Plasma-derived candidate biomarkers for detection of gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types-5'-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC.

Relevance of Neutrophil Neprilysin in Heart Failure.

Significant expression of neprilysin (NEP) is found on neutrophils, which present the transmembrane integer form of the enzyme. This study aimed to investigate the relationship of neutrophil transmembrane neprilysin (mNEP) with disease severity, adverse remodeling, and outcome in HFrEF. In total, 228 HFrEF, 30 HFpEF patients, and 43 controls were enrolled. Neutrophil mNEP was measured by flow-cytometry. NEP activity in plasma and blood cells was determined for a subset of HFrEF patients using mass-spectrometry. Heart failure (HF) was characterized by reduced neutrophil mNEP compared to controls (p < 0.01). NEP activity on peripheral blood cells was almost 4-fold higher compared to plasma NEP activity (p = 0.031) and correlated with neutrophil mNEP (p = 0.006). Lower neutrophil mNEP was associated with increasing disease severity and markers of adverse remodeling. Higher neutrophil mNEP was associated with reduced risk for mortality, total cardiovascular hospitalizations, and the composite endpoint of both (p < 0.01 for all). This is the first report describing a significant role of neutrophil mNEP in HFrEF. The biological relevance of neutrophil mNEP and exact effects of angiotensin-converting-enzyme inhibitors (ARNi) at the neutrophil site have to be determined. However, the results may suggest early initiation of ARNi already in less severe HF disease, where effects of NEP inhibition may be more pronounced.

Serum neprilysin levels are elevated in preeclampsia.

OBJECTIVE: To evaluate the possible associations between serum Neprilysin (NEP) levels and preeclampsia and mild and severe preeclampsia subgroups. MATERIALS AND METHODS: Fifty-five consecutive women with mild preeclampsia and fifty-five consecutive women with severe preeclampsia were compared with 110 approximately gestational age-matched (±1 week) women with an uncomplicated pregnancy. RESULTS: Mean serum NEP was significantly higher in women with preeclampsia compared to that of the gestational age-matched-controls (231.62 ± 65.30 pg/mL vs. 187.75 ± 84.38 pg/mL, p < 0.001). Mean serum NEP was significantly higher in the mild preeclampsia group compared to its gestational age-matched control group (228.84 ± 67.26 pg/mL vs. 186.14 ± 85.09 pg/mL, p = 0.008); and in the severe preeclampsia group compared to its gestational age-matched control group (234.45 ± 63.85 pg/mL vs. 189.29 ± 84.59 pg/mL, p = 0.004). Serum NEP was positively correlated with systolic and diastolic blood pressure, BUN, uric acid, and creatinine. CONCLUSION: Mean serum NEP was significantly higher in women with preeclampsia than women with an uncomplicated pregnancy. Further studies are needed to elucidate the possible therapeutic role of NEP inhibitors to treat preeclampsia.

Circulating neprilysin hypothesis: A new opportunity for sacubitril/valsartan in patients with heart failure and preserved ejection fraction?

BACKGROUND: Circulating Neprilysin (sNEP) has emerged as a potential prognostic biomarker in heart failure (HF). In PARAGON-HF benefit of sacubitril/valsartan was only observed in patients with left ventricular ejection fraction (LVEF) ≤57%. We aimed to assess the prognostic value of sNEP in outpatients with HF and LVEF >57%, in comparison with patients with LVEF ≤57%. METHODS: Consecutive HF outpatients were included from May-2006 to February-2016. The primary endpoint was the composite of all-cause death or HF hospitalization and the main secondary endpoint was the composite of cardiovascular death or HF hospitalization. For the later competing risk methods were used. RESULTS: sNEP was measured in 1428 patients (age 67.7±12.7, 70.3% men, LVEF 35.8% ±14), 144 of which had a LVEF >57%. sNEP levels did not significantly differ between LVEF groups (p = 0.31). During a mean follow-up of 6±3.9 years, the primary endpoint occurred in 979 patients and the secondary composite endpoint in 714 (in 111 and 84 of the 144 patients with LVEF >57%, respectively). sNEP was significantly associated with both composite endpoints. Age- and sex- adjusted Cox regression analyses showed higher hazard ratios for sNEP in patients with LVEF >57%, both for the primary (HR 1.37 [1.16-1.61] vs. 1.04 [0.97-1.11]) and the secondary (HR 1.38 [1.21-1.55] vs. 1.11 [1.04-1.18]) composite endpoints. CONCLUSIONS: sNEP prognostic value in patients with HF and LVEF >57% outperforms that observed in patients with lower LVEF. Precision medicine using sNEP may identify HF patients with preserved LVEF that may benefit from treatment with sacubitril/valsartan.

