Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

The amount of hyaluronic acid and airway remodelling increase with the severity of inflammation in neutrophilic equine asthma.

BACKGROUND: Equine asthma (EA) is a chronic lower airway inflammation that leads to structural and functional changes. Hyaluronic acid (HA) has crucial functions in the extracellular matrix homeostasis and inflammatory mediator activity. HA concentration in the lungs increases in several human airway diseases. However, its associations with naturally occurring EA and airway remodelling have not been previously studied. Our aim was to investigate the association of equine neutrophilic airway inflammation (NAI) severity, airway remodelling, and HA concentration in horses with naturally occurring EA. We hypothesised that HA concentration and airway remodelling would increase with the severity of NAI. HA concentrations of bronchoalveolar lavage fluid supernatant (SUP) and plasma of 27 neutrophilic EA horses, and 28 control horses were measured. Additionally, remodelling and HA staining intensity were assessed from endobronchial biopsies from 10 moderate NAI horses, 5 severe NAI horses, and 15 control horses. RESULTS: The HA concentration in SUP was higher in EA horses compared to controls (p = 0.007). Plasma HA concentrations were not different between the groups. In the endobronchial biopsies, moderate NAI horses showed epithelial hyperplasia and inflammatory cell infiltrate, while severe NAI horses also showed fibrosis and desquamation of the epithelium. The degree of remodelling was higher in severe NAI compared to moderate NAI (p = 0.048) and controls (p = 0.016). Intense HA staining was observed in bronchial cell membranes, basement membranes, and connective tissue without significant differences between the groups. CONCLUSION: The release of HA to the airway lumen increases in naturally occurring neutrophilic EA without clear changes in its tissue distribution, and significant airway remodelling only develops in severe NAI.

Review of chemical models and applications in Geant4-DNA: Report from the ESA BioRad III Project.

A chemistry module has been implemented in Geant4-DNA since Geant4 version 10.1 to simulate the radiolysis of water after irradiation. It has been used in a number of applications, including the calculation of G-values and early DNA damage, allowing the comparison with experimental data. Since the first version, numerous modifications have been made to the module to improve the computational efficiency and extend the simulation to homogeneous kinetics in bulk solution. With these new developments, new applications have been proposed and released as Geant4 examples, showing how to use chemical processes and models. This work reviews the models implemented and application developments for modeling water radiolysis in Geant4-DNA as reported in the ESA BioRad III Project.

Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

OBJECTIVE: Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. DESIGN: We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. RESULTS: The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. CONCLUSIONS: Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

Osteological profiling of femoral diaphysis and neck in aquatic, semiaquatic, and terrestrial carnivores and rodents: effects of body size and locomotor habits.

The increased limb bone density documented previously for aquatic tetrapods has been proposed to be an adaptation to overcome buoyancy during swimming and diving. It can be achieved by increasing the amount of bone deposition or by reducing the amount of bone resorption, leading to cortical thickening, loss of medullary cavity, and compaction of trabecular bone. The present study examined the effects of locomotor habit, body size, and phylogeny on the densitometric, cross-sectional, and biomechanical traits of femoral diaphysis and neck in terrestrial, semiaquatic, and aquatic carnivores, and in terrestrial and semiaquatic rodents (12 species) by using peripheral quantitative computed tomography, three-point bending, and femoral neck loading tests. Groupwise differences were analyzed with the univariate generalized linear model and the multivariate linear discriminant analysis supplemented with hierarchical clustering. While none of the individual features could separate the lifestyles or species adequately, the combinations of multiple features produced very good or excellent classifications and clusterings. In the phocid seals, the aquatic niche allowed for lower femoral bone mineral densities than expected based on the body mass alone. The semiaquatic mammals mostly had high bone mineral densities compared to the terrestrial species, which could be considered an adaptation to overcome buoyancy during swimming and shallow diving. Generally, it seems that different osteological properties at the levels of mineral density and biomechanics could be compatible with the adaptation to aquatic, semiaquatic, or terrestrial niches.

Tetraspanin profiles of serum extracellular vesicles reflect functional limitations and pain perception in knee osteoarthritis.

