Abstract Sepsis-associated acute kidney injury (SA-AKI) portends severe health burden due to significant morbidity and mortality, while early diagnosis remains challenging. In this study, proximity-dependent barcoding assay (PBA) was established to profile the surface proteome of single urinary extracellular vesicle (uEV). Principle uEV clusters with unique function and origination were profiled in SA-AKI. Reduction of complement receptor CD35 on single uEV (CD35-uEV) was revealed as a novel biomarker from one of the main EV clusters with significant proportional differences. CD35-uEV demonstrated high diagnostic accuracy for SA-AKI (receiver operating characteristic-area under the curve (ROC-AUC), 0.98 in screening cohort (n=16), and 0.89 in validation cohort (n=134)). Besides, CD35-uEV enables identification of subclinical AKI (ROC-AUC, 0.84 in prospective cohort (n=72)) which was independent of other clinical parameters as validated by multivariate analysis (p<0.001). Moreover, CD35-uEV correlated closely with AKI severity which also predicts persistent AKI (ROC-AUC, 0.77), progression to AKD (ROC-AUC, 0.66), and mortality risks (ROC-AUC, 0.70). Integrative single-cell and spatial transcriptomics analysis identified that CD35-uEV originated from injured podocyte characterized with diminished CD35 expression. The combination of CD35-uEV with tubular injury biomarkers (TIMP2*IGFBP7) showed improved accuracy in identifying subclinical SA-AKI and prediction of severe stages. Overall, this study identified a novel biomarker on single uEV related to injured podocyte for early diagnosis and risk stratification of SA-AKI.

Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells.
© 2014 John Wiley & Sons Ltd.

Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.
Copyright © 2013 S. Karger AG, Basel.