Analytical performance and user-friendliness of four commercially available point-of-care devices for C-reactive protein.

INTRODUCTION: Proper implementation of Point-of-Care testing (POCT) for C-reactive protein (CRP) in primary care can decrease the inappropriate use of antibiotics, thereby tackling the problem of growing antimicrobial resistance. OBJECTIVE: The analytical performance and user-friendliness of four POCT-CRP assays were evaluated: QuikRead go easy, LumiraDx, cobas b 101 and Afinion 2. MATERIALS AND METHODS: Imprecision was evaluated using plasma pools in addition to manufacturer-specific control material. Trueness was assessed by verification of traceability to ERM-DA474/IFCC in parallel to method comparison towards the central laboratory CRP method (cobas c 503) using i) retrospectively selected plasma samples (n = 100) and ii) prospectively collected capillary whole blood samples (n = 50). User-friendliness was examined using a questionnaire. RESULTS: Between-day imprecision on plasma pools varied from 4.5 % (LumiraDx) to 11.5 % (QuikRead). Traceability verification revealed no significant difference between cobas c 503 CRP results and the ERM-DA474/IFCC certified value. cobas b 101 and Afinion achieved the best agreement with the central laboratory method. LumiraDx and QuikRead revealed a negative mean difference, with LumiraDx violating the criterion of > 95 % of POCT-CRP-results within ± 20 % of the comparison method. Regarding user-friendliness, Afinion obtained the highest Likert-scores. CONCLUSION: The analytical performance and user-friendliness of POCT-CRP devices varies among manufacturers, emphasizing the need for quality assurance supervised by a central laboratory.

The influence of undetected hemolysis on POCT potassium results in the emergency department.

OBJECTIVES: This study aimed to evaluate discrepancies in potassium measurements between point-of-care testing (POCT) and central laboratory (CL) methods, focusing on the impact of hemolysis on these measurements and its impact in the clinical practice in the emergency department (ED). METHODS: A retrospective analysis was conducted using data from three European university hospitals: Technische Universitat München (Germany), Hospital Universitario La Paz (Spain), and Erasmus University Medical Center (The Netherlands). The study compared POCT potassium measurements in EDs with CL measurements. Data normalization was performed in categories for potassium levels (kalemia) and hemolysis. The severity of discrepancies between POCT and CL potassium measurements was assessed using the reference change value (RCV). RESULTS: The study identified significant discrepancies in potassium between POCT and CL methods. In comparing POCT normo- and mild hypokalemia against CL results, differences of -4.20 % and +4.88 % were noted respectively. The largest variance in the CL was a +4.14 % difference in the mild hyperkalemia category. Additionally, the RCV was calculated to quantify the severity of discrepancies between paired potassium measurements from POCT and CL methods. The overall hemolysis characteristics, as defined by the hemolysis gradient, showed considerable variation between the testing sites, significantly affecting the reliability of potassium measurements in POCT. CONCLUSIONS: The study highlighted the challenges in achieving consistent potassium measurement results between POCT and CL methods, particularly in the presence of hemolysis. It emphasised the need for integrated hemolysis detection systems in future blood gas analysis devices to minimise discrepancies and ensure accurate POCT results.

Failure Mode and Effects Analysis (FMEA) at the preanalytical phase for POCT blood gas analysis: proposal for a shared proactive risk analysis model.

OBJECTIVES: Proposal of a risk analysis model to diminish negative impact on patient care by preanalytical errors in blood gas analysis (BGA). METHODS: Here we designed a Failure Mode and Effects Analysis (FMEA) risk assessment template for BGA, based on literature references and expertise of an international team of laboratory and clinical health care professionals. RESULTS: The FMEA identifies pre-analytical process steps, errors that may occur whilst performing BGA (potential failure mode), possible consequences (potential failure effect) and preventive/corrective actions (current controls). Probability of failure occurrence (OCC), severity of failure (SEV) and probability of failure detection (DET) are scored per potential failure mode. OCC and DET depend on test setting and patient population e.g., they differ in primary community health centres as compared to secondary community hospitals and third line university or specialized hospitals. OCC and DET also differ between stand-alone and networked instruments, manual and automated patient identification, and whether results are automatically transmitted to the patient's electronic health record. The risk priority number (RPN = SEV × OCC × DET) can be applied to determine the sequence in which risks are addressed. RPN can be recalculated after implementing changes to decrease OCC and/or increase DET. Key performance indicators are also proposed to evaluate changes. CONCLUSIONS: This FMEA model will help health care professionals manage and minimize the risk of preanalytical errors in BGA.

