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.

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.

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.

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.

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.

Metabolomics of Synovial Fluid and Infrapatellar Fat Pad in Patients with Osteoarthritis or Rheumatoid Arthritis.

Osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA) are inflammatory joint diseases with complex and insufficiently understood pathogeneses. Our objective was to characterize the metabolic fingerprints of synovial fluid (SF) and its adjacent infrapatellar fat pad (IFP) obtained during the same surgical operation from OA and RA knees. Non-targeted metabolite profiling was performed for 5 non-inflammatory trauma controls, 10 primary OA (pOA) patients, and 10 seropositive RA patients with high-resolution mass spectrometry-based techniques, and metabolites were matched with known metabolite identities. Groupwise differences in metabolic features were analyzed with the univariate Welch's t-test and the multivariate linear discriminant analysis (LDA) and principal component analysis (PCA). Significant discrimination of metabolite profiles was discovered by LDA for both SF and IFP and by PCA for SF based on diagnosis. In addition to a few drug-derived substances, there were 16 and 13 identified metabolites with significant differences between the diagnoses in SF and IFP, respectively. The pathways downregulated in RA included androgen, bile acid, amino acid, and histamine metabolism, and those upregulated included biotin metabolism in pOA and purine metabolism in RA and pOA. The RA-induced downregulation of androgen and bile acid metabolism was observed for both SF and IFP. The levels of 11 lipid metabolites, mostly glycerophospholipids and fatty acid amides, were also altered by these inflammatory conditions. The identified metabolic pathways could be utilized in the future to deepen our understanding of the pathogeneses of OA and RA and to develop not only biomarkers for their early diagnosis but also therapeutic targets.

Synovial Fluid Fatty Acid Profiles Are Differently Altered by Inflammatory Joint Pathologies in the Shoulder and Knee Joints.

Anomalies of fatty acid (FA) metabolism characterize osteoarthritis (OA) and rheumatoid arthritis (RA) in the knee joint. No previous study has investigated the synovial fluid (SF) FA manifestations in these aging-related inflammatory diseases in the shoulder. The present experiment compared the FA alterations between the shoulder and knee joints in patients with end-stage OA or end-stage RA. SF samples were collected during glenohumeral or knee joint surgery from trauma controls and from OA and RA patients (n = 42). The FA composition of SF total lipids was analyzed by gas chromatography with flame ionization and mass spectrometric detection and compared across cohorts. The FA signatures of trauma controls were mostly uniform in both anatomical locations. RA shoulders were characterized by elevated percentages of 20:4n-6 and 22:6n-3 and with reduced proportions of 18:1n-9. The FA profiles of OA and RA knees were relatively uniform and displayed lower proportions of 18:2n-6, 22:6n-3 and total n-6 polyunsaturated FAs (PUFAs). The results indicate location- and disease-dependent differences in the SF FA composition. These alterations in FA profiles and their potential implications for the production of PUFA-derived lipid mediators may affect joint lubrication, synovial inflammation and pannus formation as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases.

Fatty Acids and Oxylipins in Osteoarthritis and Rheumatoid Arthritis-a Complex Field with Significant Potential for Future Treatments.

PURPOSE OF REVIEW: Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterized by abnormal lipid metabolism manifested as altered fatty acid (FA) profiles of synovial fluid and tissues and in the way dietary FA supplements can influence the symptoms of especially RA. In addition to classic eicosanoids, the potential roles of polyunsaturated FA (PUFA)-derived specialized pro-resolving lipid mediators (SPM) have become the focus of intensive research. Here, we summarize the current state of knowledge of the roles of FA and oxylipins in the degradation or protection of synovial joints. RECENT FINDINGS: There exists discordance between the large body of literature from cell culture and animal experiments on the adverse and beneficial effects of individual FA and the lack of effective treatments for joint destruction in OA and RA patients. Saturated 16:0 and 18:0 induce mostly deleterious effects, while long-chain n-3 PUFA, especially 20:5n-3, have positive influence on joint health. The situation can be more complex for n-6 PUFA, such as 18:2n-6, 20:4n-6, and its derivative prostaglandin E2, with a combination of potentially adverse and beneficial effects. SPM analogs have future potential as analgesics for arthritic pain. Alterations in FA profiles and their potential implications in SPM production may affect joint lubrication, synovial inflammation, pannus formation, as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases. Further research directions include high-quality randomized controlled trials on dietary FA supplements and investigations on the significance of lipid composition of microvesicle membrane and cargo in joint diseases.

Extracellular Vesicles and Their Potential Significance in the Pathogenesis and Treatment of Osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease characterized by inflammation, gradual destruction of articular cartilage, joint pain, and functional limitations that eventually lead to disability. Join tissues, including synovium and articular cartilage, release extracellular vesicles (EVs) that have been proposed to sustain joint homeostasis as well as to contribute to OA pathogenesis. EVs transport biologically active molecules, and OA can be characterized by altered EV counts and composition in synovial fluid. Of EV cargo, specific non-coding RNAs could have future potential as diagnostic biomarkers for early OA. EVs may contribute to the propagation of inflammation and cartilage destruction by transporting and enhancing the production of inflammatory mediators and cartilage-degrading proteinases. In addition to inducing OA-related gene expression patterns in synoviocytes and articular chondrocytes, EVs can induce anti-OA effects, including increased extracellular matrix deposition and cartilage protection. Especially mesenchymal stem cell-derived EVs can alleviate intra-articular inflammation and relieve OA pain. In addition, surgically- or chemically-induced cartilage defects have been repaired with EV therapies in animal models. While human clinical trials are still in the future, the potential of actual cures to OA by EV products is very promising.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Toxic Potential of Traditionally Consumed Mushroom Species-A Controversial Continuum with Many Unanswered Questions.

