Calprotectin as a new inflammatory marker of abdominal aortic aneurysm: A pilot study.

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a relevant clinical problem due to the risk of rupture of progressively dilated infrarenal aorta. It is characterized by degradation of elastic fibers, extracellular matrix, and inflammation of the arterial wall. Though neutrophil infiltration is a known feature of AAA, markers of neutrophil activation are scarcely analyzed; hence, the main objective of this study. METHODS: Plasma levels of main neutrophil activation markers were quantified in patients with AAA and a double control group (CTL) formed by healthy volunteers (HV) and patients with severe atherosclerosis submitted for carotid endarterectomy (CE). Calprotectin, a cytoplasmic neutrophil protein, was quantified, by Western blot, in arterial tissue samples from patients with AAA and organ donors. Colocalization of calprotectin and neutrophil elastase was assessed by immunofluorescence. RESULTS: Plasma calprotectin and IL-6 were both elevated in patients with AAA compared with CTL (p ⩽ 0.0001) and a strong correlation was found between both molecules (p < 0.001). This difference was maintained when comparing with HV and CE for calprotectin but only with HV for IL-6. Calprotectin was also elevated in arterial tissue samples from patients with AAA compared with organ donors (p < 0.0001), and colocalized with neutrophils in the arterial wall. CONCLUSIONS: Circulating calprotectin could be a specific AAA marker and a potential therapeutical target. Calprotectin is related to inflammation and neutrophil activation in arterial wall and independent of other atherosclerotic events.

"In Vitro" Evaluation of Energy-Based Sealing of Graft Side Branches in Bypass Surgery.

INTRODUCTION: Our objective was to compare the in vitro efficacy of electrothermal bipolar [EB] vessel sealing and ultrasonic harmonic scalpel [HS] versus mechanical interruption, with conventional ties or surgical clips (SC), in sealing saphenous vein (SV) collaterals, during its eventual preparation for bypass surgery. METHODS: Experimental in vitro study on 30 segments of SV. Each fragment included two collaterals at least 2 mm in diameter. One of them was sealed by ligation with 3/0 silk ties (control) and the other one with EB (n = 10), HS (n = 10) or medium-6 mm SC (n = 10). After incorporation in a closed circuit with pulsatile flow, the pressure was progressively increased until causing rupture. Collateral diameter, burst pressure, leak point, and histological study were recorded. RESULTS: Burst pressure was higher for SC (1320.20 ± 373.847 mmHg) as compared with EB (942.2 ± 344.9 mmHg, p = 0.065), and especially with HS (637.00 ± 320.61 mmHg, p = 0.0001). No statistically significant difference between EB and HS was found, and bursting always happened at supraphysiological pressures. The leak point for HS was always detected in the sealing zone (10/10), while for EB and SC, it occurred in the sealing zone only in 6/10(60%) and 4/10(40%), respectively (p = 0.015). CONCLUSIONS: Energy delivery devices showed similar efficacy and safety in sealing of SV side branches. Although bursting pressure was lower than with tie ligature or SC, non-inferiority efficacy was shown at the range of physiological pressures in both, EB and HS. Due to their speed and easy handling, they may be useful in the preparation of the venous graft during revascularization surgery. However, remaining questions about healing process, potential spread of tissue damage and sealing durability, will require further analysis.

Bladder cancer patients have increased NETosis and impaired DNaseI-mediated NET degradation that can be therapeutically restored in vitro.