Organisation and quality monitoring for point-of-care testing (POCT) in Belgium: proposal for an expansion of the legal framework for POCT into primary health care.

BACKGROUND: There is a trend towards decentralisation of laboratory tests by means of Point-of-Care testing (POCT). Within hospitals, Belgian law requires a POCT policy, coordinated by the clinical laboratory. There is however no legal framework for POCT performed outside the hospital: no reimbursement, no compulsory quality monitoring and no limits nor control on the prices charged to the patient. Uncontrolled use of POCT can have negative consequences for individual and public health. PROPOSAL: We propose that POCT outside hospitals would only be reimbursed for tests carried out within a legal framework, requiring evidence-based testing and collaboration with a clinical laboratory, because clinical laboratories have procedures for test validation and quality monitoring, are equipped for electronic data transfer, are familiar with logistical processes, can provide support when technical issues arise and can organise and certify training. Under these conditions the government investment will be offset by health benefits, e.g. fall in antibiotic consumption with POCT for CRP in primary care, quick response to SARS-CoV2-positive cases in COVID-19 triage centres. PRIORITIES: 1° extension of the Belgian decree on certification of clinical laboratories to decentralised tests in primary care; 2° introduction of a separate reimbursement category for POCT; 3° introduction of reimbursement for a limited number of specified POCT; 4° setup of a Multidisciplinary POCT Advisory Council, the purpose of which is to draw up a model for reimbursement of POCT, to select tests eligible for reimbursement and to make proposals to the National Institute for Health and Disability Insurance (RIZIV/INAMI).

Belgian rare diseases plan in clinical pathology: identification of key biochemical diagnostic tests and establishment of reference laboratories and financing conditions.

BACKGROUND: One objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories. METHODS: A feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019. RESULTS: In 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium. CONCLUSIONS: In the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests.

Biomarkers and Donor Selection in Heart Transplantation.

BACKGROUND: Previously, we showed that B-type natriuretic peptide (BNP) measured in the donor was related to cardiac performance after cardiac transplantation. The present study assesses the value of 3 biomarkers in the selection of donor hearts in a larger cohort. METHODS: Blood samples were prospectively obtained in 105 brain-dead patients scheduled for heart donation. BNP, soluble suppressor of tumorigenicity 2 (ST2), and troponin of heart donors were correlated with hemodynamic parameters early after transplantation as well as with the mortality of the recipients. RESULTS: A significant inverse relationship was found between donor BNP measured at the time of donation and recipient cardiac index and cardiac output at day 13 post-transplantation (r = -0.31, P = .005, and r = -0.34, P = .0016, respectively). Logistic regression analysis-including BNP, ST2, and troponin-showed that donor BNP was a predictor of a poor cardiac index (< 2.2 L/min/m2) in the recipient (P = .04). A donor BNP > 132 pg/mL has a sensitivity of 56% (95% confidence interval 21-86) and a specificity of 86% (95% confidence interval 77-93) to predict poor cardiac performance in the recipient. When the donor BNP is ≤ 132 pg/mL, the risk of a poor cardiac function in the recipient is very low (negative predictive value 94%). Mortality at 30 days was also correlated to donor BNP (r = 0.29, P = .0029). Long-term survival of the recipient was not correlated to the biomarkers measured in the donor. CONCLUSION: Donor BNP, but not donor ST2 or high-sensitivity troponin, provides information on the donor heart and early post-transplant performance, including 1-month mortality.

Rapid Ventricular Pacing for Neurovascular Surgery: A Study on Cardiac and Cerebral Effects.