Mushroom poisonings remain a significant cause of emergency medicine. While there are well-known species, such as Amanita phalloides, causing life-threatening poisonings, there is also accumulating evidence of poisonings related to species that have been considered edible and are traditionally consumed. In particular, the Tricholoma equestre group was reported to cause myotoxicity. In addition, particular wild mushrooms that are traditionally consumed especially in Asia and Eastern Europe have been subject to suspicion due to possible mutagenicity. Hitherto, the causative agents of these effects often remain to be determined, and toxicity studies have yielded contradictory results. Due to this, there is no consensus about the safety of these species. The issue is further complicated by difficulties in species identification and other possible sources of toxicity, such as microbiological contamination during storage, leading to sometimes opposite conclusions about the edibility of a species. This review focuses on existing data about these types of mushroom poisonings, including the still sparse knowledge about the causative chemical agents. In addition, the aim is to initiate a meta-discussion about the issue and to give some suggestions about how to approach the situation from the viewpoint of the collector, the researcher, and the practicing physician.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

Distinct fatty acid signatures in infrapatellar fat pad and synovial fluid of patients with osteoarthritis versus rheumatoid arthritis.

BACKGROUND: Infrapatellar fat pad (IFP) has recently emerged as a potential source of inflammation in knee arthropathies. It has been proposed to be one source of adipocytokines, fatty acids (FA), and FA-derived lipid mediators that could contribute to the pathophysiological processes in the knee joint. Alterations in synovial fluid (SF) lipid composition have been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA). The aim of the present study was to compare the FA signatures in the IFP and SF of RA and OA patients. METHODS: Pairs of IFP and SF samples were collected from the same knees of RA (n = 10) and OA patients (n = 10) undergoing total joint replacement surgery. Control SF samples (n = 6) were harvested during diagnostic or therapeutic arthroscopic knee surgery unrelated to RA or OA. The FA composition in the total lipids of IFP and SF was determined by gas chromatography with flame ionization and mass spectrometric detection. RESULTS: Arthropathies resulted in a significant reduction in the SF proportions of n-6 polyunsaturated FA (PUFA), more pronouncedly in OA than in RA. OA was also characterized with reduced percentages of 22:6n-3 and lower product/precursor ratios of n-3 PUFA. The proportions of total monounsaturated FA increased in both RA and OA SF. Regarding IFP, RA patients had lower proportions of 20:4n-6, total n-6 PUFA, and 22:6n-3, as well as lower product/precursor ratios of n-3 PUFA compared to OA patients. The average chain length of SF FA decreased in both diagnoses and the double bond index in OA. CONCLUSIONS: The observed complex alterations in the FA signatures could have both contributed to but also limited the inflammatory processes and cartilage destruction in the RA and OA knees.

Anterior cruciate ligament transection alters the n-3/n-6 fatty acid balance in the lapine infrapatellar fat pad.

BACKGROUND: The infrapatellar fat pad (IFP) of the knee joint has received lots of attention recently due to its emerging role in the pathogenesis of osteoarthritis (OA), where it displays an inflammatory phenotype. The aim of the present study was to examine the infrapatellar fatty acid (FA) composition in a rabbit (Oryctolagus cuniculus) model of early OA created by anterior cruciate ligament transection (ACLT). METHODS: OA was induced randomly in the left or right knee joint of skeletally mature New Zealand White rabbits by ACLT, while the contralateral knee was left intact. A separate group of unoperated rabbits served as controls. The IFP of the ACLT, contralateral, and control knees were harvested following euthanasia 2 or 8 weeks post-ACLT and their FA composition was determined with gas chromatography-mass spectrometry. RESULTS: The n-3/n-6 polyunsaturated FA (PUFA) ratio shifted in a pro-inflammatory direction after ACLT, already observed 2 weeks after the operation (0.20 ± 0.008 vs. 0.18 ± 0.009). At 8 weeks, the FA profile of the ACLT group was characterized with increased percentages of 20:4n-6 (0.44 ± 0.064 vs. 0.98 ± 0.339 mol-%) and 22:6n-3 (0.03 ± 0.014 vs. 0.07 ± 0.015 mol-%) and with decreased monounsaturated FA (MUFA) sums (37.19 ± 1.586 vs. 33.20 ± 1.068 mol-%) and n-3/n-6 PUFA ratios (0.20 ± 0.008 vs. 0.17 ± 0.008). The FA signature of the contralateral knees resembled that of the unoperated controls in most aspects, but had increased proportions of total n-3 PUFA and reduced MUFA sums. CONCLUSIONS: These findings provide novel information on the effects of early OA on the infrapatellar FA profile in the rabbit ACLT model. The reduction in the n-3/n-6 PUFA ratio of the IFP is in concordance with the inflammation and cartilage degradation in early OA and could contribute to disease pathogenesis.