Infrared to terahertz identification of chemical substances used for the production of IEDs.

In our modern times, improvised explosive devices (IEDs) have become more sophisticated than ever, capable of causing destruction and loss of life. The creative use of homemade substances for IEDs manufactures has led to efforts in developing sensitive detection methods that can anticipate, identify and protect against improvised attacks. Laser-based spectroscopic techniques provide rapid and accurate detection of chemicals in improvised explosives, but no single method can detect all components of all explosives. In this study, two spectroscopic methods are used for the sensitive identification of 8 explosive chemical substances in the form of powders and vapors. Absorption spectra of benzene, toluene, acetone and ethylene glycol were examined with CO2 laser photoacoustic spectroscopy. The photoacoustic signals of the samples were recorded in the CO2 laser emission range from 9.2 to 10.8 µm and a different spectral behavior was observed for each analyzed substance. Time-domain spectroscopy with THz radiation was used to analyze ammonium nitrate, potassium chlorate, dinitrobenzene, hexamethylenetetramine transmission spectra in the 0.1-3 THz range, and it was observed that they have characteristic THz fingerprint spectra. CO2 laser photoacoustic spectroscopy and THz time domain spectroscopy have met the criterion of proven effectiveness in identifying explosive components. The combination of these spectroscopic methods is innovative, giving a promising new approach for detection of a large number of IED components.

Trophic dynamics of methylmercury and trace elements in a remote Amazonian Lake.

Information on pollutant trophodynamics can be crucial for public health, as contaminated food consumption may lead to deleterious effects. This study was performed in Puruzinho Lake, a remote body of water in the Brazilian Amazon from which a riparian human population obtains an important part of its animal protein intake. Samples from 92 individuals, comprising 13 species and four trophic guilds (iliophagous, planktivorous, omnivorous, and piscivorous fish) were analysed for the determination of trace elements (Fe, Cr, Mn, Ni, Zn, Ca, Sr, Cd, Sn, Tl and Pb) and methylmercury concentrations. Samples from the same individuals had already been analysed for stable isotope (SI) measurements (δ13C and δ15N) in a previous investigation and the SI data have been statistically treated with those generated in this study for the evaluation of trophic dynamics of contaminants. Methylmercury was the only analyte that biomagnified, presenting TMF values of 4.65 and 4.55 for total and resident ichthyofauna, respectively. Trace elements presented either trophic dilution or independence from the trophic position, constituting a behaviour that was coherent with that found in the scientific literature. The similarity between Ni behaviour through the trophic web to that of essential elements contributes to the discussion on the essentiality of this metal to fish. Considering the Non-cancer Risk Assessment, the calculated Target Hazard Quotient (THQ) values were higher than 1.0 for all analysed individuals for methylmercury, as well as for only one individual for nickel. No other analyte rendered THQ values higher than 1.0.

Mercury methylation upon coastal sediment resuspension: a worst-case approach under dark conditions.

Mercury behavior upon resuspension of sediments from two impacted areas of Guanabara Bay was evaluated to assess worst-case methylmercury (MeHg) responses, under dark experimental conditions to prevent demethylation by photolysis. Study areas include the Rio de Janeiro Harbor (RJH) and the chlor-alkali plant-affected Meriti River (MR) estuary. Total mercury (THg) and MeHg concentrations were determined along 24-h experiments of sediment resuspension in the bay water in dark conditions. Fine-grained Meriti River (MR) estuary sediments had 8 times higher MeHg initial concentrations than sandy Rio de Janeiro Harbor (RJH) sediments (3.4 ± 0.29 vs. 0.41 ± 0.1 ng g-1, respectively). Though THg contents were uncorrelated with resuspension time, statistically significant correlations of MeHg (rs = 0.78) and %MeHg in relation to THg (rs = 0.86) with resuspension time were observed for RJH sediments, indicating net methylation only for this study site. These positive correlation trends correspond to a 2.8 times MeHg concentration increase (ΔMeHg = 0.75 ng g-1) and 4.4 times increase in %MeHg (Δ%MeHg = 1.0%), after 24 h of resuspension. This suggests that assessments of factors affecting the MeHg spatial-temporal variability and associated toxicity risks can be limited in some sites if concentration changes due to sediment resuspension-redeposition processes are not considered. Therefore, the inclusion of MeHg evaluation before and after sediment resuspension events is recommendable for the improvement of dredging licensing and monitoring activities.