BACKGROUND AND OBJECTIVE: Intraoperative rupture of a cerebral aneurysm during neurosurgery can be a devastating event that increases perioperative morbidity and mortality. Rapid ventricular pacing (RVP) is a technique to obtain flow arrest for short periods of time during dissection or rupture of the aneurysm. The objective of this study was to evaluate the neurological and cardiac effects of repetitive periods of RVP during cerebrovascular surgery. METHODS: Data from patients who underwent repetitive RVP during craniotomy for cerebrovascular disorders were retrospectively analyzed from a single-center medical records database (Cegeka Medical Health Care Systems). We compared preoperative and postoperative troponin levels (cTnI) to assess cardiac ischemia. Preoperative and postoperative magnetic resonance imaging (MRI) results were screened for RVP-induced infarcts by evaluating diffusion restriction in the hemisphere contralateral to the operated side and the fossa posterior. RESULTS: A total of 37 patients were analyzed. An immediate decrease of systolic arterial blood pressure (<60 mm Hg) was achieved after initiation of RVP. Postoperative MRI did not reveal areas of diffusion restriction. RVP tended to be a predictor of postoperative cTnI increase. Preoperative and postoperative cTnI levels were higher in patients with a subarachnoid hemorrhage compared to patients without one. After 24 hours cTnI jevels normalized to preoperative values. CONCLUSIONS: Systolic arterial blood pressure decreased after RVP but normalized immediately when pacing stopped. MRI of the brain did not show RVP-related ischemia and the limited cTnI level increase normalized to preoperative values. Therefore, RVP during short periods of time seems to be a safe blood pressure-lowering technique for the brain and heart.

Bi-allelic Loss-of-Function Mutations in the NPR-C Receptor Result in Enhanced Growth and Connective Tissue Abnormalities.

The natriuretic peptide signaling pathway has been implicated in many cellular processes, including endochondral ossification and bone growth. More precisely, different mutations in the NPR-B receptor and the CNP ligand have been identified in individuals with either short or tall stature. In this study we show that the NPR-C receptor (encoded by NPR3) is also important for the regulation of linear bone growth. We report four individuals, originating from three different families, with a phenotype characterized by tall stature, long digits, and extra epiphyses in the hands and feet. In addition, aortic dilatation was observed in two of these families. In each affected individual, we identified a bi-allelic loss-of-function mutation in NPR3. The missense mutations (c.442T>C [p.Ser148Pro] and c.1088A>T [p.Asp363Val]) resulted in intracellular retention of the NPR-C receptor and absent localization on the plasma membrane, whereas the nonsense mutation (c.1524delC [p.Tyr508∗]) resulted in nonsense-mediated mRNA decay. Biochemical analysis of plasma from two affected and unrelated individuals revealed a reduced NTproNP/NP ratio for all ligands and also high cGMP levels. These data strongly suggest a reduced clearance of natriuretic peptides by the defective NPR-C receptor and consequently increased activity of the NPR-A/B receptors. In conclusion, this study demonstrates that loss-of-function mutations in NPR3 result in increased NPR-A/B signaling activity and cause a phenotype marked by enhanced bone growth and cardiovascular abnormalities.

Analytical performance of a platform for point-of-care CRP testing in adults consulting for lower respiratory tract infection in primary care.

C-reactive protein (CRP) is a biomarker widely used for disease severity assessment and treatment of inflammatory conditions. Point-of-care testing (POCT) devices should ideally be rapid and provide similar results to standard tests done in laboratories. Two thousand nine hundred twenty-two serum samples were obtained from adult patients presenting to primary care with symptoms of lower respiratory infection in a European diagnostic study. The analytic performance of the CRP QuikRead POCT device (Orion Diagnostica) was evaluated by comparing results with a central laboratory method (Dimension Vista, Siemens), with both tests performed in a laboratory setting. For a CRP cut-off concentration of ≥ 30 mg/L, the QuikRead test had a sensitivity of 92.2%, and specificity of 99.4%. The mean difference between the QuikRead and the central lab test was 0.4 mg/L. The slope of the Passing-Bablok regression was 0.94 (95% CI 0.93-0.95) indicating an underestimation of CRP levels of 6% by QuikRead. CRP estimates obtained from the QuikRead test correlate well with a central laboratory assay and the measurement displays low inter-assay variation. Therefore, the QuikRead test is a good candidate for CRP testing in primary care.

