Cerebrospinal fluid extracellular vesicle enrichment for protein biomarker discovery in neurological disease; multiple sclerosis.

The discovery of disease biomarkers, along with the use of "liquid biopsies" as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential "biomarker treasure chests". However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory diseases.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Monocytes and γδ T cells control the acute-phase response to intravenous zoledronate: insights from a phase IV safety trial.

Aminobisphosphonates (NBPs) are used widely against excessive bone resorption in osteoporosis and Paget's disease as well as in metastatic bone disease and multiple myeloma. Intravenous NBP administration often causes mild to severe acute-phase responses (APRs) that may require intervention with analgesics and antipyretics and lead to treatment noncompliance and nonadherence. We here undertook a phase IV safety trial in patients with osteoporosis to investigate the APR of otherwise healthy individuals to first-time intravenous treatment with the NBP zoledronate. This study provides unique insight into sterile acute inflammatory responses in vivo, in the absence of confounding factors such as infection or cancer. Our data show that both peripheral γδ T cells and monocytes become rapidly activated after treatment with zoledronate, which ultimately determines the clinical severity of the APR. Our study highlights a key role for IFN-γ in the zoledronate-induced APR and identifies pretreatment levels of monocytes and central/memory Vγ9/Vδ2 T cells as well as their responsiveness to zoledronate in vitro as predictive risk factors for the occurrence of subclinical and clinical symptoms. These findings have diagnostic and prognostic implications for patients with and without malignancy and are relevant for Vγ9/Vδ2 T-cell-based immunotherapy approaches.

In vivo evaluation of defined polished titanium surfaces to prevent soft tissue adhesion.

Soft tissue-implant adhesion is often required for implant integration into the body; however, in some situations, the tissue is required to glide freely over an implant. In the case of distal radius fracture treatment, current literature describes how titanium and its alloys tend to lead to more intra-tendon inflammatory reactions compared with stainless steel. This leads to tendon-implant adhesion and damage possibly causing limited palmar flexion and even tendon rupture. The goal of this study was to analyze the effect of different surface polishings of titanium and titanium molybdenum implants on soft tissue reactions in vivo, with the aim to prevent direct soft tissue adhesion. Using a nonfracture model, to allow for study of the soft-tissue-implant surface interactions only, six surface variants of the same plate design were implanted onto the tibia of 24 New Zealand white rabbits and left in situ for 12 weeks. Results indicate that paste polished commercially pure titanium and titanium molybdenum alloy had the least soft tissue adhesion, with the concomitant development of a soft tissue capsule. Surface topography did not appear influence the thickness of the connective tissue surrounding the plate. Therefore, suitable surface polishing could be applied to plates for clinical use, where free gliding of tissues is required.

Proteomics analysis of bladder cancer exosomes.

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.