BACKGROUND: Emerging evidence suggests that extracellular vesicles (EVs) can play roles in inflammatory processes and joint degradation in primary osteoarthritis (OA), a common age-associated joint disease. EV subpopulations express tetraspanins and platelet markers that may reflect OA pathogenesis. The present study investigated the associations between these EV surface markers and articular cartilage degradation, subjectively and objectively assessed pain, and functional limitations in primary knee OA (KOA). METHODS: Serum EVs were determined by high-sensitivity flow cytometry (large CD61+ EVs) and single particle interferometric reflectance imaging sensor (small CD41+, CD63+, CD81+, and CD9+ EVs) from end-stage KOA patients and controls (n = 8 per group). Knee pain and physical functions were assessed with several health- and pain-related questionnaires, established measurements of physical medicine, and neuromuscular examination. The obtained data were analyzed using supervised and unsupervised univariate and multivariate models. RESULTS: With the combined dataset of cartilage thickness, knee function, pain, sensation, and EV molecular signatures, we identified highly correlated groups of variables and found several EV markers that were statistically significant predictors of pain, physical limitations, and other aspects of well-being for KOA patients, for instance CD41+/CD63+/CD9+ small EVs associated with the range of motion of the knee, physical performance, and pain sensitivity. CONCLUSIONS: Particular serum EV subpopulations showed clear associations with KOA pain and functional limitations, suggesting that their implications in OA pathophysiology warrant further study.

Fatty acid fingerprints in bronchoalveolar lavage fluid and its extracellular vesicles reflect equine asthma severity.

Equine asthma (EA) is an inflammatory disease of the lower airways driven by mediators released from cells. Extracellular vesicles (EVs) are vehicles for lipid mediators, which possess either pro-inflammatory or dual anti-inflammatory and pro-resolving functions. In this study, we investigated how the respiratory fatty acid (FA) profile reflects airway inflammatory status. The FA composition of bronchoalveolar lavage fluid (BALF), BALF supernatant, and bronchoalveolar EVs of healthy horses (n = 15) and horses with mild/moderate EA (n = 10) or severe EA (SEA, n = 5) was determined with gas chromatography and mass spectrometry. The FA profiles distinguished samples with different diagnoses in all sample types, yet they were insufficient to predict the health status of uncategorized samples. Different individual FAs were responsible for the discrimination of the diagnoses in different sample types. Particularly, in the EVs of SEA horses the proportions of palmitic acid (16:0) decreased and those of eicosapentaenoic acid (20:5n-3) increased, and all sample types of asthmatic horses had elevated dihomo-γ-linolenic acid (20:3n-6) proportions. The results suggest simultaneous pro-inflammatory and resolving actions of FAs and a potential role for EVs as vehicles for lipid mediators in asthma pathogenesis. EV lipid manifestations of EA can offer translational targets to study asthma pathophysiology and treatment options.

Increased n-6 Polyunsaturated Fatty Acids Indicate Pro- and Anti-Inflammatory Lipid Modifications in Synovial Membranes with Rheumatoid Arthritis.

Emerging evidence suggests that fatty acids (FAs) and their lipid mediator derivatives can induce both beneficial and detrimental effects on inflammatory processes and joint degradation in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA). The present study characterized the detailed FA signatures of synovial membranes collected during knee replacement surgery of age- and gender-matched OA and RA patients (n = 8/diagnosis). The FA composition of total lipids was determined by gas chromatography and analyzed with univariate and multivariate methods supplemented with hierarchical clustering (HC), random forest (RF)-based classification of FA signatures, and FA metabolism pathway analysis. RA synovium lipids were characterized by reduced proportions of shorter-chain saturated FAs (SFAs) and elevated percentages of longer-chain SFAs and monounsaturated FAs, alkenyl chains, and C20 n-6 polyunsaturated FAs compared to OA synovium lipids. In HC, FAs and FA-derived variables clustered into distinct groups, which preserved the discriminatory power of the individual variables in predicting the RA and OA inflammatory states. In RF classification, SFAs and 20:3n-6 were among the most important FAs distinguishing RA and OA. Pathway analysis suggested that elongation reactions of particular long-chain FAs would have increased relevance in RA. The present study was able to determine the individual FAs, FA groups, and pathways that distinguished the more inflammatory RA from OA. The findings suggest modifications of FA elongation and metabolism of 20:4n-6, glycerophospholipids, sphingolipids, and plasmalogens in the chronically inflamed RA synovium. These FA alterations could have implications in lipid mediator synthesis and potential as novel diagnostic and therapeutic tools.

Space radiation quality factor for Galactic Cosmic Rays and typical space mission scenarios using a microdosimetric approach.