Background: Neutrophils, key players of the immune system, also promote tumor development through the formation of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are extracellular networks of DNA, histones and cytoplasmic and granular proteins (calprotectin, myeloperoxidase, elastase, etc.) released by neutrophils upon activation. NETs regulate tumor growth while promoting angiogenesis and invasiveness, and tumor cells also stimulate NETosis. Although NETosis seems to be increased in cancer patients, an increase of NETs in plasma may also be mediated by an impaired degradation by plasma DNaseI, as evidenced in several immunological disorders like lupus nephritis. However, this has never been evidenced in bladder cancer (BC) patients. Herein, we aimed to evaluate the occurrence of increased NETosis in plasma and tumor tissue of BC patients, to ascertain whether it is mediated by a reduced DNaseI activity and degradation, and to in vitro explore novel therapeutic interventions. Methods: We recruited 71 BC patients from whom we obtained a plasma sample before surgery and a formalin-fixed paraffin embedded tumor tissue sample, and 64 age- and sex-matched healthy controls from whom we obtained a plasma sample. We measured NETs markers (cell-free fDNA, calprotectin, nucleosomes and neutrophil elastase) and the DNaseI activity in plasma with specific assays. We also measured NETs markers in BC tissue by immunofluorescence. Finally, we evaluated the ability of BC and control plasma to degrade in vitro-generated NETs, and evaluated the performance of the approved recombinant human DNaseI (rhDNaseI, Dornase alfa, Pulmozyme®, Roche) to restore the NET-degradation ability of plasma. In vitro experiments were performed in triplicate. Statistical analysis was conducted with Graphpad (v.8.0.1). Results: NETosis occurs in BC tissue, more profusely in the muscle-invasive subtype (P<0.01), that with the worst prognosis. Compared to controls, BC patients had increased NETosis and a reduced DNaseI activity in plasma (P<0.0001), which leads to an impairment to degrade NETs (P<0.0001). Remarkably, this can be therapeutically restored with rhDNaseI to the level of healthy controls. Conclusion: To the best of our knowledge, this is the first report demonstrating that BC patients have an increased NETosis systemically and in the tumor microenvironment, in part caused by an impaired DNaseI-mediated NET degradation. Remarkably, this defect can be therapeutically restored in vitro with the approved Dornase alfa, thus Pulmozyme® could become a potential therapeutic tool to locally reduce BC progression.

LC-MS metabolomics of urine reveals distinct profiles for non-muscle-invasive and muscle-invasive bladder cancer.

PURPOSE: Bladder cancer (BC) is among the most frequent malignancies worldwide. Novel non-invasive markers are needed to diagnose and stage BC with more accuracy than invasive procedures like cystoscopy. To date, no study has identified urine metabolites characteristic of all BC stages. To discover novel urine metabolomic profiles to diagnose and stage non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) patients using mass spectrometry-based metabolomics. METHODS: We prospectively recruited 198 BC patients and 98 age- and sex-matched healthy volunteers without evidence of renal or bladder condition confirmed by ultrasound, from whom we collected a first morning urine sample (before surgery in patients). In a discovery stage, an untargeted metabolomic analysis was conducted in urine samples of a selection of 64 BC patients (19 TaG1, 11 TaG3, 20 T1G3, 12 T2G3, 1 T2G2, 1 T3G3) and 20 controls to identify dysregulated metabolites. Next, after exhaustive multivariate analysis, confirmed dysregulated metabolites were validated in an independent cohort of 134 BC patients (19 TaG1, 62 TaG2, 9 TaG3, 15 T1G2, 16 T1G3, 4 T2G2, 9 T2G3) and 78 controls. RESULTS: We validated p-cresol glucuronide as potential diagnostic biomarker for BC patients compared to controls (AUC = 0.79). For NMIBC, p-cresol glucuronide was valuable as staging biomarker (AUC = 0.803). And among NMIBCs, p-coumaric acid may be a potential specific staging biomarker for the TaG1 NMIBC; however, future validation experiments should be conducted once the precise version of the standard is commercially available. Remarkably, for MIBC we validated spermine as potential specific staging biomarker (AUC = 0.882). CONCLUSION: Ours is the first metabolomics study conducted in urine of a thoroughly characterized cohort comprising all stages of NMIBC, MIBC and healthy controls in which we identified non-invasive diagnostic and staging biomarkers. These may improve BC management, thus reducing the use of current harmful diagnostic techniques.

Comparison of Three Workstations For Abdominal Aortic Aneurysm Sizing: Impact in Decision Making and Graft Selection.

