Performance of a Novel Research-Use-Only Secretoneurin ELISA in Patients with Suspected Acute Coronary Syndrome: Comparison with an Established Secretoneurin Radioimmunoassay.

BACKGROUND: Circulating secretoneurin (SN) concentrations, as measured by established radioimmunoassay (RIA), risk stratify patients with cardiovascular disease. We now report data for a recently developed research-use-only SN enzyme-linked immunosorbent assay (ELISA) in patients with suspected acute coronary syndrome (ACS). METHODS: SN ELISA was developed according to industry standards and tested in 401 unselected chest pain patients. Blood samples were drawn <24 h from admission, and we adjudicated all hospitalizations as ACS or non-ACS. The mean follow-up was 6.2 years. RESULTS: SN ELISA with 2 monoclonal sheep anti-SN antibodies has a measuring range of 10-250 pmol/L and demonstrates excellent analytical precision and accuracy across the range of SN concentrations. SN measured by ELISA and RIA correlated in the chest pain patients: rho = 0.39, p < 0.001. SN concentrations were higher in ACS patients (n = 161 [40%]) than in non-ACS patients (n = 240) for both assays, with an area under the curve (AUC) of 0.66 (95% CI: 0.61-0.71) for ELISA and 0.59 (0.54-0.65) for RIA. SN concentrations were also higher in nonsurvivors (n = 65 [16%]) than survivors, with an AUC of 0.72 (0.65-0.79) for ELISA versus 0.64 (0.56-0.72) for RIA, p = 0.007, for difference between assays. Adjusting for age, sex, blood pressure, previous myocardial infarction, atrial fibrillation, and heart failure in multivariable analysis, SN concentrations as measured by ELISA, but not RIA, remained associated with mortality, with a hazard ratio of 1.71 (1.03-2.84), p = 0.038. CONCLUSIONS: The novel SN ELISA has excellent performance, higher AUC for diagnosis, and superior prognostic accuracy compared to the established RIA in chest pain patients.

Identification of novel loci affecting circulating chromogranins and related peptides.

Chromogranins are pro-hormone secretory proteins released from neuroendocrine cells, with effects on control of blood pressure. We conducted a genome-wide association study for plasma catestatin, the catecholamine release inhibitory peptide derived from chromogranin A (CHGA), and other CHGA- or chromogranin B (CHGB)-related peptides, in 545 US and 1252 Australian subjects. This identified loci on chromosomes 4q35 and 5q34 affecting catestatin concentration (P = 3.40 × 10-30 for rs4253311 and 1.85 × 10-19 for rs2731672, respectively). Genes in these regions include the proteolytic enzymes kallikrein (KLKB1) and Factor XII (F12). In chromaffin cells, CHGA and KLKB1 proteins co-localized in catecholamine storage granules. In vitro, kallikrein cleaved recombinant human CHGA to catestatin, verified by mass spectrometry. The peptide identified from this digestion (CHGA360-373) selectively inhibited nicotinic cholinergic stimulated catecholamine release from chromaffin cells. A proteolytic cascade involving kallikrein and Factor XII cleaves chromogranins to active compounds both in vivo and in vitro.

Circulating Secretoneurin Concentrations After Cardiac Surgery: Data From the FINNish Acute Kidney Injury Heart Study.