Coronary artery calcifications and diastolic dysfunction versus visceral fat area in type 1 diabetes: VISCERA study.

AIMS: Type 1 diabetic patients (T1DM) experience a higher cardiovascular disease and mortality risk than controls. We investigated whether visceral adipose tissue (VAT) contributes to coronary artery calcifications (CAC) and cardiac dysfunction in T1DM. METHODS: A cross-sectional study of 118 T1DM patients without a history of cardiovascular disease (men/women: 68/50, age 46±12years, HbA1c 7.6±0.9%, BMI 25.8±4.1kg/m2) was conducted. CAC and VAT were measured using a CT scan. CAC was scored using the Agatston method. Cardiac functional abnormalities were assessed by echocardiography. RESULTS: CAC scored ≥10 in 42% of patients. Systolic function was normal in all, but diastolic dysfunction was present in 75%. Forty-six percent had VAT≥100cm2. CAC score≥10 occurred more often in subjects with VAT≥100cm2 (54% vs 31%; p=0.01). Age (OR=1.10; p<0.0001), diabetes duration (OR=1.10; p=0.008), gender (OR=4.28; p=0.016), LDL-cholesterol (OR=1.03; p=0.009) and metabolic syndrome (OR=5.79; p=0.005) were independently associated with a CACS≥10. Subjects with CACS≥10 were more prone to have diastolic dysfunction (84 vs 54%; p=0.03). Factors independently associated with diastolic dysfunction were age (OR=1.11; p=0.002), waist circumference (OR=1.10; p=0.016) and VAT (OR=0.99; p=0.035). CONCLUSIONS: Excess VAT in T1DM, present in 46%, is associated with diastolic dysfunction and CAC, present in respectively 75% and 42% of patients. Timely detection might improve future cardiovascular risk.

Accuracy, user-friendliness and usefulness of the Cobas h232 point-of-care test for NT-proBNP in primary care.

AIMS: N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been shown to be useful for ruling out heart failure in primary care. In this study, we examined the accuracy of the Cobas h232 point-of-care (POC) instrument in primary care compared with an in-hospital measurement. Furthermore, we investigated the user-friendliness and usefulness of the POC device. METHODS: Five general practitioner (GP) groups were asked to evaluate adult patients who were suspected of having heart failure and to test NT-proBNP with the Cobas h232. The samples were subsequently delivered to and analysed at a central hospital laboratory by the Cobas e602 using conventional transport and storage. Difference between the paired measurements was analysed using a percentage difference plot, and correlation was assessed using Passing-Bablok linear regression analysis. User-friendliness and usefulness were assessed using semistructured questionnaires. RESULTS: Nineteen GPs studied 94 patients. Passing-Bablok analysis showed a slope of 1.05 (95% CI 1.00 to 1.11) (R2=0.97). The percentage difference plot showed a mean difference of 15.7% (95% CI -46.0% to -77.4%). User-friendliness and usefulness had median scores of 4 or 5 on a five-point Likert scale. Eighteen out of 19 GPs confirmed that the device influenced their clinical practice. During the study, GPs' confidence in using NT-proBNP increased significantly from a mean score of 4.4 (95% CI 3.2 to 5.6) to 7.6 out of 10 (95% CI 7.1 to 8.2). CONCLUSIONS: The Cobas h232 NT-proBNP POC test proved to be an accurate, user-friendly and useful test in primary care. Nearly all participating GPs were convinced that the test could benefit clinical decision making.

Failed Downregulation of Circulating MicroRNA-155 in the Early Phase after ST Elevation Myocardial Infarction Is Associated with Adverse Left Ventricular Remodeling.

