 9 years at recurrence. Median interval between events was 18 months. Mean National Institutes of Health Stroke Scale (first/second event) was 4/9 at admission and 2/8 at discharge. Over 30% of patients developed symptomatic epilepsy. Etiologic distribution was (first/second event) the following: probable cerebral amyloid angiopathy (CAA) (12/20), possible CAA (3/0), hypertensive (5/4), anticoagulation (4/3), vascular malformation (2/4), ischemia with secondary hemorrhage (4/0), vasculitis (0/1), undetermined (4/0). <b><i>Conclusions:</i></b> Recurrent ICH is rare, CAA being its most common etiology. Etiology of ICH may differ between the first/second event in about 10%. The findings indicate the need of a complete and distinct work-up including MRI in every instance of ICH recurrence.</p><br

Supplementary Material for: Individual and Complementary Effects of Human Papillomavirus Oncogenes on Epithelial Cell Proliferation and Differentiation

Previous studies on human papillomavirus (HPV) type 16 protein functions have established the oncogenic nature of three viral proteins: E5, E6 and E7. Here we have studied the functions of these proteins by functional deletion of the individual E5, E6 or E7, or both E6 and E7 oncogenes in the context of the whole viral genome. These mutants, or the intact wild-type genome, were expressed from the natural viral promoters along with differentiation of epithelial HaCaT cells in three-dimensional collagen raft cultures. High episomal viral copy numbers were obtained using a transfection-based loxp-HPV16-eGFP-N1 vector system. All epithelial equivalents carrying the different HPV type 16 genomes showed pronounced hyperplastic and dysplastic morphology. Particularly the E7 oncogene, with contribution of E6, was shown to enhance cell proliferation. Specifically, the crucial role of E7 in HPV-associated hyperproliferation was clearly manifested. Based on morphological characteristics, immunohistochemical staining for differentiation and proliferation markers, and low expression of E1^E4, we propose that our raft culture models produce cervical intraepithelial neoplasia (CIN)1 and CIN2-like tissue. Our experimental setting provides an alternative tool to study concerted functions of HPV proteins in the development of epithelial dysplasia

Supplementary Material for: Cognition and Incident Dementia Hospitalization: Results from the Atherosclerosis Risk in Communities Study

<b><i>Background/Aims:</i></b> Cognitive decline is a defining feature of dementia. We sought to determine if a single baseline cognitive test score or change in test score over time is more strongly associated with risk of dementia hospitalization. We also sought to compare short- and long-term dementia risk. <b><i>Methods:</i></b> Prospective cohort study of 9,399 individuals from the Atherosclerosis Risk in Communities Study (median 10 years of follow-up). Cognition was assessed at two time points (6 years apart) using three tests: Delayed Word Recall Test (DWRT), Digit Symbol Substitution Test (DSST), and Word Fluency Test. Dementia hospitalizations were determined using ICD-9 codes. <b><i>Results:</i></b> Baseline cognitive test scores were associated with both short-term and long-term risk of dementia. The association of 6-year change in cognitive test score with dementia risk was stronger than that of individual test scores at a single visit [change from highest to lowest tertile, DWRT: hazard ratio = 6.45 (95% confidence interval = 1.80–23.08); DSST: hazard ratio = 10.94 (95% confidence interval = 3.07–38.97)]. <b><i>Conclusions:</i></b> In this community-based population, 6-year changes in cognitive scores were more strongly associated with risk of incident dementia hospitalization than baseline scores, although single DWRT and DSST scores were predictive. Our findings support the contention that cognitive changes may precede clinical dementia by a decade or more

Erratum: A Synergistic Association of ACE I/D and eNOS G894T Gene Variants with the Progression of Immunoglobulin A Nephropathy – A Pilot Study

