The Rostock International Parkinson's Disease (ROPAD) Study: Protocol and Initial Findings.

BACKGROUND: Genetic stratification of Parkinson's disease (PD) patients facilitates gene-tailored research studies and clinical trials. The objective of this study was to describe the design of and the initial data from the Rostock International Parkinson's Disease (ROPAD) study, an epidemiological observational study aiming to genetically characterize ~10,000 participants. METHODS: Recruitment criteria included (1) clinical diagnosis of PD, (2) relative of participant with a reportable LRRK2 variant, or (3) North African Berber or Ashkenazi Jew. DNA analysis involved up to 3 successive steps: (1) variant (LRRK2) and gene (GBA) screening, (2) panel sequencing of 68 PD-linked genes, and (3) genome sequencing. RESULTS: Initial data based on the first 1360 participants indicated that the ROPAD enrollment strategy revealed a genetic diagnostic yield of ~14% among a PD cohort from tertiary referral centers. CONCLUSIONS: The ROPAD screening protocol is feasible for high-throughput genetic characterization of PD participants and subsequent prioritization for gene-focused research efforts and clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Validation of a monoclonal antibody directed against the human sphingosine 1-phosphate receptor type 1.

The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.

Rapid Large-Scale COVID-19 Testing During Shortages.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in economic and social lockdowns in most countries all over the globe. Early identification of infected individuals is regarded as one of the most important prerequisites for fighting the pandemic and for returning to a 'New Normal'. Large-scale testing is therefore crucial, but is facing several challenges including shortage of sample collection tools and of molecular biological reagents, and the need for safe electronic communication of medical reports. We present the successful establishment of a holistic SARS-CoV-2 testing platform that covers proband registration, sample collection and shipment, sample testing, and report issuing. The RT-PCR-based virus detection, being central to the platform, was extensively validated: sensitivity and specificity were defined as 96.8% and 100%, respectively; intra-run and inter-run precision were <3%. A novel type of sample swab and an in-house-developed RNA extraction system were shown to perform as good as commercially available products. The resulting flexibility guarantees independence from the current bottlenecks in SARS-CoV-2 testing. Based on our technology, we offered testing at local, national, and global levels. In the present study, we report the results from approx. 18,000 SARS-CoV-2 tests in almost 10,000 individuals from a low-frequency SARS-CoV-2 pandemic area in a homogenous geographical region in north-eastern Germany for a period of 10 weeks (21 March to 31 May 2020). Among the probands, five SARS-CoV-2 positive cases were identified. Comparative analysis of corresponding virus genomes revealed a diverse origin from three of the five currently recognized SARS-CoV-2 phylogenetic clades. Our study exemplifies how preventive SARS-CoV-2 testing can be set up in a rapid and flexible manner. The application of our test has enabled a safe maintenance/resume of critical local infrastructure, e.g., nursing homes where more than 5000 elderlies and caretakers got tested. The strategy outlined by the present study may serve as a blueprint for the implementation of large-scale preventive SARS-CoV-2 testing elsewhere.

Inflammatory Conditions Disrupt Constitutive Endothelial Cell Barrier Stabilization by Alleviating Autonomous Secretion of Sphingosine 1-Phosphate.

The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.