OBJECTIVES: Secretoneurin is associated with cardiomyocyte Ca handling and improves risk prediction in patients with acute myocardial dysfunction. Whether secretoneurin improves risk assessment on top of established cardiac biomarkers and European System for Cardiac Operative Risk Evaluation II in patients undergoing cardiac surgery is not known. DESIGN: Prospective, observational, single-center sub-study of a multicenter study. SETTING: Prospective observational study of survival in patients undergoing cardiac surgery. PATIENTS: A total of 619 patients undergoing cardiac surgery. INTERVENTIONS: Patients underwent either isolated coronary artery bypass graft surgery, single noncoronary artery bypass graft surgery, two procedures, or three or more procedures. Procedures other than coronary artery bypass graft were valve surgery, surgery on thoracic aorta, and other cardiac surgery. MEASUREMENTS AND MAIN RESULTS: We measured preoperative and postoperative secretoneurin concentrations and adjusted for European System for Cardiac Operative Risk Evaluation II, N-terminal pro-B-type natriuretic peptide, and cardiac troponin T concentrations in multivariate analyses. During 961 days of follow-up, 59 patients died (9.5%). Secretoneurin concentrations were higher among nonsurvivors compared with survivors, both before (168 pmol/L [quartile 1-3, 147-206 pmol/L] vs 160 pmol/L [131-193 pmol/L]; p = 0.039) and after cardiac surgery (173 pmol/L [129-217 pmol/L] vs 143 pmol/L [111-173 pmol/L]; p < 0.001). Secretoneurin concentrations decreased from preoperative to postoperative measurements in survivors, whereas we observed no significant decrease in secretoneurin concentrations among nonsurvivors. Secretoneurin concentrations were weakly correlated with established risk indices. Patients with the highest postoperative secretoneurin concentrations had worse outcome compared with patients with lower secretoneurin concentrations (p < 0.001 by the log-rank test) and postoperative secretoneurin concentrations were associated with time to death in multivariate Cox regression analysis: hazard ratio lnsecretoneurin 2.96 (95% CI, 1.46-5.99; p = 0.003). Adding postoperative secretoneurin concentrations to European System for Cardiac Operative Risk Evaluation II improved patient risk stratification, as assessed by the integrated discrimination index: 0.023 (95% CI, 0.0043-0.041; p = 0.016). CONCLUSIONS: Circulating postoperative secretoneurin concentrations provide incremental prognostic information to established risk indices in patients undergoing cardiac surgery.

Prognostic Value of Secretoneurin in Patients With Severe Sepsis and Septic Shock: Data From the Albumin Italian Outcome Sepsis Study.

OBJECTIVES: Secretoneurin directly influences cardiomyocyte calcium handling, and circulating secretoneurin levels seem to improve risk prediction in patients with myocardial dysfunction by integrating information on systemic stress, myocardial function, and renal function. Accordingly, in this study, we hypothesized that secretoneurin would improve risk prediction in patients with sepsis and especially in patients with septic shock as these patients are more hemodynamically unstable. DESIGN: Multicenter, interventional randomized clinical trial. SETTING: Multicenter, pragmatic, open-label, randomized, prospective clinical trial testing fluid administration with either 20% human albumin and crystalloids or crystalloid solutions alone in patients with severe sepsis or septic shock (The Albumin Italian Outcome Sepsis). PATIENTS OR SUBJECTS: In total, 540 patients with septic shock and 418 patients with severe sepsis. INTERVENTIONS: Either 20% human albumin and crystalloids or crystalloid solutions alone. MEASUREMENTS AND MAIN RESULTS: We measured secretoneurin on days 1, 2, and 7 after randomization and compared the prognostic value of secretoneurin for ICU and 90-day mortality with established risk indices and cardiac biomarkers in septic shock and severe sepsis. High secretoneurin levels on day 1 were associated with age and serum concentrations of lactate, bilirubin, creatinine, and N-terminal pro-B-type natriuretic peptide. Adjusting for established risk factors and cardiovascular biomarkers, secretoneurin levels on day 1 were associated with ICU (odds ratio, 2.27 [95% CI, 1.05-4.93]; p = 0.04) and 90-day mortality (2.04 [1.02-4.10]; p = 0.04) in patients with septic shock, but not severe sepsis without shock. Secretoneurin levels on day 2 were also associated with ICU (3.11 [1.34-7.20]; p = 0.008) and 90-day mortality (2.69 [1.26-5.78]; p = 0.01) in multivariate regression analyses and improved reclassification in patients with septic shock, as assessed by the net reclassification index. Randomized albumin administration did not influence the associations between secretoneurin and outcomes. CONCLUSIONS: Secretoneurin provides early and potent prognostic information in septic patients with cardiovascular instability.

Chromogranin A and neuron-specific enolase in neuroblastoma: Correlation to stage and prognostic factors.