The influence of sex and body mass index on the association between soluble neprilysin and risk of heart failure hospitalizations.

A higher neprilysin activity has been suggested in women. In this retrospective analysis, we evaluated the association of sex and body mass index (BMI) with soluble neprilysin (sNEP) and recurrent admissions among 1021 consecutive HF outpatients. The primary and secondary endpoints were the number of HF hospitalizations and all-cause mortality, respectively. The association between sNEP with either endpoint was evaluated across sex and BMI categories (≥ 25 kg/m2 vs. < 25 kg/m2). Bivariate count regression (Poisson) was used, and risk estimates were expressed as incidence rates ratio (IRR). During a median follow-up of 6.65 years (percentile 25%-percentile 75%:2.83-10.25), 702 (68.76%) patients died, and 406 (40%) had at least 1 HF hospitalization. Median values of sNEP and BMI were 0.64 ng/mL (0.39-1.22), and 26.9 kg/m2 (24.3-30.4), respectively. Left ventricle ejection fraction was < 40% in 78.9% of patients, and 28% were women. In multivariable analysis, sNEP (main effect) was positively associated with HF hospitalizations (p = 0.001) but not with mortality (p = 0.241). The predictive value of sNEP for HF hospitalizations varied non-linearly across sex and BMI categories (p-value for interaction = 0.003), with significant and positive effect only on women with BMI ≥ 25 kg/m2 (p = 0.039). For instance, compared to men, women with sNEP of 1.22 ng/mL (percentile 75%) showed a significantly increased risk (IRRs: 1.26; 95% CI: 1.05-1.53). The interaction analysis for mortality did not support a differential prognostic effect for sNEP (p = 0.072). In conclusion, higher sNEP levels in overweight women better predicted an increased risk of HF hospitalization.

Altered serum levels of neprilysin in heart failure patients with reduced ejection fraction.

OBJECTIVE: In addition to the recent success of neprilysin inhibition in treatment of heart failure, elevated soluble neprilysin (sNEP) in circulation has been suggested to be a prognostic biomarker in heart failure with a reduced ejection fraction (HFrEF). However, the diagnostic performance of sNEP is nebulous and its levels in HFrEF have not been compared with controls. For the purpose of this study, we determined the role of sNEP levels as a biomarker in routine ambulatory care of HFrEF patients, when compared to the control subjects. METHODS: Ambulant patients with chronic HFrEF (n = 18) were included. Apparently healthy volunteers - hospital physicians (n = 9) were included as the controls. Besides standard diagnostic tools (echocardiographic examination and laboratory biochemical diagnostic tests including NT-proBNP assessment), we analysed serum levels of neprilysin with a commercially available human soluble neprilysin ultrasensitive ELISA kit (Aviscera Bioscience, USA). RESULTS: Concentrations of sNEP were significantly reduced in HFrEF patients (average ± S.E.M.=1038 ± 464 pg/ml) when compared to the controls (1947 ± 613 pg/ml; p < 0.05). Two of eighteen HFrEF samples were below, while two of ten control samples were above the detection limit of the immunoassay. We documented a lack of significant correlation between sNEP and left ventricular ejection fraction (LVEF) and other echocardiographic features as well as NT-proBNP. However, sNEP significantly negatively correlated to serum natrium levels (Spearman r = ‒0.6112, p < 0.05) and to systolic blood pressure (Spearman r = ‒0.4746, p < 0.05) in HFrEF. CONCLUSION: Levels of sNEP were significantly reduced in HFrEF, when compared to the controls, with absent correlations to relevant HF-related features (e.g. LVEF). These findings might contribute to clarification of the diagnostic value of sNEP in HF (Tab. 2, Fig. 2, Ref. 30) Keywords: soluble neprilysin, heart failure, reduced ejection fraction, pharmacotherapy.