BACKGROUND: MicroRNA are noncoding RNA that have a significant role in both inflammatory and cardiovascular diseases. AIMS: We aimed to assess whether the inflammation-related microRNA-155 is associated with the development of adverse left ventricular (LV) remodeling following ST elevation myocardial infarction (STEMI). METHODS: Peripheral blood samples were collected in the inflammatory (day 2), proliferative (day 5), and maturation phases (6 months) after STEMI (n = 20). Granulocytes, monocytes, and lymphocytes were enumerated with flow cytometry. The changes in LV volumes were assessed with 3-D echocardiography on day 1 and after 6 months. Adverse remodeling was defined as a >20% increase in end-diastolic volume. Healthy subjects were recruited as controls. RESULTS: MicroRNA-155 measured on day 5 correlated positively with the relative change in end-diastolic volume (ρ = 0.490, p = 0.028). MicroRNA-155 (day 5) was significantly higher in patients with compared to patients without adverse LV remodeling. The expression level was similar in healthy subjects (n = 8) and in patients with LV remodeling. There was a positive correlation between microRNA-155 and the amount of monocytes (day 5, ρ = 0.463, p = 0.046). CONCLUSION: Impaired downregulation of microRNA-155 during the second phase of the post- STEMI inflammatory response is a determinant of the development of adverse LV remodeling.

Accelerated cellular senescence as underlying mechanism for functionally impaired bone marrow-derived progenitor cells in ischemic heart disease.

BACKGROUND AND AIMS: Bone marrow (BM)-derived progenitor cells are functionally impaired in patients with ischemic heart disease (IHD), thereby hampering the outcome of autologous stem cell therapy. In search for underlying mechanisms for this BM dysfunction, accelerated cellular senescence was explored. METHODS: We analysed telomere length of BM-derived mononuclear cells (MNC) by MMqPCR in patients with coronary artery disease (n = 12), ischemic heart failure (HF; n = 9), non-ischemic HF (n = 7) and controls (n = 10), and related it to their myeloid differentiation capacity. Expressions of senescence-associated genes p53, p21Cip1 and p16lnk4A; and telomere maintenance genes TERT, TRF1/2, Sirt1 in BM-MNC were evaluated using qPCR. Pro-inflammatory cytokine levels (TNFα, IFNy, IL-6) in BM were measured by MSD. RESULTS: BM-MNC telomere length was shortened in patients with IHD, irrespective of associated cardiomyopathy, and shortened further with increasing angiographic lesions. This telomere shortening was associated with reduced myeloid differentiation capacity of BM-MNC, suggesting accelerated senescence as underlying cause for progenitor cell dysfunction in IHD. Both p16lnk4A and p21Cip1 were activated in IHD and inversely related to myeloid differentiation capacity of BM-MNC; hence, the BM-MNC functional impairment worsens with increasing senescence. While BM-MNC telomere attrition was not related with alterations in TERT, TRF1/2 and Sirt1 expression, IFNy levels were associated with p21Cip1/p16lnk4A upregulation, suggesting a link between inflammation and cellular senescence. Still, the trigger for telomere shortening in IHD needs to be elucidated. CONCLUSIONS: Accelerated replicative senescence is associated with a functional impairment of BM-derived progenitor cells in IHD and could be targeted to improve efficacy of stem cell therapy.

Caspase-3 Deletion Promotes Necrosis in Atherosclerotic Plaques of ApoE Knockout Mice.

Apoptosis of macrophages and vascular smooth muscle cells (VSMCs) in advanced atherosclerotic plaques contributes to plaque progression and instability. Caspase-3, a key executioner protease in the apoptotic pathway, has been identified in human and mouse atherosclerotic plaques but its role in atherogenesis is not fully explored. We therefore investigated the impact of caspase-3 deletion on atherosclerosis by crossbreeding caspase-3 knockout (Casp3-/-) mice with apolipoprotein E knockout (ApoE-/-) mice. Bone marrow-derived macrophages and VSMCs isolated from Casp3-/-ApoE-/- mice were resistant to apoptosis but showed increased susceptibility to necrosis. However, caspase-3 deficiency did not sensitize cells to undergo RIP1-dependent necroptosis. To study the effect on atherosclerotic plaque development, Casp3+/+ApoE-/- and Casp3-/-ApoE-/- mice were fed a western-type diet for 16 weeks. Though total plasma cholesterol, triglycerides, and LDL cholesterol levels were not altered, both the plaque size and percentage necrosis were significantly increased in the aortic root of Casp3-/-ApoE-/- mice as compared to Casp3+/+ApoE-/- mice. Macrophage content was significantly decreased in plaques of Casp3-/-ApoE-/- mice as compared to controls, while collagen content and VSMC content were not changed. To conclude, deletion of caspase-3 promotes plaque growth and plaque necrosis in ApoE-/- mice, indicating that this antiapoptotic strategy is unfavorable to improve atherosclerotic plaque stability.

Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

Autophagy and the unfolded protein response (UPR) are key cellular homeostatic mechanisms and are both involved in liver diseases, including nonalcoholic fatty liver disease (NAFLD). Although increasing but conflicting results link these mechanisms to lipid metabolism, their role and potential cross talk herein have been poorly investigated. Therefore, we assessed the effects of hepatocyte-specific autophagy deficiency on liver parenchyma, the UPR, and lipid metabolism. Adult hepatocellular-specific autophagy-deficient mice (Atg7F/FAlb-Cre+) were compared with their autophagy-competent littermates (Atg7+/+Alb-Cre+). Livers were analyzed by electron microscopy, histology, real-time qPCR, and Western blotting. Atg7F/FAlb-Cre+ mice developed hepatomegaly with significant parenchymal injury, as shown by inflammatory infiltrates, hepatocellular apoptosis, pericellular fibrosis, and a pronounced ductular reaction. Surprisingly, the UPR exhibited a pathway-selective pattern upon autophagy deficiency. The activity of the adaptive activating transcription factor 6 (ATF6) pathway was abolished, whereas the proapoptotic protein kinase RNA-like ER kinase pathway was increased compared with Atg7+/+Alb-Cre+ mice. The inositol-requiring enzyme-1α signal was unaltered. Fasting-induced steatosis was absent in Atg7F/FAlb-Cre+ mice. Remarkably, some isolated islands of fat-containing and autophagy-competent cells were observed in these livers. Hepatocellular autophagy is essential for parenchymal integrity in mice. Moreover, in the case of autophagy deficiency, the three different UPR branches are pathway selectively modulated. Attenuation of the ATF6 pathway might explain the observed impairment of fasting-induced steatosis. Finally, autophagy and lipid droplets are directly linked to each other.

Bone Marrow-Derived Progenitor Cells Are Functionally Impaired in Ischemic Heart Disease.

To determine whether the presence of ischemic heart disease (IHD) per se, or rather the co-presence of heart failure (HF), is the primum movens for less effective stem cell products in autologous stem cell therapy, we assessed numbers and function of bone marrow (BM)-derived progenitor cells in patients with coronary artery disease (n = 17), HF due to ischemic cardiomyopathy (n = 8), non-ischemic HF (n = 7), and control subjects (n = 11). Myeloid and erythroid differentiation capacity of BM-derived mononuclear cells was impaired in patients with underlying IHD but not with non-ischemic HF. Migration capacity decreased with increasing IHD severity. Hence, IHD, with or without associated cardiomyopathy, is an important determinant of progenitor cell function. No depletion of hematopoietic and endothelial progenitor cells (EPC) within the BM was observed, while circulating EPC numbers were increased in the presence of IHD, suggesting active recruitment. The observed myelosuppression was not driven by inflammation and thus other mechanisms are at play.

NecroX-7 reduces necrotic core formation in atherosclerotic plaques of Apoe knockout mice.