<i>Background:</i> Individual variability in the natural history and response to therapy of immunoglobulin A nephropathy (IgAN) suggests a complex multifactorial pathogenesis. We investigated whether single nucleotide polymorphisms (SNPs) involved in the non-immunologic progression of renal disease are related with disease progression. <i>Methods:</i> This is a pilot historic cohort study of 64 Caucasian patients with biopsy-proven IgAN and a median follow-up of 70 months. Three SNPs of the renin-angiotensin system genes (angiotensin I converting enzyme insertion/deletion (ACE I/D), angiotensinogen (AGT) M235T, and angiotensin II type 1 receptor (AT₁R) 1166A/C), 2 of the endothelial nitric oxide synthase (eNOS), 4a/b and G894T, and 1 of the bradykinin 1 receptor, G–699C, were genotyped. The primary outcome was ‘kidney survival’ defined as a 30% decrease of baseline creatinine clearance; annualized decrease of glomerular filtration rate was also calculated. <i>Results:</i> Proteinuria, histological lesions, and mean arterial pressure were related to an unfavorable outcome. The simultaneous presence of the DD and GG variants of the ACE and eNOS genes was related to an unfavorable outcome as compared with other combinations [hazard ratio ranging from 4.7 (95% CI 1.52–14.33) to 8.4 (95% CI 2.45–29.10)] after controlling for proteinuria, mean arterial pressure and baseline histological lesions. <i>Conclusion:</i> This study suggests that in our population with IgAN, an interaction between ACE and eNOS polymorphisms may be a prognostic factor for renal function deterioration

Supplementary Material for: The Metabolic Syndrome and Cognitive Decline in the Atherosclerosis Risk in Communities Study (ARIC)

<b><i>Background:</i></b> Midlife metabolic syndrome (MetS) may impact cognitive health as a construct independently of hypertension, hyperlipidemia and other components. <b><i>Methods:</i></b> 10,866 participants aged 45-64 years at baseline were assessed for MetS and completed cognitive testing at two later time points (3 and 9 years from the baseline visit). <b><i>Results:</i></b> MetS is associated with increased odds of low cognitive performance in the domains of executive function and word fluency, but not with 6-year cognitive decline. Individual MetS components explained this association (hypertension, diabetes, low HDL, elevated triglycerides and increased waist circumference). <b><i>Conclusions:</i></b> A focus on the individual risk factors as opposed to MetS during midlife is important to reduce the incidence of cognitive impairment in later life

Supplementary Material for: Serum Vitamin D Concentrations and Cognitive Change Over 20 Years: The Atherosclerosis Risk in Communities Neurocognitive Study

<b><i>Background/Aims:</i></b> 25-hydroxyvitamin D (25[OH]D) concentrations have been associated with cognitive decline and incident dementia in elderly populations; however, these relationships are susceptible to reverse causation. Less is known about the association of midlife 25(OH)D with long-term cognitive decline. <b><i>Methods:</i></b> This was a prospective cohort study of 13,044 participants (mean age 57 years at baseline) in the Atherosclerosis Risk in Communities Study. 25(OH)D was measured from serum collected at baseline (1990–1992) using liquid chromatography tandem high-sensitivity mass spectrometry. Cognition was assessed using 3 neuropsychological tests at 3 time points, which were combined into a composite cognitive <i>Z</i>-score. Multivariable-adjusted linear mixed-effects models with random intercepts and slopes were used to estimate associations between 25(OH)D and cognitive change over 20 years. <b><i>Results:</i></b> Compared to persons with sufficient 25(OH)D (≥30 ng/mL), those with deficient (< 20 ng/mL) and intermediate (20–< 30 ng/mL) 25(OH)D concentrations had similar cognitive decline in composite cognitive <i>Z</i>-scores (deficient versus sufficient: –0.035 [95% CI –0.104 to 0.033] and intermediate versus sufficient: –0.029 [95% CI –0.080 to 0.023]). <b><i>Conclusions:</i></b> Lower concentrations of 25(OH)D measured in midlife were not significantly associated with more rapid cognitive decline over a 20-year follow-up period. The results of this prospective study are less susceptible to reverse causation than prior studies