Immune Response After Rabies Vaccination in Owned Free-Roaming Domestic Dogs in Flores Island, Indonesia.

Vaccination is the main tool to prevent the circulation of rabies in dog populations. The development of an immune response after vaccination differs between individual dogs and depends on many factors such as dog characteristics, management, or genetics. Here, we first investigated the level of, and associated factors for, the presence of binding antibodies in 130 healthy dogs from Flores Island, Indonesia. Secondly, we identified factors associated with the development of binding antibodies within 30 days after vaccination among a subsample of dogs that had a binding antibody titre <0.5 EU/ml at the day of vaccination (D0, N = 91). Blood samples were collected from the individual dogs immediately before vaccination at D0 and 30 days after vaccination (D30). The rabies antibody titres were determined using ELISAs. Information on potential risk factors such as the dog's age and sex, history of vaccination, type and frequency of feeding, and BCS (body condition score) were gathered during interviews at D0. Regression analyses were performed to identify the risk factors associated with the presence of binding antibody titre ≥0.5 EU/ml at D0 for the 130 dogs and the development of binding antibody titre ≥0.5EU/ml at D30 for the 91 dogs. The results showed that the proportion of dogs with antibody titre ≥0.5 EU/ml was 30% (39/130) at D0. The only factors found to be significantly influencing the presence of binding antibodies titres ≥0.5 EU/ml was previous vaccination within 1 year before D0 [46.8 vs. 14.7%, Odds ratio (OR) = 3.6, 95%CI 1.5-9.3; p-value = 0.006], although the same trend was found for dogs of higher age and better BCS. Eighty-six percent (79/91) of dogs whose rabies binding antibody level was <0.5 EU/ml at D0 had developed an adequate immune response (≥0.5 EU/ml) at D30. Almost a significantly higher proportion developed an adequate immune response in dogs of good BCS compared to those of poor BCS (95.3% vs. 79.2%, OR = 4.7, 95%CI 1.1-32.5; p-value = 0.057. Twelve (13.2%) dogs retain binding antibody level <0.5 EU/ml at D30, indicating poor immune response after vaccination. A majority of them did not receive vaccine before D0 according to the owner and had poor BCS (83.3%; 10/12). Our findings show the high effectiveness of rabies vaccine in under field conditions to develop measurable immunity and the importance of a good BCS, often achievable by good dog keeping conditions, for developing efficient immunity after parenteral vaccination in dogs.

Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island, Indonesia.

Effective parenteral vaccines are available to control rabies in dogs. While such vaccines are successfully used worldwide, the period between vaccine boosters required to guarantee protection of the population against rabies varies between vaccines and populations. In Flores Island, Indonesia, internationally and locally produced rabies vaccines are used during annual vaccination campaigns of predominantly free-roaming owned domestic dogs. The study objective was to identify the duration of the presence and factors associated with the loss of adequate level of binding antibodies (≥0.5 EU/ml) following rabies vaccination in a domestic dog population on Flores Island. A total of 171 dogs that developed an antibody titre higher or equal to 0.5 EU/ml 30 days after vaccination (D30), were repeatedly sampled at day 90, 180, 270, and 360 after vaccination. On the day of vaccination (D0), an interview was performed with dog owners to collect information on dog characteristics (age, sex, body condition score (BCS)), history of rabies vaccination, kind of daily food, frequency of feeding, and origin of the dog. Serum samples were collected and the level of antibodies was quantitatively assessed using ELISA tests. Dogs were categorized as having an adequate level of binding antibodies (≥0.5 EU/ml) or inadequate level of binding antibodies (<0.5 EU/ml) at each time points examined. A total of 115, 72, 23, and 31 dogs were sampled at D90, D180, D270, and D360, respectively, with the highest proportion of antibodies ≥ 0.5 EU/ml (58%, 95% CI, 49-67%) at D90, which reduced gradually until D360 (35%, 95% CI, 19-52%). Multivariable logistic regression models showed that loss of adequate level of binding antibodies is significantly associated with dogs having no history of vaccination or vaccination applied more than 12 months before D0, being less than 12 months of age, and having a poor BCS. These results highlight the importance of BCS regarding the immune response duration and provide insights into frequency of vaccination campaigns required for the internationally available vaccine used on Flores Island. For dogs without vaccination history or vaccination being applied more than 12 months before D0, a booster is recommended within 3 months (a largest drop of antibodies was detected within the first 90 days) after the first vaccination to guarantee measurable protection of the population that lasts at least for one year.