Space radiation exposure from omnipresent Galactic Cosmic Rays (GCRs) in interplanetary space poses a serious carcinogenic risk to astronauts due to the-limited or absent-protective effect of the Earth's magnetosphere and, in particular, the terrestrial atmosphere. The radiation risk is directly influenced by the quality of the radiation, i.e., its pattern of energy deposition at the micron/DNA scale. For stochastic biological effects, radiation quality is described by the quality factor, [Formula: see text], which can be defined as a function of Linear Energy Transfer (LET) or the microdosimetric lineal energy ([Formula: see text]). In the present work, the average [Formula: see text] of GCR for different mission scenarios was calculated using a modified version of the microdosimetric Theory of Dual Radiation Action (TDRA). NASA's OLTARIS platform was utilized to generate the radiation environment behind different aluminum shielding (0-30 g/cm2) for a typical mission scenario in low-earth orbit (LEO) and in deep space. The microdosimetric lineal energy spectra of ions ([Formula: see text]) in 1 µm liquid water spheres were calculated by a generalized analytical model which considers energy-loss fluctuations and δ-ray transport inside the irradiated medium. The present TDRA-based [Formula: see text]-values for the LEO and deep space missions were found to differ by up to 10% and 14% from the corresponding ICRP-based [Formula: see text]-values and up to 3% and 6% from NASA's [Formula: see text]-model. In addition, they were found to be in good agreement with the [Formula: see text]-values measured in the International Space Station (ISS) and by the Mars Science Laboratory (MSL) Radiation Assessment Detector (RAD) which represent, respectively, a LEO and deep space orbit.

Equine osteoarthritis modifies fatty acid signatures in synovial fluid and its extracellular vesicles.

BACKGROUND: Individual fatty acids (FAs) and their derivatives (lipid mediators) with pro-inflammatory or dual anti-inflammatory and pro-resolving properties have potential to influence the health of joint tissues. Osteoarthritis (OA) is an age-associated chronic joint disease that can be featured with altered FA composition in the synovial fluid (SF) of human patients. The counts and cargo of extracellular vesicles (EVs), membrane-bound particles released by synovial joint cells and transporting bioactive lipids, can also be modified by OA. The detailed FA signatures of SF and its EVs have remained unexplored in the horse - a well-recognized veterinary model for OA research. METHODS: The aim of the present study was to compare the FA profiles in equine SF and its ultracentrifuged EV fraction between control, contralateral, and OA metacarpophalangeal joints (n = 8/group). The FA profiles of total lipids were determined by gas chromatography and the data compared with univariate and multivariate analyses. RESULTS: The data revealed distinct FA profiles in SF and its EV-enriched pellet that were modified by naturally occurring equine OA. Regarding SFs, linoleic acid (generalized linear model, p = 0.0006), myristic acid (p = 0.003), palmitoleic acid (p < 0.0005), and n-3/n-6 polyunsaturated FA ratio (p < 0.0005) were among the important variables that separated OA from control samples. In EV-enriched pellets, saturated FAs palmitic acid (p = 0.020), stearic acid (p = 0.002), and behenic acid (p = 0.003) indicated OA. The observed FA modifications are potentially detrimental and could contribute to inflammatory processes and cartilage degradation in OA. CONCLUSIONS: Equine OA joints can be distinguished from normal joints based on their FA signatures in SF and its EV-enriched pellet. Clarifying the roles of SF and EV FA compositions in the pathogenesis of OA and their potential as joint disease biomarkers and therapeutic targets warrants future studies.

Dihomo-γ-Linolenic Acid (20:3n-6)-Metabolism, Derivatives, and Potential Significance in Chronic Inflammation.

Dihomo-γ-linolenic acid (DGLA) has emerged as a significant molecule differentiating healthy and inflamed tissues. Its position at a pivotal point of metabolic pathways leading to anti-inflammatory derivatives or via arachidonic acid (ARA) to pro-inflammatory lipid mediators makes this n-6 polyunsaturated fatty acid (PUFA) an intriguing research subject. The balance of ARA to DGLA is probably a critical factor affecting inflammatory processes in the body. The aim of this narrative review was to examine the potential roles of DGLA and related n-6 PUFAs in inflammatory conditions, such as obesity-associated disorders, rheumatoid arthritis, atopic dermatitis, asthma, cancers, and diseases of the gastrointestinal tract. DGLA can be produced by cultured fungi or be obtained via endogenous conversion from γ-linolenic acid (GLA)-rich vegetable oils. Several disease states are characterized by abnormally low DGLA levels in the body, while others can feature elevated levels. A defect in the activity of ∆6-desaturase and/or ∆5-desaturase may be one factor in the initiation and progression of these conditions. The potential of GLA and DGLA administrations as curative or ameliorating therapies in inflammatory conditions and malignancies appears modest at best. Manipulations with ∆6- and ∆5-desaturase inhibitors or combinations of long-chain PUFA supplements with n-3 PUFAs could provide a way to modify the body's DGLA and ARA production and the concentrations of their pro- and anti-inflammatory mediators. However, clinical data remain scarce and further well-designed studies should be actively promoted.

Injury-related cell death and proteoglycan loss in articular cartilage: Numerical model combining necrosis, reactive oxygen species, and inflammatory cytokines.