Diagnostic performance of the ClearLLab 10C B cell tube.

INTRODUCTION: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice. METHODS: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls. RESULTS: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good. CONCLUSIONS: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

The changing features of serum adropin, copeptin, neprilysin and chitotriosidase which are associated with vascular endothelial function in type 2 diabetic retinopathy patients.

AIMS: Adropin (AD), copeptin (CP), neprilysin (NEP) and chitotriosidase (CHIT1) have been associated with the regulation of vascular endothelial function. In this work, we analyzed the plasma concentrations of cytokines (AD, CP, NEP and CHIT1) in type 2 diabetic patients with or without retinopathy (DR) to predict the risk of DR for diabetic patients. METHOD: A total of 392 patients diagnosed as type 2 diabetes mellitus (T2DM) and 120 healthy volunteers as a control group were enrolled in this study. T2DM patients were divided into three groups: diabetes without retinopathy (NDR, n = 174) group, non-proliferative diabetic retinopathy (NPDR, n = 118) group and proliferative diabetic retinopathy (PDR, n = 100) group. The serum AD, CP, NEP and CHIT1 levels of subjects were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: We reported a significant decrease in AD and a significant increase in CP, NEP and CHIT1 in NDR as well as DR patients when compared with controls (p < 0.05), the lower level of AD and significantly higher levels of CP, NEP and CHIT1 were seen in DR patients compared to NDR group (p < 0.05), at the same time, we observed the lowest level of AD and the highest levels of CP, NEP and CHIT1 in the PDR group. Logistic regression analysis showed that AD was a protective factor for DR, conversely, CP, NEP and CHIT1 were the independent risk factors (p < 0.05). Moreover, receiver operating characteristic curve analyses indicated that CP had greater diagnosis capacity with an AUC (the areas under the ROC curve) of 0.901 than AD, NEP, CHIT1 for DR patients. CONCLUSION: The decreased AD level and the elevated CP, NEP and CHIT1 levels involved in vascular endothelial function may be evidence facilitating the presence of DR. Thereby they can be explored to use as promising non-invasive biomarkers for prediction of DR severity, distinguishing DR from diabetic patients.

Soluble neprilysin and long-term clinical outcomes in patients with coronary artery disease undergoing percutaneous coronary intervention: a retrospective cohort study.

BACKGROUND: Neprilysin has an essential role in regulating fluid balance and vascular resistance, and neprilysin inhibitors have shown beneficial effects in patients with heart failure. However, the potential predictive value of neprilysin levels as a biomarker for cardiovascular risk remains unclear. The aim of this study was to assess the prognostic value of soluble neprilysin (sNEP) levels in patients with ischemic heart disease. METHODS: Neprilysin levels were measured in 694 consecutive patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). These patients were classified into two groups according to their serum levels of neprilysin and categorized into the lower neprilysin group (n = 348) and the higher neprilysin group (n = 346). The primary clinical endpoint was all-cause mortality, and the secondary endpoint was a composite of major adverse cardiac events (MACE). RESULTS: The median sNEP level was 76.0 pg/ml. The median sNEP levels were higher in patients with left ventricular ejection fraction (LVEF) ≥40% (77.6 pg/ml, interquartile range 46.6-141.3) than in those with LVEF < 40% (70.0 pg/ml, interquartile range 47.1-100.6; P = 0.032). Among all patients, each clinical outcome and MACE did not differ significantly according to the groups divided into median, tertile, or quartile of sNEP levels during a median follow-up of 28.4 months. We did not find a significant relationship between sNEP levels and clinical outcomes in multivariate Cox regression analysis. Among patients with LVEF < 40%, an increased sNEP level was associated with a higher rate of all-cause death (adjusted hazard ratio 2.630, 95% confidence interval 1.049-6.595, P = 0.039). CONCLUSION: Serum sNEP levels are not associated with long-term mortality or cardiovascular outcomes after PCI in patients with CAD. In the LVEF < 40% group, increased sNEP levels may be associated with a higher risk of all-cause death.

