Mortality prediction in stable hemodialysis patients is refined by YKL-40, a 40-kDa glycoprotein associated with inflammation.

Chronic inflammation contributes to increased mortality in hemodialysis (HD) patients. YKL-40 is a novel marker of inflammation, tissue remodeling, and highly expressed in macrophages inside vascular lesions. Elevated levels of YKL-40 have been reported for HD patients but how it integrates into the proinflammatory mediator network as a predictor of mortality remains elusive. We studied serum YKL-40, Interleukin-6 (IL-6), high-sensitivity C-reactive protein, monocyte chemotactic protein-1 (MCP-1), and interferon-gamma induced protein-10 (IP-10) in 475 chronic hemodialysis patients. Patients were followed for mortality for a median of 37 [interquartile range: 25-49] months and checked for interrelation of the measured mediators. To plot cumulative incidence functions, patients were stratified into terciles per YKL-40, IL-6, MCP-1, and IP-10 levels. Multivariable Cox regression models were built to examine associations of YKL-40, IP-10, and MCP-1 with all-cause and cause-specific mortality. Net reclassification improvement was calculated for the final models containing YKL-40 and IL-6. Increased YKL-40 was independently associated with age, IP-10, and IL-6 serum levels. After adjustment for demographic and laboratory parameters, comorbidities, and IL-6, only YKL-40 significantly improved risk prediction for all-cause (hazard ratio 1.4; 95% confidence interval 1.1-1.8) and cardiovascular mortality (hazard ratio 1.5; 95% confidence interval 1.03-2.2). Thus, in contrast to other biomarkers of aberrant macrophage activation, YKL-40 reflects inflammatory activity, which is not covered by IL-6. Mechanistic and prospective studies are needed to test for causal involvement of YKL-40 and whether it might qualify as a therapeutic target.

Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs.

Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.

Magnetic bead fluorescent immunoassay for the rapid detection of the novel inflammation marker YKL40 at the point-of-care.

Pneumonia is one of the leading causes of death worldwide.We present a magnetic bead fluorescent sandwich immunoassay that allows rapid serum measurement of the novel inflammation marker YKL40 (CHI3L1) at the point of care (POC) that could aid pneumonia diagnosis. The magnetic beads serve as the solid phase for separation of YKL40 from serum. The readout is performed using a small and robust fluorescence reader,which detects the turnover of a fluorescent substrate. The assay procedure, from sample addition to data retrieval, consists of three steps and is performed in less than 20 min. The presented assay has a linear range from 3 to 111 ng/mL, with a limit of detection of 2.9 ng/mL. The average recoveries were found between 101 and 111%. The developed method was applied in sera from healthy subjects (n= 14; c(YKL40)= 50 ± 49 ng/mL) and from pneumonia patients (n = 14; c(YKL40) = 333.6 ± 225 ng/mL). The elevated YKL40 concentrations in pneumonia-diseased patients are in good agreement with previously published data. The POC-ready device provides a simple immunoassay that could help to optimize pneumonia inflammation diagnostics in low-resource settings.

Biomimetic biosensor to distinguish between inhibitory and non-inhibitory factor VIII antibodies.

Patients with hereditary or acquired haemophilia A may develop inhibitory factor VIII (FVIII) antibodies. These disrupt FVIII activity predominantly by preventing the formation of the tenase complex, leading to a serious bleeding disorder. Antibodies without inhibiting activity, however, can also be found when screening patients with haemophilia A under FVIII supplementation. Therefore, the detection of only these allo- or autoantibodies from plasma is not sufficient. Rather, the characterization of the antibody-induced effects on the coagulation cascade should be considered due to its great diagnostic importance. Currently, inhibitory activities are detected by the functional Bethesda assay, which directly measures the delay in clotting time by the patient plasma. However, this assay does not provide information on the cause of the inhibition. Here, we report the development of a surface plasmon resonance (SPR) biosensor that has the potential to integrate both quantitative and functional information on patient antibody characteristics in one measurement. Recombinant FVIII protein was immobilized on the sensor surface to detect antibodies from patient plasma. The interaction of the FIX- and FXa-clotting proteins with the formed anti-FVIII/FVIII complex could be detected subsequently within the same SPR measurement cycle. Inhibitory antibodies led to the prevention of these interactions. Thus, discrimination between the clinically relevant inhibitory and non-inhibitory antibodies was enabled. In a group of 16 patients with inhibitory antibodies (both ELISA- and Bethesda-positive), 5 patients with non-inhibitory antibodies (ELISA-positive but Bethesda-negative) and 12 healthy controls, diagnostic sensitivity and specificity data of 100% for the FIX interaction were achieved using this biomimetic biosensor approach. The new method allows for detection and quantification, as well as for evaluation of inhibitory activity of allo- and autoantibodies, using small sample volume and short analysis time.