A novel multi-ancestry proteome-wide Mendelian randomization study implicates extracellular proteins, tubular cells, and fibroblasts in estimated glomerular filtration rate regulation.

Estimated glomerular filtration rate (eGFR) impacts the concentration of plasma biomarkers confounding biomarker association studies of eGFR with reverse causation. To identify biomarkers causally associated with eGFR, we performed a proteome-wide Mendelian randomization study. Genetic variants nearby biomarker coding genes were tested for association with plasma concentration of 1,161 biomarkers in a multi-ancestry sample of 12,066 participants from the Prospective Urban and Rural Epidemiological (PURE) study. Using two-sample Mendelian randomization, individual variants' effects on biomarker concentration were correlated with their effects on eGFR and kidney traits from published genome-wide association studies (GWAS). Genetically altered concentrations of 22 biomarkers were associated with eGFR above a Bonferroni-corrected significance threshold. Five biomarkers were previously identified by GWAS (UMOD, FGF5, LGALS7, NINJ1, COL18A1). Nine biomarkers were within 1 Mb of the lead GWAS variant but the gene for the biomarker was unidentified as the candidate for the GWAS signal (INHBC, TNFRSF11A, TCN2, PXN1, PRTN3, PSMD9, TFPI, ITGB6, CA3). Single-cell transcriptomic data indicated the 22 biomarkers are expressed in kidney tubules, collecting duct, fibroblasts, and immune cells. Pathway analysis showed significant enrichment of identified biomarkers in the extracellular kidney parenchyma. Thus, using genetic regulators of biomarker concentration via proteome-wide Mendelian randomization, we identified 22 biomarkers that appear to causally impact eGFR in either a beneficial or adverse manner. The current study provides rationale for novel therapeutic targets for eGFR and emphasized a role for extracellular proteins produced by tubular cells and fibroblasts for impacting eGFR.

Analyzing the capability of PSP, PCT and sCD25 to support the diagnosis of infection in cancer patients with febrile neutropenia.

Background Early diagnosis of infection is essential for the initial management of cancer patients with chemotherapy-associated febrile neutropenia (FN). In this study, we have evaluated two emerging infection biomarkers, pancreatic stone protein (PSP) and soluble receptor of interleukin 2, known as soluble cluster of differentiation 25 (sCD25), for the detection of an infectious cause in FN, in comparison with other commonly used infection biomarkers, such as procalcitonin (PCT). Methods A total of 105 cancer patients presenting to the emergency department were prospectively enrolled. We observed 114 episodes of chemotherapy-associated FN. At presentation, a blood sample was collected for the measurement of PCT, PSP and sCD25. In order to evaluate the discriminatory ability of these markers for the diagnosis of infection, the area under the curve (AUC) of the receiver operating characteristic curves was calculated. Results Infection was documented in 59 FN episodes. PCT, PSP and sCD25 levels were significantly higher in infected patients. PCT was the biomarker with the highest diagnostic accuracy for infection (AUC: 0.901), whereas PSP and sCD25 showed a similar performance, with AUCs of 0.751 and 0.730, respectively. In a multivariable analysis, PCT and sCD25 were shown to be independently associated with infection. Conclusions Two novel biomarkers, PSP and sCD25, correlated with infection in cancer patients with chemotherapy-associated FN, but neither PSP nor sCD25 improved the performance of PCT. Based on the results obtained, the introduction of these novel biomarkers as a tool for the diagnosis of infection in this patient group is not recommended.

Pancreatic stone protein and soluble CD25 for infection and sepsis in an emergency department.

BACKGROUND: Infection is a common problem in emergency departments (EDs) and is associated with high mortality, morbidity and costs. Identifying infection in ED patients can be challenging. Biomarkers can facilitate its diagnosis, enabling an early management and improving outcomes. In the critical care setting, two emerging biomarkers, pancreatic stone protein (PSP) and soluble CD25 (sCD25), have demonstrated to be useful for diagnosis of sepsis. We aimed to assess the diagnostic value of these biomarkers, in comparison with procalcitonin (PCT), for infection and sepsis in an ED population with suspected infection. MATERIALS AND METHODS: Through a prospective, observational study, we investigated the utility of serum PCT, PSP and sCD25 levels, measured on admission, for diagnosis of infection and sepsis, defined according to the recently updated for sepsis (Sepsis-3), in patients presenting to the ED for suspected infection. Diagnostic accuracy was evaluated by using receiver operating characteristic curves (ROC) analysis. RESULTS: Of the 152 patients enrolled in this study, 129 had a final diagnosis of infection, including 82 with noncomplicated infection and 47 with sepsis. Median PCT, PSP and sCD25 levels were significantly higher in patients with infection and sepsis. The ROC curve analysis revealed a similar diagnostic accuracy for infection (ROC area under the curve (AUC) PCT: 0·904; sCD25: 0·869 and PSP: 0·839) and for sepsis (ROC AUC: PCT: 0·820; sCD25: 0·835 and PSP: 0·872). CONCLUSIONS: Pancreatic stone protein and sCD25 perform well as infection and sepsis biomarkers, with a similar performance than PCT, in ED patients with suspected infection. Further larger studies investigating use of PSP and sCD25 are needed.