Dysregulation of the OGF-OGFr pathway correlates with elevated serum OGF and ocular surface complications in the diabetic rat.

IMPACT STATEMENT: This research extends our knowledge about the presence and role of the OGF-OGFr regulatory axis in type 1 diabetes (T1D) and demonstrates specific targets within the pathway that are dysregulated. Serum levels of OGF, an inhibitory growth factor, are significantly elevated in male T1D rats, and OGFr serum values are increased in T1D. The onset of elevated OGF corresponds to the onset of ocular surface complications including dry eye, delayed corneal epithelial repair, and abnormal corneal surface sensitivity in T1D. Systemic insulin does not protect against elevated OGF levels or the onset of dry eye and sensitivity. These data are the first to associate some ocular surface defects in T1D with alterations in the OGF-OGFr pathway.

Serum neprilysin levels are associated with myocardial stunning after ST-elevation myocardial infarction.

BACKGROUND: Left ventricular remodeling following ST-elevation myocardial infarction (STEMI) is associated with poor outcome, including heart failure (HF). Neprilysin inhibition leads to improved outcome in patients with altered left ventricular ejection fraction (LVEF). METHODS: We aimed to assess the association between serum levels of neprilysin and left ventricular (LV) volumes, function and remodeling in STEMI patients with successful myocardial reperfusion and no clinical sign of HF. Sixty-eight patients were admitted for STEMI and had both plasma neprilysin measurement at baseline and 3D transthoracic echocardiogram at baseline and after a median follow-up of 7 months. We compared 3 groups: a group with a low-level of plasma neprilysin (< 125 pg/mL, i.e. the lower limit of detection of the assay) and the two other groups were defined as being below or above the median value of the remaining samples. RESULTS: Median age was 58.5 ± 12.8 years and 56 (82.4%) were men. Median LVEF was 45.0 ± 8.5%. Baseline characteristics were comparable between groups (low-level of neprilysin group [≤125 pg/mL, n = 38], medium-level of neprilysin group [126-450 pg/mL, n = 15] and a high-level group [> 450 pg/mL, n = 15]). At baseline there was a non-significant trend towards lower end-diastolic volume (p = 0.07) but significantly lower LVEF in the high neprilysin group (46.4 ± 8.3%, 47.1 ± 8.1% and 39.1 ± 6.9%, p < 0.01). At follow-up, the magnitude of LVEF increase was significantly more important in the high neprilysin group compared to the other groups (p = 0.022 for relative change in LVEF and 6.6 ± 7.3%, 3.6 ± 9.0% and 11.3 ± 8.4%, p = 0.031 for absolute change in LVEF) resulting in similar LVEF levels at follow-up between all groups (53.0 ± 8.9%, 50.6 ± 9.7% and 50.4 ± 9.9%, p = 0.55). CONCLUSIONS: Initial high neprilysin levels may identify patients with stunned myocardium early after STEMI, with a recovery of contractility leading to improved LVEF at follow-up. Future studies will have to assess the role of neprilysin in the setting of STEMI and the potential benefit of its blockade.

Significance of CD10 protein expression in the diagnostics of follicular lymphoma: A comparison of conventional immunohistochemistry with flow cytometry supported by the establishment of BCL2 and BCL6 rearrangements.