PURPOSE: The aim of this study was to assess the agreement of multiplanar reconstruction (MPR) and semiautomated central lumen line (CLL) analysis of abdominal aortic aneurysms (AAA), with 3 different software workstations (WS1, WS2, WS3) and 2 experienced practitioners as well as to analyze its eventual impact in graft selection. MATERIALS AND METHODS: Twenty computed tomography (CT) angiography data sets were randomly chosen from a series of 100 consecutive studies. Measurements were performed twice by each reader, in random order, and included 8 parameters (5 diameters and 3 lengths). Each observer performed a complete set of 60 studies. Intra-observer and interobserver variability for every WS was assessed. Measurements were evaluated using Bland-Altman analysis, correlation coefficients (r), and concordance correlation coefficients (CCC [95% confidence interval (CI)]). RESULTS: A high overall agreement between repeated measurements for both observers was obtained (r=0.989; CCC=0.988 [0.982-0.992] and r=0.998; CCC=0.996 [0.994-0.997], for observers 1 and 2, respectively). However, reproducibility for individual parameters was excellent for observer 2 and only moderate for observer 1. A high overall agreement was obtained for interobserver concordance (r=0.987; CCC=0.986 [0.982-0.989]). When analyzing for individual parameters, greatest interobserver differences were found at CLL measurement of the diameter of aortic neck (WS2) and bifurcation (WS1 and WS2) as well as iliac diameter in all 3 WS for both CLL and MPR. Similar differences were observed in paired comparison between WS when involving these parameters. Careful inspection of Bland-Altman charts revealed some cases of disagreement between WS and observers that would affect decision making on graft selection, changing the neck diameter to a different size, in 2 cases when measuring with WS1, and iliac diameter in 4 cases (2 of them with WS1 and 2 with WS2). Greatest discordance was observed regarding ipsilateral iliac length affecting 7 measurements that would lead to change the length of the selected limb graft (2 with WS1, 3 with WS2, and 2 with WS3). CONCLUSIONS: Although a high agreement between different observers using different WS for AAA measurements is to be expected, small differences may lead to the selection of a different graft size. The use of a single software by experienced users, and double check by a different one, may be advisable. CLINICAL IMPACT: Influence of inter and intraobserver variability in CT measurements during planning of endovascular aneurysm repair (EVAR) has been extensively reviewed. However, its impact in graft selection (final choose of diameter and lengths) has been scarcely analyzed. The results of this study suggest that, although a high agreement between different observers using different workstations for AAA measurements is to be expected, small differences may lead to the selection of a different graft size. The use of a single software by experienced users, and double check by a different one, may be advisable.

Intraplaque calcium and its relation with the progression of carotid atheromatous disease.

BACKGROUND: Calcification and progression of atheromatous disease (AD) both have been independently related with the risk of stroke. However, the link between the two phenomena is still unclear. The main objective of this study was to analyze the temporal evolution of Ca content of carotid atheromatous plaques and its relation with the progression of carotid AD using quantitative CT Angiography (CTA). METHODS: Forty-three asymptomatic patients with stenosis of the internal carotid artery (ICA)>50% completed the study. Contrast mold volume and calcium (Ca) content by quantitative CTA and Modified Agatston Score (Ca volume × radiological density) were assessed at baseline and after 12±2 months. Biochemical parameters, including main markers of Ca/Phosphorus (P) metabolism, were determined. RESULTS: CTA measurement showed an increase of volumetric stenosis (volume decrease of the contrast mold), compared to baseline (475.45 [155.6] mm3 × U.H vs. 501.3 [171.9] mm3 × U.H; P=0.04) as well as an increase of intraplaque Ca (64.58 [57.8] mm3 × U.H. vs. 56.8 [52.3] P=0.002). An inverse correlation between baseline Ca content and volumetric stenosis progression (r=-0.481; P<0.001), as well as between the increase of carotid Ca and plasma levels of vitamin D (r=0.4; P=0.025) were also found. Multiple regression analysis found a model with baseline intraplaque Ca, adjusted by Body Mass Index (BMI) as most predictive of carotid AD progression. CONCLUSIONS: These results suggest that a higher content of Ca confers greater stability against the progression of carotid AD and, eventually, its ability to generate symptomatology.

Thrombospondins in human aortic aneurysms.

Thrombospondins are a family of matricellular proteins with a multimeric structure that is known to be involved in several biological and pathological processes. Their relationship with vascular disorders has raised special interest recently. Aortic aneurysms are related to the impairment of vascular remodeling, in which extracellular matrix proteins seem to play an important role. Thus, research in thrombospondins, and their potential role in aneurysm development is progressively gaining importance. Nevertheless, studies showing thrombospondin dysregulation in human samples are still scarce. Although studies performed in vitro and in vivo models are essential to understand the molecular mechanisms and pathways underlying the disorder, descriptive studies in human samples are also necessary to ascertain their real value as biomarkers and/or novel therapeutic targets. The present article reviews the latest findings regarding the role of thrombospondins in aortic aneurysm development, paying particular attention to the studies performed in human samples.