Human Mast Cell Line HMC1 Expresses Functional Mas-Related G-Protein Coupled Receptor 2.

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and ß-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.

Behçet's Disease Under Microbiotic Surveillance? A Combined Analysis of Two Cohorts of Behçet's Disease Patients.

Background: In Behçet's disease (BD), an auto-inflammatory vasculitis, an unbalanced gut microbiota can contribute to pro-inflammatory reactions. In separate studies, distinct pro- and anti-inflammatory bacteria associated with BD have been identified. Methods: To establish disease-associated determinants, we performed gut microbiome profiling in BD patients from the Netherlands (n = 19) and Italy (n = 13), matched healthy controls (HC) from the Netherlands (n = 17) and Italy (n = 15) and oral microbiome profiling in Dutch BD patients (n = 18) and HC (n = 15) by 16S rRNA gene sequencing. In addition, we used fecal IgA-SEQ analysis to identify specific IgA coated bacterial taxa in Dutch BD patients (n = 13) and HC (n = 8). Results: In BD stool samples alpha-diversity was conserved, whereas beta-diversity analysis showed no clustering based on disease, but a significant segregation by country of origin. Yet, a significant decrease of unclassified Barnesiellaceae and Lachnospira genera was associated with BD patients compared to HC. Subdivided by country, the Italian cohort displays a significant decrease of unclassified Barnesiellaceae and Lachnospira genera, in the Dutch cohort this decrease is only a trend. Increased IgA-coating of Bifidobacterium spp., Dorea spp. and Ruminococcus bromii species was found in stool from BD patients. Moreover, oral Dutch BD microbiome displayed increased abundance of Spirochaetaceae and Dethiosulfovibrionaceae families. Conclusions: BD patients show decreased fecal abundance of Barnesiellaceae and Lachnospira and increased oral abundance of Spirochaetaceae and Dethiosulfovibrionaceae. In addition, increased fecal IgA coating of Bifidobacterium, Ruminococcus bromii and Dorea may reflect retention of anti-inflammatory species and neutralization of pathosymbionts in BD, respectively. Additional studies are warranted to relate intestinal microbes with the significance of ethnicity, diet, medication and response with distinct pro- and inflammatory pathways in BD patients.

Evaluation of the bioaccumulation kinetics of toxic metals in fish (A. brasiliensis) and its application on monitoring of coastal ecosystems.

This study proposes a pro-active approach for evaluations of methylmercury (MeHg), total mercury (THg), arsenic (As), cadmium (Cd) and lead (Pb) in situ bioaccumulation in fish (Atherinella brasiliensis) muscles, using specimens from the external sector of Guanabara Bay as a study case. This approach included an hierarchical sequence: analysis of the pollutants concentrations and their comparison to safety criteria; correlations between specimens concentrations vs length (as a proxy of exposure time); projections of concentrations in key lengths (sexual maturation, asymptotic, length limits for fishing and median of fish population) through polynomial regressions, dose-response analysis (Probit), decreasing curves and incorporation rates (using only three length intervals). The incorporation rates were ascending for MeHg and THg (continued bioaccumulation) and descending for As, Pb and Cd (possible biological dilution). The projections were satisfactory, evidencing their use for an improvement on the risks monitoring of fishing and fish consumption by humans in coastal environments.

