EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Acute joint inflammation induces a sharp increase in the number of synovial fluid EVs and modifies their phospholipid profile.

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.

Sperm motility activation, sperm heterogeneity and sperm-female tract interactions in Bennett's wallaby (Macropus rufogriseus rufogriseus).

Sperm-oviduct interactions in Bennett's wallaby (Macropus rufogriseus rufogriseus) were investigated using in vitro cocultures of cauda epididymal spermatozoa and oviducal epithelial cells. Kidney epithelial cells were used as non-reproductive control tissues. Spermatozoa attached to epithelial cells of both origins, but sperm survival and activity was higher when cocultured with oviducal cells. New findings during live sperm-epithelial interactions included: (1) a high frequency of reversible head movements, from linear (streamlined configuration) to T shape (thumbtack configuration) in swimming spermatozoa immediately after the start of coculture; (2) the loss of sperm tails (tail shedding) increasing with time; and (3) interrupted swimming patterns, where periods of fast movement were interspersed with slower swimming while the spermatozoa interacted with the epithelial cell surface. Sperm motility activation responses were characterised after diluting the epididymal samples in phosphate-buffered saline, medium M199 and Tyrode's medium. The results confirmed that the marsupial oviduct is able to support the viability and motility of a sperm subpopulation for at least 20 h in vitro and suggest that some spermatozoa shed their tails after binding, possibly as a result of a selective process.

Cohort profile: The Applied Public-Private Research enabling OsteoArthritis Clinical Headway (IMI-APPROACH) study: a 2-year, European, cohort study to describe, validate and predict phenotypes of osteoarthritis using clinical, imaging and biochemical markers.

PURPOSE: The Applied Public-Private Research enabling OsteoArthritis Clinical Headway (APPROACH) consortium intends to prospectively describe in detail, preselected patients with knee osteoarthritis (OA), using conventional and novel clinical, imaging, and biochemical markers, to support OA drug development. PARTICIPANTS: APPROACH is a prospective cohort study including 297 patients with tibiofemoral OA, according to the American College of Rheumatology classification criteria. Patients were (pre)selected from existing cohorts using machine learning models, developed on data from the CHECK cohort, to display a high likelihood of radiographic joint space width (JSW) loss and/or knee pain progression. FINDINGS TO DATE: Selection appeared logistically feasible and baseline characteristics of the cohort demonstrated an OA population with more severe disease: age 66.5 (SD 7.1) vs 68.1 (7.7) years, min-JSW 2.5 (1.3) vs 2.1 (1.0) mm and Knee injury and Osteoarthritis Outcome Score pain 31.3 (19.7) vs 17.7 (14.6), except for age, all: p<0.001, for selected versus excluded patients, respectively. Based on the selection model, this cohort has a predicted higher chance of progression. FUTURE PLANS: Patients will visit the hospital again at 6, 12 and 24 months for physical examination, pain and general health questionnaires, collection of blood and urine, MRI scans, radiographs of knees and hands, CT scan of the knee, low radiation whole-body CT, HandScan, motion analysis and performance-based tests.After two years, data will show whether those patients with the highest probabilities for progression experienced disease progression as compared to those wit lower probabilities (model validation) and whether phenotypes/endotypes can be identified and predicted to facilitate targeted drug therapy. TRIAL REGISTRATION NUMBER: NCT03883568.

Extracellular Vesicles in Joint Disease and Therapy.

The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and their linking medium, the synovial fluid, in particular. Further insight is urgently needed in order to appreciate the full potential of EVs and to exploit these in EV-mediated therapies. Physiologic joint homeostasis is a prerequisite for proper functioning of joints and we postulate that EVs play a key role in the regulation of joint homeostasis and hence can have an important function in re-establishing disturbed joint homeostasis, and, in parallel, in the regeneration of articular tissues. In this mini-review EVs in the joint are explained from a historical perspective in both health and disease, including the potential niche for EVs in articular tissue regeneration. Furthermore, the translational potential of equine models for human joint biology is discussed. Finally, the use of MSC-derived EVs that is recently gaining ground is highlighted and recommendations are given for further EV research in this field.