Identification of miR-20a-5p as Robust Normalizer for Urine microRNA Studies in Renal Cell Carcinoma and a Profile of Dysregulated microRNAs.

Renal cell carcinoma (RCC) is the third most frequent urinary malignancy and one of the most lethal. Current diagnostic and follow-up techniques are harmful and unspecific in low-grade tumors. Novel minimally invasive markers such as urine microRNAs (miRNAs) are under study. However, discrepancies arise among studies in part due to lack of consent regarding normalization. We aimed to identify the best miRNA normalizer for RCC studies performed in urine samples together with a miRNA profile with diagnostic value and another for follow-up. We evaluated the performance of 120 candidate miRNAs in the urine of 16 RCC patients and 16 healthy controls by RT-qPCR followed by a stability analysis with RefFinder. In this screening stage, miR-20a-5p arose as the most stably expressed miRNA in RCC and controls, with a good expression level. Its stability was validated in an independent cohort of 51 RCC patients and 32 controls. Using miR-20a-5p as normalizer, we adjusted and validated a diagnostic model for RCC with three miRNAs (miR-200a-3p, miR-34a-5p and miR-365a-3p) (AUC = 0.65; Confidence Interval 95% [0.51, 0.79], p = 0.043). let-7d-5p and miR-205-5p were also upregulated in patients compared to controls. Comparing RCC samples before surgery and fourteen weeks after, we identified let-7d-5p, miR-152-3p, miR-30c-5p, miR-362-3p and miR-30e-3p as potential follow-up profile for RCC. We identified validated targets of most miRNAs in the renal cell carcinoma pathway. This is the first study that identifies a robust normalizer for urine RCC miRNA studies, miR-20a-5p, which may allow the comparison of future studies among laboratories. Once confirmed in a larger independent cohort, the miRNAs profiles identified may improve the non-invasive diagnosis and follow-up of RCC.

Circulating Cell-Free DNA in Liquid Biopsies as Potential Biomarker for Bladder Cancer: A Systematic Review.

Bladder cancer (BC) is among the most frequent cancer types in the world and is the most lethal urological malignancy. Presently, diagnostic and follow-up methods for BC are expensive and invasive. Thus, the identification of novel predictive biomarkers for diagnosis, progression, and prognosis of BC is of paramount importance. To date, several studies have evidenced that cell-free DNA (cfDNA) found in liquid biopsies such as blood and urine may play a role in the particular scenario of urologic tumors, and its analysis may improve BC diagnosis report about cancer progression or even evaluate the effectiveness of a specific treatment or anticipate whether a treatment would be useful for a specific patient depending on the tumor characteristics. In the present review, we have summarized the up-to-date studies evaluating the value of cfDNA as potential diagnostic, prognostic, or monitoring biomarker for BC in several biofluids.

Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer.

Bladder cancer (BC) is among the most frequent malignancies worldwide, being the most expensive cancer to treat and monitor and the most lethal urological cancer. Urine microRNAs (miRNAs) have been proposed as novel non-invasive biomarkers to early diagnose and monitor BC patients in order to avoid the performance of current aggressive diagnostic techniques. However, huge discrepancies arise among studies mainly due to the lack of standardization in the normalization, a crucial step in all miRNA studies. Our aim was to identify the best miRNA normalizer for miRNA studies in urine of BC patients. We evaluated the performance of 110 candidate miRNAs in urine of 35 BC patients and 15 healthy controls by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) followed by a stability analysis with RefFinder. In this screening stage, miR-29c-3p arose as the most stably expressed miRNA in BC and controls, with a good expression level. Stability of miR-29c-3p expression was validated in an independent cohort of 153 BC patients and 57 controls. Finally, we evaluated the robustness of miR-29c-3p as normalizer in the expression study of miR-200c-3p, a potential diagnostic marker for BC. We propose miR-29c-3p as a normalizer for miRNA studies in BC urine. This is the first study that characterizes a reliable normalizer that may allow the comparison of future urine miRNA studies as non-invasive biomarkers for BC diagnosis and monitoring.

Increase of Neutrophil Activation Markers in Venous Thrombosis-Contribution of Circulating Activated Protein C.