Soluble levels of sCD40L and s4-1BB are associated with a poor prognosis in elderly patients with colorectal cancer.

BACKGROUND AND OBJECTIVES: The prognosis of colorectal cancer (CRC) has improved in the last decades, however, a lower overall survival persists in the elderly. The understanding of immunity changes in the elderly with CRC will allow the emergence of new treatments with higher response rates. 4-1BB and CD40L, an immune checkpoint stimulator, play an important role in T-cell responses and platelets. Our aim was to characterize the soluble levels of CD40L and 4-1BB in CRC elderly patients. METHODS: A cross-sectional study was performed in 41 patients with CRC and 35 healthy elderly controls. Patients with CRC were divided into three groups according to staging: 13 patients with advanced tumor restricted to the organ (stages II); 16 patients with lymph node metastasis (stage III); and 12 patients with distant metastasis (stage IV). RESULTS: There were higher levels of soluble s4-1BB and sCD40L in CRC elderly stage II patients when compared with healthy controls (P = .0009 and P < .0001, respectively), stage III patients (P = .008 and P < .0001, respectively) and stage IV patients (P = .007 and P < .0001, respectively). CONCLUSIONS: We concluded that sCD40L and s4-1BB molecules may be prognostic biomarkers, since the reduction in plasma levels of these molecules was associated with disease progression.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Discriminative Power of the HEART Score for Obstructive Coronary Artery Disease in Acute Chest Pain Patients Referred for CCTA.

OBJECTIVE: To investigate the ability of the HEART score to predict the presence of obstructive coronary artery disease (CAD) determined by coronary computed tomography angiography (CCTA) and its ability to predict the occurrence of major adverse cardiac events (MACE) in patients referred for CCTA after emergency department (ED) presentation. METHODS: From December 2011 to August 2014, 710 ED patients with chest pain who underwent CCTA within 30 days were included. The HEART score was retrospectively calculated and patients were followed for MACE, comprised of death, myocardial infarction, and revascularization. Association of CAD at CCTA in the different categories of the HEART score was analyzed using χ test. The performance of the HEART score in discriminating between those with and without obstructive CAD was evaluated by receiver operating characteristics. Kaplan-Meier analysis was used to assess MACE-free survival stratified by HEART-score categories. RESULTS: During median follow-up of 826 days (interquartile range: 563-1056), MACE occurred in 46 (6.5%) patients; 3 (0.4%) myocardial infarction, 8 (1.1%) death, and 36 (5.1%) revascularizations. A low HEART score was a significant predictor for MACE-free survival (P = 0.010). CCTA revealed obstructive CAD in 11.7% of patients, with no significant difference between patients with a low and intermediate/high HEART score, respectively 10.7% and 13.2% (P = 0.29). The ability of the HEART score to identify obstructive CAD was poor with an AUC of the receiver operating characteristics curve of 0.53. CONCLUSION: The HEART score does not adequately identify patients with obstructive CAD at CCTA. It does however predict occurrence of MACE in medium-term follow-up. Excluding patients from additional testing based solely on a low HEART score may lead to suboptimal patient management. CCTA had important implications on patient management and may be a more appropriate tool to further stratify risk in ED chest pain patients.

The 11q Terminal Deletion Disorder Jacobsen Syndrome is a Syndromic Primary Immunodeficiency.