INTRODUCTION: Histopathological examination and immunohistochemistry (IHC) with a crucial role of CD10 expression remain a standard diagnostic tool in follicular lymphoma (FL). The results of IHC CD10 detection with different primary antibodies are not fully reproducible, but some reports show that flow cytometry (FCM) can be a reliable method of CD10 identification. METHODS: The aim of the study was to compare results of CD10 expression in FL by IHC and FCM including immunophenotypic features in the context of the BCL2 and BCL6 alterations. RESULTS: Out of 76 histopathologically diagnosed FL, a group of 25 cases had simultaneously FCM. Immunohistochemically 77.6% of cases were CD10-positive with comparable and reproducible results to FCM. Differences between the FCM expression of CD5/CD10/CD11c/CD25/CD43 and BCL2 overexpression (BCL2(+)higher ) correlated with the BCL2 and BCL6 rearrangements (R) status. Lack of CD10 expression corresponded with the absence of BCL2R and higher MUM1 expression by IHC results but had no clinical impact on the long-time outcomes. CONCLUSIONS: Immunohistochemistry staining is a comparable method to FCM assessment in the evaluation of CD10 expression and diagnosis of FL. Fine-needle aspiration biopsy/FCM (FNAB/FCM) could be a useful tool for verifying FL diagnosis and CD10 detection. Despite its heterogeneity, FL has a characteristic immunophenotype. BCL2R and BCL6R FL cases differ mainly in levels of BCL2 and CD10 with CD43 co-expression; BCL2(+)higher by FCM correlates with BCL2R. Moreover, FNAB plays an important role in material provision for supportive karyotyping and BCL2R, BCL6R assessed by FISH.

Plasma Neprilysin Displays No Relevant Association With Neurohumoral Activation in Chronic HFrEF.

Background Neprilysin is a transmembrane endopeptidase involved in the breakdown of a variety of vasoactive peptides and serves as a therapeutic target in heart failure with reduced ejection fraction (HFrEF). This study aimed to investigate the relationship of circulating neprilysin with neurohumoral activation and the impact of plasma neprilysin activity on prognosis in HFrEF. Methods and Results A total of 369 chronic HFrEF patients were enrolled prospectively. Plasma neprilysin concentration and activity were determined by a specific ELISA and a fluorometric method. The association between plasma neprilysin and heart failure (HF) severity, neurohumoral activation, ie norepinephrine and absolute renin concentration, as well as all-cause mortality was assessed. Median plasma neprilysin concentrations and activity levels were 413 pg/mL (interquartile range 0-4111) and 2.36 nmol/mL per minute (interquartile range 1.16-4.59). No correlation could be shown between plasma neprilysin concentrations and activity (rs=0.09, P=0.088). Plasma neprilysin activity correlated with HF severity reflected by New York Heart Association stage (P=0.003) and tertiles of N-terminal pro-B-type natriuretic peptide (P<0.001), whereas neprilysin concentrations did not (P=0.220; P=0.849). There was no relevant relationship between plasma neprilysin concentrations and activity, with neurohumoral activation reflected by absolute renin concentration (rs=-0.02, P=0.648; rs=0.03, P=0.574) or norepinephrine levels (rs=-0.06, P=0.248; rs=0.20, P<0.001). Neither circulating neprilysin concentrations nor activity were associated with outcome. Conclusions Plasma neprilysin concentrations and activity are not directly related to neurohumoral activation, indicating that neprilysin regulation is either more complex or not correctly mirrored by circulating neprilysin as a biomarker. Circulating neprilysin concentrations and activity were not associated with overall survival, implicating limited prognostic value of plasma neprilysin measurements in HFrEF patients.

Soluble neprilysin: A versatile biomarker for heart failure, cardiovascular diseases and diabetic complications-A systematic review.

The potential role of soluble neprilysin (sNEP) as a biomarker has been poorly documented. Hence, the present systematic review emphasizes to explore sNEP as an emerging biomarker for heart failure (HF), cardiovascular diseases, diabetic kidney diseases, and so on. A systematic review was performed using an online database search in PubMed, Science Direct, Scopus, and Cochrane Library. Articles reporting biomarker's performance to diagnose various diseases in human participants were included. The results of the search outcome were 4723 articles. Based on the inclusion criteria of the systematic review, finally, 12 articles fulfilled the selection criteria. In these studies, 8 cohort study, 2 cross-sectional study, 1 case-control, and 1 prospective cohort study were identified. All these studies clearly suggested sNEP as a potential biomarker for diagnosis of various diseases (HF, cardiovascular diseases, diabetic kidney diseases, metabolic syndrome). sNEP may be a potential biomarker for HF, cardiovascular diseases, diabetic kidney disease, and so on.

Soluble neprilysin, NT-proBNP, and growth differentiation factor-15 as biomarkers for heart failure in dialysis patients (SONGBIRD).

