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The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4–6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total within-laboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80–120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptors are essential pattern-recognition receptors of the innate immune system. They recognize a range of conserved molecules of invading microorganisms. The innate immune system is developed to protect the host, but can be deleterious if activated uncontrolled or inappropriate, such as in sepsis with Gram-negative bacteria. New approaches for treatment, like inhibition of innate immune responses, may be beneficial for the outcome of such conditions. Toll-like receptor 4 associated with CD14 and MD-2, is the lipopolysaccharide (LPS)-receptor and one of the candidates for such intervention. We investigated the newly described cyanobacterial LPS analogue CyP as a potential inhibitor of Escherichia coli (E. coli) LPS-induced inflammatory response in porcine whole blood. Pro-inflammatory cytokines and soluble terminal complement complex, sC5b-9, were used as read-outs. CyP, in contrast to E. coli LPS, did not induce cytokine production using doses up to 1μg/mL whole blood, indicating a lack of agonistic effect of CyP. In contrast, CyP was an efficient LPS antagonist, dose-dependently and completely inhibiting E. coli LPS-induced TNF-α, IL-1β and IL-8 production. CyP was a modest activator of porcine complement compared to LPS from other Gram-negative bacteria. When CyP was pre-incubated in porcine whole blood before adding whole E. coli bacteria, a modest, variable and non-significant inhibition of cytokines were seen, reaching an average inhibition of 44% for IL-1β. We have demonstrated for the first time that the cyanobacterial LPS analogue, CyP, is an efficient inhibitor of E. coli LPS-induced cytokines in whole blood and may be a candidate for therapeutic LPS-inhibition. [Copyright &y& Elsevier]

The activation of resting dendritic cells (DCs) is a crucial step in the initiation of adaptive immunity because it links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. It is well recognized that a wide variety of microbial products and endogenous signals can trigger DC activation, and that different DC subsets are specialized in inducing different classes of immune responses. In this review, we will focus on how different aspects of DC maturation are regulated not only by the nature of the DC maturation stimuli, but also by their duration, combination and timing, and provide an overview of how different modes of DC activation can affect T cell responses. [Copyright &y& Elsevier]

Objectives: To investigate and further validate if two novel cancer‐related glycoproteins, discovered by a genetic‐guided proteomics approach, can distinguish benign disease from prostate cancer (PCa) in men with enlarged prostates. Patients and Methods: A retrospective study was performed that included men with a total prostate‐specific antigen (PSA) concentration of 2.0–10 ng/mL, negative digital rectal examination and enlarged prostate (volume ≥35 mL). Serum samples were collected between 2011 and 2016 at a single centre from 474 men before they underwent prostate biopsy. Serum concentrations of thrombospondin 1 (THBS1) and cathepsin D (CTSD) glycoproteins were combined with the percentage of free PSA to total PSA ratio (%fPSA) to predict any or significant cancer at biopsy. Results: The multivariable logistic regression model including THBS1, CTSD and %fPSA discriminated among biopsy‐positive and biopsy‐negative patients in the validation set with an area under the curve (AUC) of 0.86 (P < 0.001, 95% confidence interval (CI) 0.82–0.91), while %fPSA alone showed an AUC of 0.64 (P < 0.001, 95% CI 0.57–0.71). At 90% sensitivity for PCa, the specificity of the model was 62%, while %fPSA had a specificity of 23%. For high grade (Gleason score ≥ 7 in prostatectomy specimen) PCa, the specificity was 48% at 90% sensitivity, with an AUC of 0.83, (P < 0.001, 95% CI 0.77 to 0.88). Limitations of the study include the retrospective set‐up and single‐centre cohort. Conclusions: A model combining two cancer‐related glycoproteins (THBS1 and CTSD) and %fPSA can improve PCa diagnosis and may reduce the number of unnecessary prostate biopsies because of its improved specificity for PCa when compared to %fPSA alone. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses. [ABSTRACT FROM AUTHOR]

Summary APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility. [ABSTRACT FROM AUTHOR]

The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency. [ABSTRACT FROM AUTHOR]

Abstract: If we understand the structural rules governing antibody (Ab)–antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition. To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution. As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus. [Copyright &y& Elsevier]