BACKGROUND: Jacobsen syndrome (JS) is a rare contiguous gene syndrome caused by partial deletion of the long arm of chromosome 11. Clinical features include physical and mental growth retardation, facial dysmorphism, thrombocytopenia, impaired platelet function and pancytopenia. In case reports, recurrent infections and impaired immune cell function compatible with immunodeficiency were described. However, Jacobsen syndrome has not been recognized as an established syndromic primary immunodeficiency. GOAL: To evaluate the presence of immunodeficiency in a series of 6 patients with JS. METHODS: Medical history of 6 patients with JS was evaluated for recurrent infections. IgG, IgA, IgM and specific antibodies against S. pneumoniae were measured. Response to immunization with a polysaccharide vaccine (Pneumovax) was measured and B and T lymphocyte subset analyses were performed using flowcytometry. RESULTS: Five out of 6 patients suffered from recurrent infections. These patients had low IgG levels and impaired response to S. pneumoniae polysaccharide vaccination. Moreover, we also found a significant decrease in the absolute number of memory B cells, suggesting a defective germinal center function. In a number of patients, low numbers of T lymphocytes and NK cells were found. CONCLUSIONS: Most patients with JS suffer from combined immunodeficiency in the presence of recurrent infections. Therefore, we consider JS a syndromic primary immunodeficiency. Early detection of immunodeficiency may reduce the frequency and severity of infections. All JS patients should therefore undergo immunological evaluation. Future studies in a larger cohort of patients will more precisely define the pathophysiology of the immunodeficiency in JS.

Diagnostic and Therapeutic Usefulness of Coronary Computed Tomography Angiography in Out-Clinic Patients Referred for Chest Pain.

Coronary computed tomography angiography (CCTA) is widely used to exclude coronary artery disease (CAD) in patients with low-to-intermediate pretest probability (PTP) of obstructive CAD. The aim of our study was to investigate the reclassification by CCTA and the implications of CCTA results on management because limited studies exist on these subjects; 1,560 patients with chest pain without a history of CAD and with low or intermediate PTP of CAD referred for CCTA from the out-patient clinic were prospectively included. PTP was defined by the Duke Clinical Score as either low (<15%), low-intermediate (15% to 50%), or high-intermediate (50% to 85%). Distribution of CCTA results among the categories of PTP of CAD and the influence of CCTA results on management were analyzed. CCTA revealed obstructive CAD in 7%, 15%, and 23% of cases, in patients with low, low-intermediate, and high-intermediate PTP, respectively; 855 of 1,031 patients (83%) with intermediate PTP of CAD showed no obstructive CAD on CCTA and were consequently reclassified. Management changes after CCTA occurred in 689 patients (44%). In 633 patients (41%), medication was altered and 135 (9%) were referred for invasive coronary angiography. Treatment with statin was initiated in 442 (28%) and stopped in 71 patients (5%). Aspirin was initiated in 192 (12%) and stopped in 139 patients (9%). In conclusion, in a routine clinical cohort, CCTA resulted in reclassification in most patients. Furthermore, our study suggests that the Duke Clinical Score overestimates the probability of obstructive CAD compared with CCTA findings. Finally, CCTA results have implications on patient management, with medication changes in 41% of patients.

An investigation of classical swine fever virus seroprevalence and risk factors in pigs in East Nusa Tenggara, eastern Indonesia.

Classical swine fever virus (CSFV) is a highly infectious disease of pigs. It has had significant impacts on East Nusa Tenggara, eastern Indonesia since its introduction in 1997. In spite of its importance to this region, little is known about its seroprevalence and distribution, and pig-level and farmer-level factors that may have an impact on the serological status of an individual pig. To address this knowledge deficit, a cross-sectional seroprevalence survey was conducted in 2010 involving 2160 pigs and 805 farmers from four islands in the region. Farmer questionnaires and pig record forms were used to collect data about the farmers and pigs surveyed. Blood was collected from each pig to determine its CSFV serological status. Apparent and true prevalence were calculated for each island, district, subdistrict, and village surveyed. CSFV serological status was used as an outcome variable in mixed effects logistic regression analyses. Overall true CSFV seroprevalence was estimated at 17.5% (lower CI 16.0%; upper CI 19.5%). Seroprevalence estimates varied widely across the islands, districts, subdistricts, and villages. Manggarai Barat, a district on the western end of Flores Island, contained pigs that were positive for antibody to CSFV. This result was unexpected, as no clinical cases had been reported in this area. Older pigs and pigs that had been vaccinated for CSFV were more likely to test positive for antibody to CSFV. The final multivariable model accounted for a large amount of variation in the data, however much of this variation was explained by the random effects with less than 2% of the variation explained by pig age and pig CSFV vaccination status. In this study we documented the seroprevalence of CSFV across four islands in East Nusa Tenggara, eastern Indonesia. We also identified risk factors for the presence of antibody to CSFV. Further investigation is needed to understand why clinical CSFV has not been reported on the western end of Flores Island, and to identify additional risk factors that explain CSFV serological status to inform disease control strategies.

Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell lymphotropic virus-1. There is no accepted curative therapy for ATL. We have reported that certain ATL patients have increased Notch-1 signaling along with constitutive activation of the nuclear factor-κB pathway. Physical and functional interaction between these two pathways provides the rationale to combine the γ-secretase inhibitor compound E with the proteasome inhibitor bortezomib. Moreover, romidepsin, a histone deacetylase inhibitor, has demonstrated major antitumor action in leukemia/lymphoma. In this study, we investigated the therapeutic efficacy of the single agents and the combination of these agents in a murine model of human ATL, the MT-1 model. Single and double agents inhibited tumor growth as monitored by tumor size (P<0.05), and prolonged survival of leukemia-bearing mice (P<0.05) compared with the control group. The combination of three agents significantly enhanced the antitumor efficacy as assessed by tumor size, tumor markers in the serum (human soluble interleukin-2 receptor-α and ß2-microglobulin) and survival of the MT-1 tumor-bearing mice, compared with all other treatment groups (P<0.05). Improved therapeutic efficacy obtained by combining compound E, bortezomib and romidepsin supports a clinical trial of this combination in the treatment of ATL.

N-terminal pro B-type natriuretic peptide identifies patients with chest pain at high long-term cardiovascular risk.

BACKGROUND: Little is known about the long-term prognostic value of N-terminal pro B-type natriuretic peptide (NT-proBNP) and C-reactive protein (CRP) in low-risk patients with chest pain. METHODS: Between June 1997 and January 2000, a standard rule-out protocol was performed in patients presenting to the emergency department within 6 hours of onset of chest pain with a normal or nondiagnostic electrocardiogram (ECG) on admission at the Academic Medical Center Amsterdam, VU University Medical Center Amsterdam and Medical Center Alkmaar, The Netherlands. Patients with acute coronary syndrome were identified by troponin T, recurrent angina, and serial ECGs. CRP and NT-proBNP on admission were measured using standardized methods. RESULTS: A total of 524 patients were included (145 with acute coronary syndrome and 379 with rule-out acute coronary syndrome). Long-term follow-up was successfully carried out in 96% of the study population. Death occurred in 78 patients (15%), 43 (11%) in the rule-out acute coronary syndrome group and 35 (24%) in the acute coronary syndrome group (P<.001). In the rule-out acute coronary syndrome group, 21 patients (42%) died of a cardiovascular cause compared with 24 patients (69%) in the acute coronary syndrome group (P<.001). In multivariate Cox regression analysis, age more than 65 years, previous myocardial infarction, known chronic heart failure, a nondiagnostic ECG on admission, and elevated NT-proBNP levels (>87 pg/mL, as derived from the receiver operating characteristic curve) were independent predictors of long-term cardiovascular mortality in the rule-out acute coronary syndrome group. In the acute coronary syndrome group, these predictors were age more than 65 years, documented coronary artery disease, and elevated NT-proBNP levels. Elevated levels of CRP were an independent predictor for cardiovascular mortality in patients with rule-out acute coronary syndrome at 3-year follow-up only. In patients with rule-out acute coronary syndrome with normal CRP and NT-proBNP levels, the cardiovascular mortality incidence rate was 4.7 per 1000 person-years, compared with a death rate of 20 in patients with both biomarkers elevated, which was comparable to the 17.9 per 1000 person-years incidence rate in patients with acute coronary syndrome. CONCLUSION: A positive biomarker panel discriminates patients with rule-out acute coronary syndrome chest pain with a normal or nondiagnostic ECG who have a high risk for long-term cardiovascular mortality.