BACKGROUND: Dialysis patients are at increased risk of HF. However, diagnostic utility of NT-proBNP as a biomarker is decreased in patients on dialysis. GDF-15 and cNEP are biomarkers of distinct mechanisms that may contribute to HF pathophysiology in such cohorts. The aim of this study was to determine whether growth differentiation factor-15 (GDF-15) and circulating neprilysin (cNEP) improve the diagnosis of congestive heart failure (HF) in patients on dialysis. METHODS AND RESULTS: We compared circulating concentrations of NT-proBNP, GDF-15, and cNEP along with cNEP activity in patients on chronic dialysis without (n = 80) and with HF (n = 73), as diagnosed by clinical parameters and post-dialysis echocardiography. We used correlation, linear and logistic regression as well as receiver operating characteristic (ROC) analyses. Compared to controls, patients with HF had higher median values of NT-proBNP (16,216 [interquartile range, IQR = 27739] vs. 2883 [5866] pg/mL, p < 0.001), GDF-15 (7512 [7084] vs. 6005 [4892] pg/mL, p = 0.014), but not cNEP (315 [107] vs. 318 [124] pg/mL, p = 0.818). Median cNEP activity was significantly lower in HF vs. controls (0.189 [0.223] vs. 0.257 [0.166] nmol/mL/min, p < 0.001). In ROC analyses, a multi-marker model combining clinical covariates, NT-proBNP, GDF-15, and cNEP activity demonstrated best discrimination of HF from controls (AUC = 0.902, 95% CI 0.857-0.947, p < 0.001 vs. base model AUC = 0.785). CONCLUSION: We present novel comparative data on physiologically distinct circulating biomarkers for HF in patients on dialysis. cNEP activity but not concentration and GDF-15 provided incremental diagnostic information over clinical covariates and NT-proBNP and may aid in diagnosing HF in dialysis patients.

Circulating Neprilysin in Patients With Heart Failure and Preserved Ejection Fraction.

BACKGROUND: In heart failure with reduced ejection fraction (HFrEF), elevated soluble neprilysin (sNEP) levels are associated with an increased risk of cardiovascular death, and its inhibition with sacubitril/valsartan has improved survival. OBJECTIVES: This study sought to determine the relevance of sNEP as a biomarker in heart failure with preserved ejection fraction (HFpEF) and to compare circulating sNEP levels in patients with HFpEF with normal controls. METHODS: A case-control study was performed in 242 symptomatic patients with HFpEF previously enrolled in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure with Preserved Ejection Fraction (RELAX) and Nitrates's Effect on Activity Tolerance in Heart Failure With Preserved Ejection (NEAT-HFpEF) clinical trials and 891 asymptomatic subjects without HF or diastolic dysfunction (confirmed by NT-proBNP levels <200 pg/ml and echocardiography) who were enrolled in the Prevalence of Asymptomatic Left Ventricular Dysfunction study. sNEP was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) in all subjects. RESULTS: Overall, sNEP levels were lower in HFpEF compared with controls (3.5 ng/ml; confidence interval [CI]: 2.5 to 4.8 vs. 8.5 ng/ml; CI: 7.2 to 10.0; p < 0.001). After adjusting for age, gender, body mass index (BMI), and smoking history, mean sNEP levels were also lower in HFpEF compared with controls (4.0 ng/ml [CI: 2.7 to 5.4] vs. 8.2 ng/ml [CI: 6.8 to 9.7]; p = 0.002). The cohorts were propensity matched based on age, BMI, diabetes, hypertension, smoking history, and renal function, and sNEP levels remained lower in HFpEF compared with controls (median 2.4 ng/ml [interquartile range: 0.6 to 27.7] vs. 4.9 ng/ml [interquartile range: 1.2 to 42.2]; p = 0.02). CONCLUSIONS: Patients with HFpEF on average have significantly lower circulating sNEP levels compared with controls. These findings challenge our current understanding of the complex biology of circulating sNEP in HFpEF.

Serum neprilysin and the risk of death in patients with out-of-hospital cardiac arrest of non-traumatic origin.