BACKGROUND AND AIMS: A large necrotic core is a key feature of atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation, destabilization and ultimately rupture of the plaque. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In the present study, we tested the plaque stabilizing potential of NecroX-7, a novel compound with antioxidative and anti-necrotic properties. METHODS: Male apolipoprotein E (Apoe) knockout mice were treated with NecroX-7 (30 mg/kg) or vehicle, 3 times per week, via intraperitoneal injections for 16 weeks. Meanwhile, mice were fed a western-type diet to induce plaque formation. RESULTS: NecroX-7 reduced total plaque burden in the thoracic aorta as compared to vehicle-treated mice, without affecting total plasma cholesterol. Plaques in the aortic root of NecroX-7-treated mice showed a significant decrease in necrotic core area, 8-oxodG, iNOS and MMP13 expression, while collagen content and minimum fibrous cap thickness were increased. Moreover, NecroX-7 treatment reduced the expression of multiple inflammation markers such as TNFα, IL1ß, iNOS, HMGB1 and RAGE in a NF-κB-dependent manner. In vitro, NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced mitochondrial ROS formation, necrosis, iNOS expression and HMGB1 release in primary macrophages. CONCLUSIONS: NecroX-7 improves features of plaque stability in Apoe knockout mice by reducing necrotic core formation, oxidative stress and inflammation, and by increasing collagen deposition and fibrous cap thickness. Therefore, NecroX-7 could be a promising pleiotropic drug for the treatment of atherosclerosis.

Primary skeletal muscle myoblasts from chronic heart failure patients exhibit loss of anti-inflammatory and proliferative activity.

BACKGROUND: Peripheral skeletal muscle wasting is a common finding with adverse effects in chronic heart failure (HF). Whereas its clinical relevance is beyond doubt, the underlying pathophysiological mechanisms are not yet fully elucidated. We aimed to introduce and characterize the primary culture of skeletal muscle cells from individual HF patients as a supportive model to study this muscle loss. METHODS AND RESULTS: Primary myoblast and myotubes cultures were successfully propagated from the m. vastus lateralis of 6 HF patients with reduced ejection fraction (HFrEF; LVEF <45 %) and 6 age and gender-matched healthy donors. HFrEF cultures were not different from healthy donors in terms of morphology, such as myoblast size, shape and actin microfilament. Differentiation and fusion indexes were identical between groups. Myoblast proliferation in logarithmic growth phase, however, was attenuated in the HFrEF group (p = 0.032). In addition, HFrEF myoblasts are characterized by a reduced TNFR2 expression and IL-6 secretion (p = 0.017 and p = 0.016; respectively). CONCLUSION: Biopsy derived primary skeletal muscle myoblasts of HFrEF patients produce similar morphological and myogenic differentiation responses as myoblasts of healthy donors, though demonstrate loss of anti-inflammatory and proliferative activity.

A Novel Domain-Specific Mutation in a Sclerosteosis Patient Suggests a Role of LRP4 as an Anchor for Sclerostin in Human Bone.

Mutations in the LRP4 gene, coding for a Wnt signaling coreceptor, have been found to cause several allelic conditions. Among these, two are characterized by a strong skeletal involvement, namely sclerosteosis and Cenani-Lenz syndrome. In this work, we evaluated the role of LRP4 in the pathophysiology of these diseases. First, we report a novel LRP4 mutation, leading to the substitution of arginine at position 1170 in glutamine, identified in a patient with sclerosteosis. This mutation is located in the central cavity of the third ß-propeller domain, which is in line with two other sclerosteosis mutations we previously described. Reporter assays demonstrate that this mutation leads to impaired sclerostin inhibition of Wnt signaling. Moreover, we compared the effect of this novel variant to mutations causing Cenani-Lenz syndrome and show that impaired membrane trafficking of the LRP4 protein is the likely mechanism underlying Cenani-Lenz syndrome. This is in contrast to sclerosteosis mutations, previously shown to impair the binding between LRP4 and sclerostin. In addition, to better understand the biology of LRP4, we investigated the circulating sclerostin levels in the serum of a patient suffering from sclerosteosis owing to a LRP4 mutation. We demonstrate that impaired sclerostin binding to the mutated LRP4 protein leads to dramatic increase in circulating sclerostin in this patient. With this study, we provide the first evidence suggesting that LRP4 is responsible for the retention of sclerostin in the bone environment in humans. These findings raise potential concerns about the utility of determining circulating sclerostin levels as a marker for other bone-related parameters. Although more studies are needed to fully understand the mechanism whereby LRP4 facilitates sclerostin action, it is clear that this protein represents a potent target for future osteoporosis therapies and an interesting alternative for the antisclerostin treatment currently under study.