Osteoarthritis (OA) is a common musculoskeletal disease that leads to deterioration of articular cartilage, joint pain, and decreased quality of life. When OA develops after a joint injury, it is designated as post-traumatic OA (PTOA). The etiology of PTOA remains poorly understood, but it is known that proteoglycan (PG) loss, cell dysfunction, and cell death in cartilage are among the first signs of the disease. These processes, influenced by biomechanical and inflammatory stimuli, disturb the normal cell-regulated balance between tissue synthesis and degeneration. Previous computational mechanobiological models have not explicitly incorporated the cell-mediated degradation mechanisms triggered by an injury that eventually can lead to tissue-level compositional changes. Here, we developed a 2-D mechanobiological finite element model to predict necrosis, apoptosis following excessive production of reactive oxygen species (ROS), and inflammatory cytokine (interleukin-1)-driven apoptosis in cartilage explant. The resulting PG loss over 30 days was simulated. Biomechanically triggered PG degeneration, associated with cell necrosis, excessive ROS production, and cell apoptosis, was predicted to be localized near a lesion, while interleukin-1 diffusion-driven PG degeneration was manifested more globally. Interestingly, the model also showed proteolytic activity and PG biosynthesis closer to the levels of healthy tissue when pro-inflammatory cytokines were rapidly inhibited or cleared from the culture medium, leading to partial recovery of PG content. The numerical predictions of cell death and PG loss were supported by previous experimental findings. Furthermore, the simulated ROS and inflammation mechanisms had longer-lasting effects (over 3 days) on the PG content than localized necrosis. The mechanobiological model presented here may serve as a numerical tool for assessing early cartilage degeneration mechanisms and the efficacy of interventions to mitigate PTOA progression.

Towards sustainable human space exploration-priorities for radiation research to quantify and mitigate radiation risks.

Human spaceflight is entering a new era of sustainable human space exploration. By 2030 humans will regularly fly to the Moon's orbit, return to the Moon's surface and preparations for crewed Mars missions will intensify. In planning these undertakings, several challenges will need to be addressed in order to ensure the safety of astronauts during their space travels. One of the important challenges to overcome, that could be a major showstopper of the space endeavor, is the exposure to the space radiation environment. There is an urgent need for quantifying, managing and limiting the detrimental health risks and electronics damage induced by space radiation exposure. Such risks raise key priority topics for space research programs. Risk limitation involves obtaining a better understanding of space weather phenomena and the complex radiation environment in spaceflight, as well as developing and applying accurate dosimetric instruments, understanding related short- and long-term health risks, and strategies for effective countermeasures to minimize both exposure to space radiation and the remaining effects post exposure. The ESA/SciSpacE Space Radiation White Paper identifies those topics and underlines priorities for future research and development, to enable safe human and robotic exploration of space beyond Low Earth Orbit.

Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA.

PURPOSE: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. METHODS: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. RESULTS: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. CONCLUSION: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse-a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA-EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA-EV count, and HA-EV % (rs = - 0.264 to - 0.327, p = 0.012-0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA-EVs) for naturally occurring equine OA. The roles of HA-EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses.

Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell-cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.

Cyclic loading regime considered beneficial does not protect injured and interleukin-1-inflamed cartilage from post-traumatic osteoarthritis.

Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n = 406) were harvested from the patellofemoral grooves of young calves (N = 6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1 h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (35S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3-12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2-4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p < 0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95% CI: 1.23-2.70]; 1.66 [1.42-1.89]; p = 0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Minimum-Norm Estimation of TMS-Activated Motor Cortical Sites in Realistic Head and Brain Geometry.

Navigated transcranial magnetic stimulation (nTMS) is a widely used tool for motor cortex mapping. However, the full details of the activated cortical area during the mapping remain unknown due to the spread of the stimulating electric field (E-field). Computational tools, which combine the E-field with physiological responses, have potential for revealing the activated source area. We applied the minimum-norm estimate (MNE) method in a realistic head geometry to estimate the activated cortical area in nTMS motor mappings of the leg and hand muscles. We calculated the MNE also in a spherical head geometry to assess the effect of the head model on the MNE maps. Finally, we determined optimized coil placements based on the MNE map maxima and compared these placements with the initial hotspot placement. The MNE maps generally agreed well with the original motor maps: in the realistic head geometry, the distance from the MNE map maximum to the motor map center of gravity (CoG) was 8.8 ± 4.6 mm in the leg motor area and 6.6 ± 2.5 mm in the hand motor area. The head model did not have a significant effect on these distances; however, it had a significant effect on the distance between the MNE CoG and the motor map ( ). The optimized coil locations were < 1 cm from the initial hotspot in 7/10 subjects. Further research is required to determine the level of anatomical detail and the optimal mapping parameters required for robust and accurate localization.