Upon activation, neutrophils release their content through different mechanisms like degranulation and NETosis, thus prompting thrombosis. The natural anticoagulant activated protein C (APC) inhibits neutrophil NETosis and, consequently, this may lower the levels of neutrophil activation markers in plasma, further diminishing the thrombotic risk exerted by this anticoagulant. We aimed to describe the status of markers of neutrophil activation in plasma of patients with venous thrombosis, their association with the thrombotic risk and the potential contribution of APC. We quantified three markers of neutrophil activation (cell-free DNA, calprotectin, and myeloperoxidase) in 253 patients with venous thromboembolism (VTE) in a stable phase (192 lower extremity VTE and 61 splanchnic vein thrombosis) and in 249 healthy controls. In them, we also quantified plasma APC, soluble endothelial protein C receptor (EPCR), and soluble thrombomodulin (TM), and we genotyped two genetic regulators of APC: the EPCR gene (PROCR) haplotypes (H) and the TM gene (THBD) c.1418C>T polymorphism. We found a significant increase in plasma cell-free DNA (p < 0.0001), calprotectin (p = 0.0001) and myeloperoxidase (p = 0.005) in VTE patients compared to controls. Furthermore, all three neutrophil activation markers were associated with an increase in the thrombotic risk. Cell-free DNA and calprotectin plasma levels were significantly correlated (Spearman r = 0.28; p < 0.0001). As expected, the natural anticoagulant APC was significantly decreased in VTE patients (p < 0.0001) compared to controls, what was mediated by its genetic regulators PROCR-H1, PROCR-H3, and THBD-c.1418T, and inversely correlated with cell-free DNA levels. This is the largest case-control study that demonstrates the increase in markers of neutrophil activation in vivo in VTE patients and their association with an increased thrombotic risk. This increase could be mediated by low APC levels and its genetic regulators, which could also increase NETosis, further enhancing thrombosis and inflammation.

Novel contributions of neutrophils in the pathogenesis of abdominal aortic aneurysm, the role of neutrophil extracellular traps: A systematic review.

Abdominal aortic aneurysm (AAA) is a vascular disorder caused by a dilatation of the aortic diameter that can be potentially lethal in case of rupture. Molecular mechanisms underlying the development of AAA are complex and not completely understood. However, inflammation plays a pivotal role in AAA development. Infiltration of inflammatory cells, especially macrophages, has been widely observed in lesion areas. Nevertheless, neutrophils have been gaining importance in the context of AAA. The release of neutrophil extracellular traps (NETs), extracellular structures formed by DNA, histones, granular and cytoplasmic proteins, is a recently discovered mechanism of neutrophil activation that can be triggered by endogenous inflammatory stimulus. The number of studies about the role of NETs in several vascular diseases like thrombosis and atherosclerosis has increased in last decade. However, its role in AAA has been scarcely analysed. The aim of this review is to deepen in the latest advances concerning the potential role neutrophils and especially NETs in AAA development.

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients.

microRNAs (miRNAs) are small RNAs that regulate different biological processes. Our objective was to identify miRNAs dysregulated in plasma and tissue of patients with abdominal aortic aneurysm (AAA) and explore new potential targets involved in AAA. Fifty-seven subjects were recruited for a plasma study (30 AAA patients, 16 healthy volunteers and 11 patients with atherosclerosis). The expression level of 179 miRNAs was screened in plasma from a subset of samples, and dysregulated miRNAs were validated in the entire study population. Dysregulated miRNAs were also quantified in aortic tissue of 21 AAA patients and 8 organ donors. Applying a gene set enrichment analysis, an interaction map of dysregulated miRNAs and their targets was built, and selected targets were quantified in tissue samples. miR-27b-3p and miR-221-3p were overexpressed in plasma of AAA patients compared with healthy controls, 1.6 times and 1.9 times, respectively. In AAA tissue, six miRNAs (miR-1, miR-27b-3p, miR-29b-3p, miR-133a-3p, miR-133b, and miR-195-5p) were underexpressed from 1.6 to 4.8 times and four miRNAs (miR-146a-5p, miR-21-5p, miR-144-3p, and miR-103a-3p) were overexpressed from 1.3 to 7.2 times. Thrombospondin-2, a target of miR-195-5p, was increased in AAA tissue and negatively correlated with the expression of miR-195-5p, suggesting their involvement in a common regulatory mechanism.

microRNAs and Markers of Neutrophil Activation as Predictors of Early Incidental Post-Surgical Pulmonary Embolism in Patients with Intracranial Tumors.