BACKGROUND: Early risk stratification remains an unmet clinical need in patients with in out-of-hospital cardiac arrest. We hypothesised that soluble neprilysin may represent a promising biomarker in patients with out-of-hospital cardiac arrest of non-traumatic origin and provide new pathobiological insight. METHODS: This pilot study was a biomarker analysis from the Heidelberg Resuscitation Registry. Serum soluble neprilysin levels on admission were measured in 144 patients with successful return of spontaneous circulation after out-of-hospital cardiac arrest of non-traumatic origin. The primary endpoint was time to all-cause mortality. KM Event Rates are reported. Cox models were adjusted for age, bystander resuscitation, initial ECG rhythm, baseline estimated glomerular filtration rate, baseline lactate, left ventricular function at baseline, and targeted temperature management. RESULTS: In total, 90 (62.5%) patients died over a follow-up of at least 30 days. Soluble neprilysin correlated weakly with high-sensitivity troponin T (r=0.18, P=0.032) but did not correlate significantly with estimated glomerular filtration rate (r=-0.12) or lactate (r=0.11). Patients with elevated soluble neprilysin levels on admission were at significantly higher risk of all-cause mortality (Q4 69.1% vs. Q1 48.4%). After multivariable adjustment, soluble neprilysin in the top quartile (Q4) was significantly associated with all-cause mortality (Q4 vs. Q1: adjusted hazard ratio 2.48 (1.20-5.12)). In an adjusted multimarker model including high-sensitivity troponin T and high-sensitivity C-reactive protein, soluble neprilysin and high-sensitivity troponin T remained independently associated with all-cause mortality (soluble neprilysin: adjusted hazard ratio 2.27 (1.08-4.78); high-sensitivity troponin T: adjusted hazard ratio 3.40 (1.63-7.09)). CONCLUSION: Soluble neprilysin, measured as early as on hospital admission, was independently associated with all-cause mortality in patients with out-of-hospital cardiac arrest of non-traumatic origin and may prove to be useful in the estimation of risk in these patients.

Endothelin 1 Is Associated with Heart Failure Hospitalization and Long-Term Mortality in Patients with Heart Failure with Preserved Ejection Fraction and Pulmonary Hypertension.

BACKGROUND: The prevalence of pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is increasing. We aim to study the role of big endothelin 1 (Big ET1), endothelin 1 (ET1), and neprilysin (NE) in HFpEF with PH. METHOD: This was a single center prospective cohort study including 90 HFpEF patients; 30 with no PH, 30 with postcapillary PH, and 30 with combined post- and precapillary PH. After enrollment, pulmonary venous and pulmonary arterial samples of Big ET1, ET1, and NE were collected during right heart catheterization. Subjects were then followed long term for adverse outcomes which included echocardiographic evidence of right ventricular dysfunction, heart failure hospitalization, and all-cause mortality. RESULTS: Patients with HFpEF-PH were found to have increased ET1 in pulmonary veins (endothelin from the wedge; ET1W) compared to controls (2.3 ± 1.4 and 1.6 ± 0.9 pg/mL, respectively). ET1W levels were associated with both PH (OR 2.7, 95% CI 1.5-4.7, p = 0.01) and pulmonary vascular resistance (OR 1.6, 95% CI 1.04-2.3, p = 0.03). No evidence of right ventricular dysfunction was observed after 1 year of follow-up. ET1W (OR 1.8, 95% CI 1.2-2.6, p = 0.01) and ET1 gradient (ET1G; OR 1.4, 95% CI 1.04-2, p = 0.03) were predictive of 1-year hospitalization. ET1G ≥0.2 pg/mL was associated with long-term mortality (log-rank 4.8, p = 0.03). CONCLUSION: In HFpEF patients, ET1W and ET1G are predictive of 1-year heart failure hospitalization, while elevated ET1G levels were found to be associated with long-term mortality in HFpEF. This study highlights the role of ET1 in developing PH in HFpEF patients and also explores the potential of ET1 as a prognostic biomarker.

Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes.

INTRODUCTION: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. METHODS: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. RESULTS: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) p = .058, CD11b: 2.13× (1.6×-2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. CONCLUSION: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15-20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.