Extracellular vesicles ­ new tool for joint repair and regeneration.

Cell-derived extracellular vesicles (EVs), present in synovial fluid and cartilage extracellular matrix (ECM), are involved in joint development and in the regulation of joint homeostasis. Although the exact function of EVs in these processes remains incompletely defined, the knowledge already acquired in this field suggests a role for these EVs as biomarkers of joint disease, and as a new tool to restore joint homeostasis and enhance articular tissue regeneration. In addition to direct injection of therapeutic EVs into the target site, surface coating of scaffolds and embedding of EVs in hydrogels might also lead to novel therapeutic possibilities. Based on the existing literature of EVs in synovial fluid and articular tissues, and investigation of the molecular factors (including microRNAs) active in joint homeostasis (or during its disturbance), we postulate novel perspectives for the implementation of EVs as a regenerative medicine approach in joint repair.

TrkB in the hippocampus and nucleus accumbens differentially modulates depression-like behavior in mice.

Brain-derived neurotrophic factor (BDNF) exerts antidepressant-like effects in the hippocampus and pro-depressant effects in the nucleus accumbens (NAc). It is thought that downstream signaling of the BDNF receptor TrkB mediates the effects of BDNF in these brain structures. Here, we evaluate how TrkB regulates affective behavior in the hippocampus and NAc. We overexpressed TrkB by electroporating a non-viral plasmid in the NAc or hippocampus in mice. Depression- and anxiety-like behaviors were evaluated in the sucrose test (anhedonia), the forced swim test (despair) and the elevated zero maze (anxiety). Targeted brain tissue was biochemically analyzed to identify molecular mechanisms responsible for the observed behavior. Overexpressing TrkB in the NAc increased the number of young neuronal cells and decreased despair and basal corticosterone levels. TrkB overexpression in the hippocampus increased astrocyte production and activation of the transcription factor CREB, yet without altering affective behavior. Our data suggest antidepressant effects of BDNF-TrkB in the NAc, which could not be explained by activation of the transcription factors CREB or ß-catenin. The effects TrkB has on depression-related behavior in different brain regions appear to critically depend on the targeted cell type.

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles.

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

Differential BDNF responses of triple versus dual reuptake inhibition in neuronal and astrocytoma cells as well as in rat hippocampus and prefrontal cortex.

Monoamine reuptake inhibitors increase brain-derived neurotrophic factor (BDNF) activity, and this growth factor is regarded as an interesting target for developing new antidepressant drugs. The aims of this study were to evaluate whether monoaminergic reuptake inhibition increases BDNF in vivo and in vitro as predicted by the neurotrophic hypothesis of depression, and whether triple reuptake inhibition has a superior BDNF response compared to dual reuptake inhibition. Twenty-one days of oral treatment (30 mg/kg) with the dual serotonin/noradrenaline reuptake inhibitor duloxetine or the triple serotonin/noradrenaline/dopamine reuptake inhibitor DOV 216,303 restored BDNF protein levels in the rat hippocampus, which were initially decreased due to injection stress. The prefrontal cortex contained increased BDNF levels only after DOV 216,303 treatment. In vitro, neither duloxetine nor DOV 216,303 altered intracellular BDNF levels in murine HT22 neuronal cells. In contrast, BDNF release was more effectively decreased following treatment with DOV 216,303 in these cells. In rat C62B astrocytomas, both antidepressants increased intracellular BDNF levels at their highest nontoxic concentration. C62B astrocytomas did not release BDNF, even after antidepressant treatment. Increased BDNF levels support the neurotrophic hypothesis of depression, but our findings do not clearly evidence that the BDNF response after triple reuptake inhibitors is more effective than after dual reuptake inhibitors. Moreover, the data suggest that the role of BDNF in neurons and astrocytes is complex and likely depends on factors including specificity of cell types in different brain regions, cell-cell interactions, and different mechanisms of action of antidepressants used.