Venous thromboembolism (VTE) is a common complication of cancer that severely increases morbidity and mortality. Patients with intracranial tumors are more likely to develop VTE than patients with cancers at other sites. Conversely, limited tools exist to identify patients with high thrombotic risk. Upon activation, neutrophils release their content through different mechanisms triggering thrombosis. We explored the ability of microRNAs (miRNAs) and plasma markers of neutrophil activation measured before surgery to predict the risk of early post-surgical pulmonary embolism (PE) in glioma and meningioma patients. We recruited and prospectively followed 50 patients with glioma and 50 with meningioma, 34% of whom in each group developed an early objectively-diagnosed post-surgical PE. We measured miRNA expression and neutrophil markers (cell-free DNA, nucleosomes, calprotectin and myeloperoxidase) before surgery. In glioma patients, we adjusted and validated a predictive model for post-surgical PE with 6 miRNAs: miR-363-3p, miR-93-3p, miR-22-5p, miR-451a, miR-222-3p and miR-140-3p (AUC = 0.78; 95% Confidence Interval (CI) [0.63, 0.94]) and another with cfDNA and myeloperoxidase as predictors (AUC = 0.71; 95%CI [0.52, 0.90]). Furthermore, we combined both types of markers and obtained a model with myeloperoxidase and miR-140-3p as predictors (AUC = 0.79; 95%CI [0.64, 0.94]). In meningioma patients we fitted and validated a predictive model with 6 miRNAs: miR-29a-3p, miR-660-5p, miR-331-3p, miR-126-5p, miR-23a-3p and miR-23b-3p (AUC = 0.69; 95%CI [0.52, 0.87]). All our models outperformed the Khorana score. This is the first study that analyzes the capability of plasma miRNAs and neutrophil activation markers to predict early post-surgical PE in glioma and meningioma patients. The estimation of the thrombotic risk before surgery may promote a tailored thromboprophylaxis in a selected group of high-risk patients, in order to minimize the incidence of PE and avoid bleedings.

Urinary microRNAs: Looking for a New Tool in Diagnosis, Prognosis, and Monitoring of Renal Cancer.

PURPOSE OF REVIEW: Renal cell carcinoma (RCC) is the third most common urologic malignancy. First symptoms are usually unspecific and belated causing the stages to be high when diagnosed. As compensation, incidental detection of RCC by abdominal imaging techniques for other medical purposes is a reality that favours a decrease in the stage of new diagnosed tumours. Therefore, identifying novel predictive biomarkers for diagnosis, progression and prognosis of RCC is fundamental. RECENT FINDINGS: To date, several studies have demonstrated that microRNAs (miRNAs) play a role in the particular scenario of urologic tumors, and alterations at miRNA level are involved in the initiation, progression and metastases formation of renal cancer. In the present review, we have summarized the up­to­date preliminary clinical works on the role of urinary miRNA profiling in RCC, including an evaluation of its value as a potential biomarker for diagnosis, prognosis and follow up of RCC patients.

Urine metabolomic analysis in clear cell and papillary renal cell carcinoma: A pilot study.