Chromogranin A (CgA) and neuron specific enolase (NSE) are important markers in adult neuroendocrine tumors (NET). Neuroblastoma (NB) has certain neuroendocrine properties. The aim of this study was to correlate blood concentrations of CgA, chromogranin B (CgB), and NSE to prognostic factors and outcome in children with NB. Blood samples from 92 patients with NB, 12 patients with benign ganglioneuroma (GN), 21 patients with non-NB solid tumors, 10 patients with acute leukemias, and 69 healthy children, were analyzed. CgA concentrations were higher in neonates vs. children older than one month in the control group (p < 0.0001), and in neonates with NB vs. the control group (p < 0.01). CgA and NSE concentrations were higher in patients with stages 3 and 4 disease (p < 0.05 and p < 0.05), in patients having tumors with amplification of MYCN (p < 0.05 and p < 0.001), or chromosome 1 p deletion (p < 0.05 and p < 0.05). NSE correlated to the tumor size at diagnosis (p < 0.001) and to tumor related death (p < 0.01) in NB. CgA and NSE concentrations were elevated in patients with NB and especially in those with advanced disease. Both CgA and NSE correlated to genetic markers, while only NSE correlated to primary tumor size and outcome in NB. We found that CgA and NSE are clinically valuable tumor markers in NB and they merit prospective clinical evaluations as such.

Early invasive versus non-invasive treatment in patients with non-ST-elevation acute coronary syndrome (FRISC-II): 15 year follow-up of a prospective, randomised, multicentre study.

BACKGROUND: The FRISC-II trial was the first randomised trial to show a reduction in death or myocardial infarction with an early invasive versus a non-invasive treatment strategy in patients with non-ST-elevation acute coronary syndrome. Here we provide a remaining lifetime perspective on the effects on all cardiovascular events during 15 years' follow-up. METHODS: The FRISC-II prospective, randomised, multicentre trial was done at 58 Scandinavian centres in Sweden, Denmark, and Norway. Between June 17, 1996, and Aug 28, 1998, we randomly assigned (1:1) 2457 patients with non-ST-elevation acute coronary syndrome to an early invasive treatment strategy, aiming for revascularisation within 7 days, or a non-invasive strategy, with invasive procedures at recurrent symptoms or severe exercise-induced ischaemia. Plasma for biomarker analyses was obtained at randomisation. For long-term outcomes, we linked data with national health-care registers. The primary endpoint was a composite of death or myocardial infarction. Outcomes were compared as the average postponement of the next event, including recurrent events, calculated as the area between mean cumulative count-of-events curves. Analyses were done by intention to treat. FINDINGS: At a minimum of 15 years' follow-up on Dec 31, 2014, data for survival status and death were available for 2421 (99%) of the initially recruited 2457 patients, and for other events after 2 years for 2182 (89%) patients. During follow-up, the invasive strategy postponed death or next myocardial infarction by a mean of 549 days (95% CI 204-888; p=0·0020) compared with the non-invasive strategy. This effect was larger in non-smokers (mean gain 809 days, 95% CI 402-1175; pinteraction=0·0182), patients with elevated troponin T (778 days, 357-1165; pinteraction=0·0241), and patients with high concentrations of growth differentiation factor-15 (1356 days, 507-1650; pinteraction=0·0210). The difference was mainly driven by postponement of new myocardial infarction, whereas the early difference in mortality alone was not sustained over time. The invasive strategy led to a mean of 1128 days (95% CI 830-1366) postponement of death or next readmission to hospital for ischaemic heart disease, which was consistent in all subgroups (p<0·0001). INTERPRETATION: During 15 years of follow-up, an early invasive treatment strategy postponed the occurrence of death or next myocardial infarction by an average of 18 months, and the next readmission to hospital for ischaemic heart disease by 37 months, compared with a non-invasive strategy in patients with non-ST-elevation acute coronary syndrome. This remaining lifetime perspective supports that an early invasive treatment strategy should be the preferred option in most patients with non-ST-elevation acute coronary syndrome. FUNDING: Swedish Heart-Lung Foundation, Swedish Foundation for Strategic Research, and Uppsala Clinical Research Center.

Circulating chromogranin B levels in patients with acute respiratory failure: data from the FINNALI Study.

PURPOSE: Circulating chromogranin B (CgB) levels are increased in situations characterized by systemic and myocardial stress, but whether CgB provides prognostic information in patients with acute respiratory failure (ARF) is unknown. METHODS: We included 584 patients with ARF, defined as ventilatory support >6 h, and with blood samples available on Intensive Care Unit (ICU) admission and day 3 (n = 479). CgB levels were measured by radioimmunoassay and follow-up was 90 days. RESULTS: One-hundred-sixty-nine patients (29%) died during follow-up. Admission CgB levels separated non-survivors from survivors: median 1234 (Q1-3 989-1742) vs. 917 (753-1224) pmol/L, respectively, p < 0.001. CgB levels on ICU admission (logarithmically transformed) were associated with time to death after adjustment for established risk indices available on ICU admission, including N-terminal pro-B-type natriuretic levels: HR 2.62 (95%C.I. 1.82-3.77), p < 0.001. Admission CgB levels also improved prognostication on top of SOFA and SAPS II scores as assessed by Cox regression analyses and the category-free net reclassification index. The area under the curve (AUC) for admission CgB levels to separate survivors and non-survivors was 0.72 (95%CI 0.67-0.76), while the AUC on day 3 was 0.60 (0.54-0.66). CONCLUSIONS: CgB levels measured on ICU admission provided additional prognostic information to established risk indices in ARF patients.

Clinical implications of assay specific differences in f-calprotectin when monitoring inflammatory bowel disease activity over time.

OBJECTIVE: With several faecal calprotectin (FC) assays on the market, it has been difficult to define a uniform threshold for discriminating between remission and active disease in patients with inflammatory bowel disease (IBD). We aimed to compare the results of different FC-assays in IBD patients, followed over time. MATERIAL AND METHODS: IBD patients provided faecal samples and reported clinical activity every third month prospectively over a two year period. FC was measured with two ELISA - (Bühlmann and Immunodiagnostik) and one automated fluoroimmunoassay (Phadia). RESULTS: In total, 13 patients provided 91 faecal samples. The median (IQR) concentration of FC was higher at active disease than at remission for all assays: Bühlmann 845 (1061-226) µg/g versus 62 (224-39) µg/g, Phadia 369 (975-122) µg/g versus 11 (52-11) µg/g, and Immundiagnostik 135 (302-69) µg/g versus 8 (56-4) µg/g. The Bühlmann assay produced the largest absolute difference but the corresponding relative difference seemed to be more pronounced when analysed by the Phadia - (ratio of means 8.5; 95% CI 3.3-21.9) or the Immundiagnostik assay (ratio of means 7.4; 95% CI 3.1-17.6) than by the Bühlmann assay (ratio of means 5.3; 95% CI 2.7-10.6). Consequently, the specificity for discriminating active disease from remission varied between assays (34-75%) when the cut-off 50 µg/g was used, whereas the differences in sensitivity were less pronounced. CONCLUSIONS: Cross-comparisons revealed overall poor agreement between the assays as well as differences in the dynamics of FC. These findings suggest that standardisation of the method is needed to implement FC as a disease monitoring tool at large-scale.

Prognostic Value of Secretoneurin in Critically Ill Patients With Infections.

OBJECTIVES: Secretoneurin is produced in neuroendocrine cells, and the myocardium and circulating secretoneurin levels provide incremental prognostic information to established risk indices in cardiovascular disease. As myocardial dysfunction contributes to poor outcome in critically ill patients, we wanted to assess the prognostic value of secretoneurin in two cohorts of critically ill patients with infections. DESIGN: Two prospective, observational studies. SETTING: Twenty-four and twenty-five ICUs in Finland. PATIENTS: A total of 232 patients with severe sepsis (cohort #1) and 94 patients with infections and respiratory failure (cohort #2). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured secretoneurin levels by radioimmunoassay in samples obtained early after ICU admission and compared secretoneurin with other risk indices. In patients with severe sepsis, admission secretoneurin levels (logarithmically transformed) were associated with hospital mortality (odds ratio, 3.17 [95% CI, 1.12-9.00]; p = 0.030) and shock during the hospitalization (odds ratio, 2.17 [1.06-4.46]; p = 0.034) in analyses that adjusted for other risk factors available on ICU admission. Adding secretoneurin levels to age, which was also associated with hospital mortality in the multivariate model, improved the risk prediction as assessed by the category-free net reclassification index: 0.35 (95% CI, 0.06-0.64) (p = 0.02). In contrast, N-terminal pro-B-type natriuretic peptide levels were not associated with mortality in the multivariate model that included secretoneurin measurements, and N-terminal pro-B-type natriuretic peptide did not improve patient classification on top of age. Secretoneurin levels were also associated with hospital mortality after adjusting for other risk factors and improved patient classification in cohort #2. In both cohorts, the optimal cutoff for secretoneurin levels at ICU admission to predict hospital mortality was ≈ 175 pmol/L, and higher levels were associated with mortality also when adjusting for Simplified Acute Physiology Score II and Sequential Organ Failure Assessment scores. CONCLUSIONS: Secretoneurin levels provide incremental information to established risk indices for the prediction of mortality and shock in critically ill patients with severe infections.

Prognostic Value of Secretoneurin in Patients with Acute Respiratory Failure: Data from the FINNALI Study.

BACKGROUND: We examined whether secretoneurin (SN), a biomarker associated with cardiomyocyte Ca(2+) handling, provides prognostic information in patients with acute respiratory failure (ARF). METHODS: We included 490 patients with ARF, defined as ventilatory support >6 h, with blood samples available on admission to the intensive care unit (ICU). SN concentrations were measured by RIA. RESULTS: A total of 209 patients (43%) were hospitalized with cardiovascular (CV)-related ARF, and 90-day mortality rates were comparable between CV- and non-CV-related ARF (n = 281): 31% vs 24%, P = 0.11. Admission SN concentrations were higher in nonsurvivors than in survivors in both CV-related (median 148 [quartile 1-3, 117-203] vs 108 [87-143] pmol/L, P < 0.001) and non-CV-related ARF (139 [115-184] vs 113 [91-139] pmol/L, P < 0.001). In patients with CV-related ARF, SN concentrations on ICU admission were associated with 90-day mortality [odds ratio (OR) 1.97 (95% CI, 1.04-3.73, P = 0.04)] after adjusting for established risk indices, including N-terminal-pro-B-type natriuretic peptide (NT-proBNP) concentrations. SN also improved patient classification in CV-related ARF as assessed by the net reclassification index: 0.32 (95% CI, 0.04-0.59), P = 0.03. The area under the curve (AUC) of SN to predict mortality in patients with CV-related ARF was 0.72 (95% CI, 0.65-0.79), and the AUC of NT-proBNP was 0.64 (0.56-0.73). In contrast, SN concentrations on ICU admission did not provide incremental prognostic value to established risk indices in patients with non-CV-related ARF, and the AUC was 0.67 (0.60-0.75). CONCLUSIONS: SN concentrations measured on ICU admission provided incremental prognostic information to established risk indices in patients with CV-related ARF, but not in patients with non-CV-related ARF.

Maternal body burdens of PCDD/Fs and PBDEs are associated with maternal serum levels of thyroid hormones in early pregnancy: a cross-sectional study.

BACKGROUND: Thyroid hormones (THs) regulate many biological functions in the human body and are essential for normal brain development. Epidemiological studies have observed diverging associations between halogenated persistent organic pollutant (POP) exposure and concentrations of THs in pregnant women and their infants. We investigated whether background exposure to polybrominated diphenyl ethers (PBDEs) is related to TH status in a Swedish population of pregnant women and their infants. Furthermore, we examined associations between polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) and TH status in early pregnancy as an extension of an earlier study focusing on late pregnancy TH status. METHODS: Free thyroxine (T4), total triiodo-thyronine (T3) and thyroid stimulating hormone (TSH) were analysed in serum from first-time mothers (N = 220-281) in the first and third trimester, and in infants (N = 115-150) 3 weeks and 3 months after delivery. Antibodies to thyroid peroxidase (anti-TPO) (N = 260) were measured in maternal third trimester serum. Maternal body burdens of PCBs (N = 281) were estimated from serum lipid PCB concentrations in late pregnancy, and PCDD/F (N = 97) and PBDE (N = 186) body burdens were estimated from concentrations in mother's milk lipids 3 weeks after delivery. Linear regression models allowed for covariate adjustment of the associations between ln-transformed POP body burdens and concentrations of TH and anti-TPO. RESULTS: Maternal body burden of BDE-153 was inversely associated with first trimester total T3, otherwise no associations between PBDEs and first and second trimester THs were observed. No associations were found between maternal PBDE body burdens and infant THs. Maternal body burden of PCDD/Fs were inversely associated with first trimester total T3. No associations were observed between PCBs and first trimester THs. Third trimester anti-TPO was not associated with maternal PCBs, PCDD/Fs and PBDEs. CONCLUSIONS: Our results suggest that maternal PCDD/F and BDE-153 body burdens influence maternal TH status in early pregnancy, which is a critical period when maternal TH status influences fetal development.

Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

BACKGROUND: Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. METHODS: We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. RESULTS: A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. CONCLUSION: Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

Decreased plasma Chromogranin A361-372 (Catestatin) but not Chromogranin A17-38 (Vasostatin) in female dogs with bacterial uterine infection (pyometra).

BACKGROUND: Pyometra often induces systemic inflammatory response syndrome (SIRS) and early diagnosis is crucial for survival. Chromogranin A (CgA) is a neuroendocrine secretory protein that is co-released with catecholamines from the adrenal medulla and sympathetic nerve endings. A prognostic value of CgA has been found in humans that are critically ill or that have SIRS associated with infection. CgA has not yet been studied in dogs with bacterial infection. The aim of the study was to investigate CgA, measured by Chromogranin A361-372 (Catestatin; Cst) and Chromogranin A17-38 (Vasostatin; VS) in healthy dogs and in dogs with pyometra. RESULTS: Fifty dogs with pyometra, sampled prior to surgery and 64 healthy female dogs were included. In 19 pyometra cases, blood samples were also collected postoperatively. Concentrations of Cst and VS were measured in heparinised plasma and Cst also measured in EDTA plasma, by in-house radioimmunoassays. Student's t-test and Wilcoxon two-sample test was used to test for differences between dog groups. Pre- and postoperative samples in dogs with pyometra were analysed by paired t-test. Pearson correlation was used to investigate associations of laboratory variables and hospitalization. P < 0.05 was considered significant. Concentrations of Cst were decreased in pyometra dogs (mean ± SE, 1.01 ± 0.05 nmol/L) compared to healthy dogs (mean ± SE, 1.70 ± 0.03 nmol/L) (p ≤ 0.0001). VS concentrations did not differ significantly between dogs with pyometra (0.40 ± 0.04 nmol/L) and healthy dogs (0.42 ± 0.03 nmol/L). Mean ± SE pre- and postoperative concentration of Cst (1.0 ± 0.04 nmol/L and 0.9 ± 0.2 nmol/L) and VS (0.36 ± 0.04 nmol/L and 0.36 ± 0.04 nmol/L) in dogs with pyometra did not differ significantly. Neither Cst nor VS concentrations were associated with duration of hospitalization and were not significantly different in the four dogs with pyometra that had prolonged (≥3 d) postoperative hospitalization. CONCLUSION: Concentrations of Cst, but not VS, were decreased in pyometra. Cst and VS concentrations before and after ovariohysterectomy did not differ significantly and were not associated with duration of hospitalization. Further studies are warranted to evaluate a possible diagnostic or prognostic value for Cst and VS.

Bone metabolism in adolescent girls with eating disorders and weight loss: independent effects of weight change, insulin-like growth factor-1 and oestradiol.

Adolescents with eating disorders (ED) are at risk of developing osteoporosis if weight is not recovered. Previous investigations do not separate the effects of weight change per se from those of concomitant hormonal changes. In this investigation serum osteocalcin (OC), C-terminal telopeptide of collagen (CTX), insulin-like growth factor-1 (IGF-1) and oestradiol were measured at assessment of 498 girls with ED and during weight gain of 59 girls. At assessment, OC concentrations were associated independently with weight (change), IGF-1 and oestradiol. Low weight, a high rate of weight loss and the hormone concentrations were associated with low OC. Low weight and high rate of weight loss were associated with high CTX concentrations but there were no associations independent of weight (change) with the hormones. During weight recovery, OC and CTX were independently and positively associated with weight, weight gain, IGF-1 and oestradiol. Bone metabolism markers are related to weight change independently of IGF-1 and oestradiol during both weight loss and weight gain. During weight gain, when pubertal development and growth are resumed there is an additional independent positive association between the markers and IGF-1 and oestradiol. These relationships are strongest in premenarcheal girls.

Genetic variation at the delta-sarcoglycan (SGCD) locus elevates heritable sympathetic nerve activity in human twin pairs.

The Syrian Cardiomyopathic Hamster (BIO-14.6/53.58 strains) model of cardiac failure, resulting from naturally occurring deletion at the SGCD (delta-sarcoglycan) locus, displays widespread disturbances in catecholamine metabolism. Rare Mendelian myopathy disorders of human SGCD occur, although common naturally occurring SGCD genetic variation has not been evaluated for effects on human norepinephrine (NE) secretion. This study investigated the effect of SGCD genetic variation on control of NE secretion in healthy twin pairs. Genetic associations profiled SNPs across the SGCD locus. Trait heritability (h(2)) and genetic covariance (pleiotropy; shared h(2)) were evaluated. Sympathochromaffin exocytosis in vivo was probed in plasma by both catecholamines and Chromogranin B (CHGB). Plasma NE is substantially heritable (p = 3.19E-16, at 65.2 ± 5.0% of trait variance), sharing significant (p < 0.05) genetic determination with circulating and urinary catecholamines, CHGB, eGFR, and several cardio-metabolic traits. Participants with higher pNE showed significant (p < 0.05) differences in several traits, including increased BP and hypertension risk factors. Peak SGCD variant rs1835919 predicted elevated systemic vascular compliance, without changes in specifically myocardial traits. We used a chimeric-regulated secretory pathway photoprotein (CHGA-EAP) to evaluate the effect of SGCD on the exocytotic pathway in transfected PC12 cells; in transfected cells, expression of SGCD augmented CHGA trafficking into the exocytotic regulated secretory pathway. Thus, our investigation determined human NE secretion to be a highly heritable trait, influenced by common genetic variation within the SGCD locus. Circulating NE aggregates with BP and hypertension risk factors. In addition, coordinate NE and CHGB elevation by rs1835919 implicates exocytosis as the mechanism of release.

Autoimmunity and cystatin SA1 deficiency behind chronic mucocutaneous candidiasis in autoimmune polyendocrine syndrome type 1.

Patients with the monogenic disease autoimmune polyendocrine syndrome type I (APSI) develop autoimmunity against multiple endocrine organs and suffer from chronic mucocutaneous candidiasis (CMC), a paradoxical complication with an unknown mechanism. We report here that saliva from APSI patients with CMC is defective in inhibiting growth of Candida albicans in vitro and show reduced levels of a salivary protein identified as cystatin SA1. In contrast, APSI patients without CMC express salivary cystatin SA1 and can inhibit C. albicans to the same extent as healthy controls. We evaluated the anti-fungal activity of cystatin SA1 and found that synthesized full length cystatin SA1 efficiently inhibits growth of C. albicans in vitro. Moreover, APSI patients exhibit salivary IgA autoantibodies recognizing myosin-9, a protein expressed in the salivary glands, thus linking autoimmunity to cystatin SA1 deficiency and CMC. This data suggests an autoimmune mechanism behind CMC in APSI and provides rationale for evaluating cystatin SA1 in antifungal therapy.

Ghrelin and obestatin in human neuroendocrine tumors: expression and effect on obestatin levels after food intake.

BACKGROUND: Ghrelin and obestatin are derived from the same peptide hormone precursor and are mainly produced by the gastric mucosa. Ghrelin is involved in many biological processes, whereas the physiological function of obestatin needs further investigation. The aims of the present study were to establish the incidence of ghrelin- and obestatin-immunoreactive cells in a comprehensive panel of human neuroendocrine tumors (NETs) and to investigate if blood obestatin concentrations are influenced during a standardized meal stimulation test in healthy individuals and patients with NETs. MATERIALS AND METHODS: The expression of ghrelin and obestatin was investigated in NETs (n = 149) and other endocrine-related disorders (n = 3) using immunohistochemistry with specific polyclonal antibodies. Coexpression of the peptides was evaluated by double immunofluorescence. Concentrations of obestatin in blood were measured during a meal test in 6 healthy individuals and 5 patients with pancreatic NETs. RESULTS: Ghrelin and obestatin were expressed in 14/152 and 19/152 tumor tissues, respectively, mainly representing NETs of foregut origin and in pancreatic tissue from a nesidioblastosis patient. Double immunofluorescence staining showed colocalization of the peptides. During the meal test, obestatin levels in blood were unchanged in all patients but decreased significantly in the healthy individuals. CONCLUSION: Only a minority of NETs express ghrelin and obestatin. However, analysis of patients with tumors originating from tissues that express the peptides in normal conditions could be of importance. The results from the meal test indicate that the hormone levels are affected by food intake in healthy individuals, whereas obestatin levels remained unchanged in pancreatic NET patients.

Decreased cerebrospinal fluid secretogranin II concentrations in severe forms of bipolar disorder.

BACKGROUND: Bipolar disorder is a common psychiatric mood disorder that is defined by recurrent episodes of abnormally elevated mood and depression. Progressive structural brain changes in individuals with bipolar disorder have been suggested to be associated with defects in the secretion of neurotrophic factors. We sought to assess how the regulated secretory pathway in the brain is affected in patients with bipolar disorder by measuring chromogranin B and secretogranin II, which are 2 cerebrospinal fluid (CSF) biological markers for this process. METHODS: We measured the concentrations of chromogranin B (peptide 439-451) and secretogranin II (peptide 154-165) in the CSF of patients with well-defined bipolar disorder and healthy controls. The lifetime severity of bipolar disorder was rated using the Clinical Global Impression (CGI) scale. RESULTS: We included 126 patients with bipolar disorder and 71 healthy controls in our analysis. Concentrations of secretogranin II were significantly lower in patients with bipolar disorder type I than in healthy controls. The reduction was most pronounced in patients with high CGI scores (i.e., severe disease). LIMITATIONS: The cross-sectional design of the current study limits the ability to pinpoint the causalities behind the observed associations. CONCLUSION: This study shows that the CSF marker secretogranin II has the potential to act as a biological marker for severe forms of bipolar disorder. Our findings indicate that patients with bipolar disorder possess defects in the regulatory secretory pathway, which may be of relevance to the progressive structural brain changes seen in those with severe forms of the disease.

Altered levels of fecal chromogranins and secretogranins in IBS: relevance for pathophysiology and symptoms?

OBJECTIVES: Chromogranins (Cg) and secretogranins (Sg) are proteins ubiquitous in secretory cells of the enteric, endocrine, and immune systems, and may reflect activity of these systems. We therefore performed a hypothesis generating study to evaluate the association between fecal levels of CgA, CgB, SgII, and SgIII, with the clinical and pathophysiological phenotype of irritable bowel syndrome (IBS) patients. METHODS: Analyses of CgA, CgB, SgII, SgIII, and calprotectin in fecal samples of 82 IBS patients and 29 healthy controls were performed. All IBS subjects completed validated questionnaires to assess gastrointestinal and psychological symptom severity, and underwent rectal barostat test and colonic transit time measurement. RESULTS: IBS patients demonstrated higher levels of fecal CgA (P=0.009), SgII (P<0.001), and SgIII (P<0.001), but lower levels of CgB (P<0.001) compared with controls. SgII had good discriminative validity to positively identify IBS patients, with an area under the receiver operating characteristics (ROC) curve (AUROC) of 0.86 (95% confidence interval (CI): 0.78-0.94). SgIII and CgB both had fairly good discriminative validity to positively identify IBS patients, with an AUROC of 0.79 (95% CI: 0.71-0.87) and 0.78 (95% CI: 0.69-0.87), respectively. There were negative correlations between the colonic transit time and fecal levels of CgA (r=-0.53, P<0.001), SgII (r=-0.55, P<0.001), and SgIII (r=-0.28, P=0.03). Perceived abdominal pain was moderately associated with levels of CgA (r=0.32, P=0.004), SgII (r=0.31, P=0.006), and SgIII (r=0.24, P=0.04). Calprotectin levels were not associated with the levels of granins or with the clinical or pathophysiological phenotype of IBS patients. CONCLUSIONS: Fecal levels of Cg and Sg may be related to the underlying pathophysiology of IBS and of potential importance for symptoms of the patients. Granins also show promise to serve as future biomarkers of IBS. Further studies are needed to explore the potential role of granins in IBS patients.