Sepsis biomarkers in unselected patients on admission to intensive or high-dependency care.

INTRODUCTION: Although many sepsis biomarkers have shown promise in selected patient groups, only C-reactive protein and procalcitonin (PCT) have entered clinical practice. The aim of this study was to evaluate three promising novel sepsis biomarkers in unselected patients at admission to intensive care. We assessed the performance of pancreatic stone protein (PSP), soluble CD25 (sCD25) and heparin binding protein (HBP) in distinguishing patients with sepsis from those with a non-infective systemic inflammatory response and the ability of these markers to indicate severity of illness. METHODS: Plasma levels of the biomarkers, PCT and selected inflammatory cytokines were measured in samples taken from 219 patients during the first six hours of admission to intensive or high dependency care. Patients with a systemic inflammatory response were categorized as having sepsis or a non-infective aetiology, with or without markers of severity, using standard diagnostic criteria. RESULTS: Both PSP and sCD25 performed well as biomarkers of sepsis irrespective of severity of illness. For both markers the area under the receiver operating curve (AUC) was greater than 0.9; PSP 0.927 (0.887 to 0.968) and sCD25 0.902 (0.854 to 0.949). Procalcitonin and IL6 also performed well as markers of sepsis whilst in this intensive care unit (ICU) population, HBP did not: PCT 0.840 (0.778 to 0.901), IL6 0.805 (0.739 to 0.870) and HBP 0.607 (0.519 to 0.694). Levels of both PSP and PCT reflected severity of illness and both markers performed well in differentiating patients with severe sepsis from severely ill patients with a non-infective systemic inflammatory response: AUCs 0.955 (0.909 to 1) and 0.837 (0.732 to 0.941) respectively. Although levels of sCD25 did not correlate with severity, the addition of sCD25 to either PCT or PSP in a multivariate model improved the diagnostic accuracy of either marker alone. CONCLUSIONS: PSP and sCD25 perform well as sepsis biomarkers in patients with suspected sepsis at the time of admission to intensive or high dependency care. These markers warrant further assessment of their prognostic value. Whereas previously published data indicate HBP has clinical utility in the emergency department, it did not perform well in an intensive-care population.

Prognostic performance of pancreatic stone protein in critically ill patients with sepsis.

Aim: To assess the prognostic value for 28-day mortality of PSP in critically ill patients with sepsis. Material & methods: 122 consecutive patients with sepsis were enrolled in this study. Blood samples were collected on admission and day 2. Results: On admission, the combination of PSP and lactate achieved an area under the receiver operating characteristic (AUC-ROC) of 0.796, similar to sequential organ failure assessment score alone (AUC-ROC: 0.826). On day 2, PSP was the biomarker with the highest performance (AUC-ROC: 0.844), although lower (p = 0.041) than sequential organ failure assessment score (AUC-ROC: 0.923). Conclusion: The combination of PSP and lactate and PSP alone, on day 2, have a good performance for prognosis of 28-day mortality and could help to identify patients who may benefit most from tailored intensive care unit management.

Analytical performance of a single epitope B-type natriuretic peptide sandwich immunoassay on the Minicare platform for point-of-care diagnostics.

Point-of-care B-type natriuretic peptide (BNP) testing with adequate analytical performance has the potential to improve patient flow and provide primary care givers with easy-to-use advanced diagnostic tools in the management of heart failure. We present the analytical evaluation of the Minicare BNP immunoassay under development on the Minicare I-20 platform for point-of-care testing. Analytical performance was evaluated using EDTA venous whole blood, EDTA plasma and capillary whole blood. Method comparison with a lab-testing system was performed using samples from 187 patients. Normal values were determined based on 160 healthy adults, aging from 19 to 70 years. Limit of blank (LoB), limit of detection (LoD) were determined to be 3.3 ng/L, 5.8 ng/L. Limit of quantitation (LoQ) in whole blood at 20% and 10% coefficient of variation (CV) was found < 9 ng/L and <30 ng/L respectively without significant differences between EDTA whole blood and EDTA plasma. Total CV was found to be from 6.7% to 9.7% for BNP concentrations between 92.6 and 3984 ng/L. The sample type comparison study demonstrated correlation coefficients between 0.97 and 0.99 with slopes between 1.03 and 1.09 between the different samples. Method comparison between Minicare BNP and Siemens ADVIA Centaur BNP demonstrated a correlation coefficient of 0.92 with a slope of 1.06. The 97.5% URL of a healthy population was calculated to be 72.6 ng/L. The Minicare BNP assay is a robust, easy-to-use and sensitive test for rapid determination of BNP concentrations that can be used in a near-patient setting.

Association of ADAMDEC1 haplotype with high factor VIII levels in venous thromboembolism.

A suggestive locus on chromosome 8 could be shown to be associated with familial high factor VIII (FVIII) levels in venous thromboembolism. The ADAMDEC 1 gene is a candidate expressing an ectodomain sheddase. However, the ectodomain of the clearance receptor for FVIII, the low-density lipoprotein receptor-related protein (LRP), is subject to proteolysis by metalloproteases like ADAMDEC1. Other LRP-interacting proteins are lipoprotein lipase (LPL) and t-PA. For an association study, 165 thrombotic patients with high FVIII levels (from the MAISTHRO, i.e. Main-Isar-thrombosis register) were included. All patients with known causes for high FVIII levels had been previously excluded. The patients were compared with 214 healthy blood donors. Polymorphisms with usually a minor allele frequency >5%, i.e. 24 SNPs and two insertion/deletion polymorphisms of LPL gene, eight SNPs of the t-PA gene, and five SNPs of the ADAMDEC1 gene, were analyzed. Haplotype differences were calculated using PHASE. A new polymorphism in intron 7 of the t-PA gene with a minor allele frequency of 2.2% was identified. Analysis of each SNP by the Cochrane-Armitage trend test did not show any significant association between genotype and disease status. Interestingly, the ADAMDEC1 haplotype (rs12674766, rs10087305, rs2291577, rs2291578, rs3765124) differed between cases and controls (p = 0.04). In particular, the TGTGG haplotype showed a difference. In conclusion, the ADAMDEC 1 haplotype may indicate an underlying mechanism for high FVIII levels. The only moderate linkage disequilibrium may be due to a possible causal polymorphism in distant introns or the promoter region against a polygenic background.

Hidden population substructures in an apparently homogeneous population bias association studies.

Linkage- and association-based approaches have been applied to attempt to unravel the genetic predisposition for complex diseases. However, studies often report contradictory results even when similar population backgrounds are investigated. Unrecognized population substructures could possibly explain these inconsistencies. In an apparently homogeneous German sample of 612 patients with type 2 diabetic and end-stage diabetic nephropathy and 214 healthy controls, we tested for hidden population substructures and their possible effects on association. Using a genetic vector space analysis of genotypes of 20 microsatellite markers, we identified four distinct subsets of cases and controls. The significance of these substructures was demonstrated by subsequent association analyses, using three genetic markers (UCSNP-43,-19,-63; intron 3 of the calpain-10 gene). In the undivided sample, we found no association between individual SNPs or any haplogenotypes (ie the genotype combination of two multilocus haplotypes) and type 2 diabetes. In contrast, when analyzing the four groups separately, we found that there was evidence for association of the common C allele of UCSNP-63 with the trait in the largest group (n=547 cases/101 controls; P=0.002). In this subset haplotype 112 was more frequent in controls than in cases (P=0.006; haplogenotype 112/121: odds ratio (OR)=0.27, 95% confidence intervals (CI)=0.13-0.57), indicating a protective effect against the development of type 2 diabetes. Our study demonstrates that unconsidered population substructures (ethnicity-dependent factors) can severely bias association studies.

High factor VIII (FVIII) levels in venous thromboembolism: role of unbound FVIII.

Theoretically, von Willebrand factor (VWF) should be capable of binding all factor VIII (FVIII), but an unbound FVIII (uFVIII) plasma fraction remains. In patients' status post deep-vein thrombosis (DVT), an altered uFVIII fraction and high FVIII levels might be indicative of dysfunctional FVIII regulation. Out of 928 consecutive DVT patients, 321 were found to have high FVIII levels. After excluding 183 patients with known causes for high FVIII levels, plasma samples with unexplainably high FVIII levels were available from 84 patients. To capture the FVIII-VWF-complex, superparamagnetic polystyrene beads with covalently attached streptavidin were coated with biotinylated anti-rabbit Ig and incubated with rabbit anti-human VWF-Ig. Slowly thawed plasma samples were added to cooled beads, which were then separated by a magnetic particle concentrator. The uFVIII fraction was calculated by dividing the FVIII activity in the supernatant of the FVIII-VWF-complex-free sample by the FVIII activity in the supernatant of the control sample. Additionally, the VWF residuum in the supernatant was determined. Compared to age- and sex-matched blood donors, thrombosis patients showed a significantly higher plasma FVIII/VWF ratio (median: 1.3 vs. 1.0, p<0.001). uFVIII fraction data were adjusted for VWF residuum. After forward stepwise logistic regression, uFVIII had an odds ratio of 0.48 (95% CI 0.34-0.65), i.e. the uFVIII fraction was reduced in thrombosis patients. Analysis of covariance confirmed these results: In thrombosis patients, the estimated mean of the uFVIII fraction was significantly lower (6.34% vs. 7.58%, p<0.001). In conclusion, thrombosis patients with high FVIII levels showed a higher FVIII/VWF ratio, similar to mice with defective FVIII clearance. The clearly reduced uFVIII fraction lends further support to the hypothesis of a modified FVIII clearance.

Genetic aspects of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy from type 2 diabetes mellitus is the most common cause of end-stage renal disease (ESRD) in the United States and in Western Europe. Although patients with overt nephropathy generally experience greater cumulative glycemic exposure, the difference in glycemic control between patients developing nephropathy compared to those who did not, could not be demonstrated. This observation is consistent with the notion that perhaps other factors, in addition to glycemic control, have a bearing on the development of nephropathy. Genetic factors, which specifically increase the susceptibility to nephropathy in patients with diabetes, have been proposed. METHODS: A range of linkage and association studies has been performed for revealing the genetic background of diabetic nephropathy. RESULTS: Until now, no mutation has been identified which could explain the development of diabetic nephropathy in the majority of diabetic patients. CONCLUSIONS: Because of relatively small case numbers of all studies being performed so far, conclusions from those studies are limited. With the development of better technologies for an affordable genome-wide association study using thousands of markers, it might become possible to unravel the genetic causes of diabetic nephropathy.

Antibodies to platelet factor 4-heparin complex and outcome in hemodialysis patients with diabetes.

BACKGROUND AND OBJECTIVES: Hemodialysis patients with type 2 diabetes exhibit an excessive cardiovascular risk and regularly receive heparin. We tested whether antibodies to the platelet factor 4-heparin complex (PF4-H-AB) contribute to outcome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In 1255 hemodialysis patients with type 2 diabetes, the German Diabetes Dialysis Study evaluated the effect of atorvastatin (20 mg/d) versus placebo. In a post hoc analysis, the association among PF4-H-ABs, biochemistry, and prespecified, centrally adjudicated end points (combined cardiovascular end point [CVE], all-cause mortality, sudden death, myocardial infarction, stroke) was investigated. RESULTS: During 4 years, 460 patients reached the CVE; 605 died, 159 of sudden death. Myocardial infarction and stroke occurred in 199 and 97 patients, respectively. Positive PF4-H-AB status was found in 231 (18.7%) of 1236 tested patients and was associated with lower albumin, higher C-reactive protein, and arrhythmia. In a multivariate model adjusted for demographics, comorbidities, and biochemistry, PF4-H-ABs were associated with sudden death. No significant association between PF4-H-ABs and all-cause mortality, myocardial infarction, stroke, or the CVE was observed. Detecting an interaction between acetylsalicylic acid and PF4-H-ABs regarding sudden death and mortality, we found that the association between PF4-H-ABs and outcomes was restricted to patients with acetylsalicylic acid use, most likely because of indication bias. CONCLUSIONS: In hemodialysis patients who have type 2 diabetes and are treated with acetylsalicylic acid, PF4-H-ABs are associated with sudden and all-cause death. Further studies are needed to elucidate this association.

Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization.

We identified regulator of G-protein signaling-5 (RGS-5) as an angiogenic pericyte marker at sites of physiologic and pathologic angiogenesis. In a mouse model of pancreatic islet cell carcinogenesis, RGS-5 is specifically induced in the vasculature of premalignant lesions during the "angiogenic switch" and further elevated in tumor vessels. Similarly, RGS-5 is overexpressed in highly angiogenic astrocytomas but not in hypoxia-inducible factor-1alpha (HIF-1alpha)-deficient tumors, which grow along preexisting brain capillaries without inducing neovessels. Elevated levels of RGS-5 in pericytes are also observed during wound healing and ovulation indicating a strong correlation between RGS-5 expression and active vessel remodeling beyond tumor angiogenesis. Moreover, antitumor therapy, which reverses tumor vasculature to an almost normal morphology, results in down-regulation of RGS-5 transcription. Taken together, these data demonstrate for the first time a factor that is specific for "activated" pericytes. This further supports the notion that pericytes, like endothelial cells, undergo molecular changes during neovascularization that makes them a novel target for antiangiogenic therapy.

High factor VIII levels in venous thromboembolism show linkage to imprinted loci on chromosomes 5 and 11.

High factor VIII (FVIII) levels are known to be a risk factor for deep venous thrombosis, but the mechanisms responsible for high FVIII levels remain unclear. Here, a new phenotype "FVIII level residuum" (FVIII-R) was defined in order to eliminate the impact of common determinants on FVIII levels. We studied 13 families of patients with thrombosis and reproducibly high FVIII levels of unknown origin. Since familial clustering was evident, we looked for a possible genetic basis. A genome scan was performed with 402 evenly spaced microsatellite markers. A quantitative linkage analysis using variance component methods showed suggestive evidence for linkage of FVIII-R with a locus on chromosome 8 (logarithm of odds [LOD] = 2.1). In addition, we performed parametric exploratory linkage analysis of dichotomized FVIII-R, taking a parent-of-origin effect into account. Single-trait-locus MOD-score analysis showed suggestive evidence for linkage under an imprinting model at chromosomes 5 and 11. Furthermore, a 2-trait-locus analysis under a multiplicative model for the loci of chromosomes 5 and 11 yielded a remarkable LOD of 4.44. It confirmed the finding of paternal imprinting, obtained by single-trait-locus analysis, at both loci. Our results suggest that high FVIII levels in venous thromboembolism represent a complex trait caused by several genetic factors.

Diabetic nephropathy: an inherited disease or just a diabetic complication?

Type 2 diabetic nephropathy is the most common cause of end-stage renal disease in western Europe and the United States. Although patients with overt nephropathy generally experience greater cumulative glycemic exposure, the difference in glycemic control between patients developing nephropathy and to those who did not could not be demonstrated. This observation is consistent with the finding that factors other than glycemic control are involved in the development of nephropathy. Genetic factors which specifically increase the susceptibility to nephropathy in patients with diabetes have been proposed. A range of linkage, association, and gene expression studies have been performed for revealing the genetic background of diabetic nephropathy but were not yet successful in identifying mutations which could explain the development of diabetic nephropathy in the majority of diabetic patients. Because of relatively small case numbers of all studies being performed so far, conclusions from those studies are limited. With the development of better technologies for an affordable genomewide association study using thousands of markers, it might become possible to unravel the genetic susceptibility factors for diabetic nephropathy. Comparing the expression levels of thousands of genes in patients and controls may identify key players in the pathogenesis of diabetic nephropathy and targets for pharmacologic intervention in the future.

Cox-2 inhibition abrogates Chlamydia pneumoniae-induced PGE2 and MMP-1 expression.

Peripheral blood monocytes (PBMC) promote vascular inflammation and atherosclerosis. Chlamydia pneumoniae (Cp) infection of PBMC is found in atherosclerotic patients, appears refractory to antibiotics, and may predispose to vascular damage. In Cp-infected human PBMC we analyzed the role of cyclooxygenase-2 (Cox-2) for the proatherosclerotic key mediators prostaglandin E2 (PGE2) and interstitial collagenase (MMP-1). Cp infection resulted in rapid and sustained Cox-2 mRNA and protein stimulation depending on p38 and p44/42 MAPkinases. Subsequent upregulation of PGE synthase and MMP-1 was completely abrogated by the selective Cox-2 inhibitor NS398. Enhanced synthesis of PGE2 and MMP-1 in Cp infected PBMC is mediated through initiation of the p38 and p44/42 MAPK pathways and requires sustained Cox-2 activation. Selective Cox-2 inhibitors, currently under investigation for cardiovascular risk reduction, may represent a novel therapeutic option for patients with endovascular Cp infection as they target the actuated pathological signal transduction cascade in persistently infected PBMC.