Renal cell carcinoma (RCC) is one of the most lethal type of tumors and is twice more frequent in men than in women. Initial symptoms are unspecific and belated thus increasing mortality. Moreover, current diagnostic and monitoring tools are harmful for the patient and unspecific in low-grade tumors. Therefore, novel minimally-invasive markers are needed to diagnose and monitor RCC patients. Urine represents the ideal sample source of non-invasive biomarkers for RCC. In our study we aimed to identify a urine metabolomic profile characteristic of RCC patients with diagnostic purposes and also to identify a profile with prognostic value. By an UPLC-Q-ToF MS untargeted metabolomic analysis, we compared the metabolomic profile of 23 RCC patients (14 clear cell RCC and 9 papillary RCC) before surgery and that of 23 healthy controls. Additionally, for the first time, we compared the metabolomic profile of these RCC patients pre-nephrectomy and 3 months and one year post-nephrectomy. We identified the dysregulated metabolomic variables by querying their exact mass against those presented in the Metlin and Human Metabolome Database. Next, we experimentally confirmed their identity. Both RCC subtypes showed similar metabolomic patterns at all stages. 51 metabolomic variables were significantly increased in RCC compared to controls and, among them, 4 were selected as potential discriminant metabolites between groups. We could experimentally confirm the identity of p-cresol glucuronide thus describing for the first time an increase in this metabolite in urine of RCC patients (fold change = 2.922, P = .012). Additionally, we confirmed that no metabolomic differences occur 3 months post-nephrectomy in RRC, while 188 variables were significantly increased one year post-nephrectomy. Of the 15 most discriminant metabolomic variables, we could experimentally confirm the identity of isobutyryl-l-carnitine (fold change = 2.098, P = .004) and l-proline betaine (fold change = 3.328, P = .004), for the first time. In summary, we have identified urine p-cresol glucuronide as potential diagnostic marker for RCC and isobutyryl-l-carnitine and l-proline betaine as potential prognostic markers. When confirmed in an independent cohort of RCC patients, these markers may improve the diagnosis and monitoring of RCC patients thus reducing current harmful diagnostic procedures. SIGNIFICANCE: The high-radiation dose of current imaging techniques available to diagnose and monitor renal cell carcinoma (RCC) are harmful for the patient and unspecific in low-grade tumors. Our untargeted metabolomic analysis carried out in urine samples from RCC patients and healthy individual reveals p-cresol glucuronide as potential diagnostic marker for RCC. Additionally, the analysis of RCC urine samples one year post-nephrectomy reveals isobutyryl-l-carnitine and l-proline betaine as potential prognostic markers. These novel non-invasive urine biomarkers may improve RCC management thus reducing the use of current harmful diagnostic techniques.

MicroRNAs and Neutrophil Activation Markers Predict Venous Thrombosis in Pancreatic Ductal Adenocarcinoma and Distal Extrahepatic Cholangiocarcinoma.

Cancer-associated venous thrombosis (VTE) increases mortality and morbidity. However, limited tools are available to identify high risk patients. Upon activation, neutrophils release their content through different mechanisms, thereby prompting thrombosis. We explored plasma microRNAs (miRNAs) and neutrophil activation markers to predict VTE in pancreatic ductal adenocarcinoma (PDAC) and distal extrahepatic cholangiocarcinoma (DECC). Twenty-six PDAC and 6 DECC patients recruited at cancer diagnosis, were examined for deep vein thrombosis and pulmonary embolisms, and were then followed-up with clinical examinations, blood collections, and biCUS. Ten patients developed VTE and were compared with 22 age- and sex-matched controls. miRNA expression levels were measured at diagnosis and right before VTE, and neutrophil activation markers (cell-free DNA, nucleosomes, calprotectin, and myeloperoxidase) were measured in every sample obtained during follow-up. We obtained a profile of 7 miRNAs able to estimate the risk of future VTE at diagnosis (AUC = 0.95; 95% Confidence Interval (CI) (0.987, 1)) with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. Seven miRNAs were up- or down-regulated before VTE compared with diagnosis. We obtained a predictive model of VTE with calprotectin as predictor (AUC = 0.77; 95% CI (0.57, 0.95)). This is the first study that addresses the ability of plasma miRNAs and neutrophil activation markers to predict VTE in PDAC and DECC.

Activated protein C assays: A review.

Activated protein C (APC) acts as an "on demand" anticoagulant, reducing thrombin formation. Reduced plasma levels of APC or protein C (PC) are associated with an increased risk of venous thromboembolism. APC also displays cytoprotective functions and its therapeutic use has been evaluated in severe sepsis and is under evaluation in several diseases with an important inflammatory component. In addition, different studies have revealed a potential role of PC/APC in disorders such as obesity, pneumonia, disseminated intravascular coagulation, Alzheimer, stroke, etc. Accordingly, the therapeutic value of different recombinant APC molecules that lack anticoagulant activity but retain the cytoprotective function is being tested in clinical trials for some of these diseases. Therefore, an available method to measure circulating APC in plasma is of great interests. About 16 different methods for the quantification of APC have been reported. Here, we will review the available assays, highlighting their advantages and disadvantages as well as their different stages of implementation and the most appropriate use for each method, including their potential clinical usefulness.

Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.

Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels.