Isolation of Microvesicles from Human Circulating Neutrophils.

Neutrophil-derived microvesicles (NDMVs) are liberated by neutrophils upon cell activation by molecules. Once activated, neutrophils are primarily involved in acute inflammation; however, the microvesicles they produce are largely anti-inflammatory. NDMVs have been shown to protect cartilage during inflammatory arthritis. They exert these effects by inhibiting or affecting the function of target cells, including macrophages. NDMVs have the potential to act as disease-modifying agents, especially for inflammatory diseases. This protocol describes a method using differential centrifugation to separate neutrophils from whole human blood. Subsequently, neutrophils are identified by cytospin and Wright's staining, and then the NDMVs are isolated using differential centrifugation.

Plasma and Joint Fluid Glypican-3 Are Inversely Correlated with the Severity of Knee Osteoarthritis.

OBJECTIVE: Glypican-3 possesses a possible action in regulation of bone growth and development implicated in osteoarthritis (OA) pathology. Therefore, this study aimed to investigate glypican-3 in plasma and synovial fluid of knee OA patients and to determine the possible association between glypican-3 levels and radiographic severity. DESIGN: A total of 80 knee OA patients and 80 healthy controls were recruited. Glypican-3 levels in plasma and synovial fluid were measured using enzyme-linked immunosorbent assay. The severity of knee OA was assessed by radiographic grading according to the Kellgren-Lawrence classification. Relative mRNA expression of glypican-3 in 10 inflamed synovial tissues from OA patients and 10 noninflamed synovial controls was quantified using real-time polymerase chain reaction. RESULTS: Plasma glypican-3 levels were significantly lower in OA patients than in healthy controls (P = 0.03), whereas synovial fluid glypican-3 levels were remarkably greater than in paired plasma samples of OA patients (P < 0.001). Subsequent analysis demonstrated that plasma and synovial fluid glypican-3 levels were inversely associated with the radiographic severity of knee OA (r = -0.691, P < 0.001; r = -0.646, P < 0.001, respectively). Furthermore, there was a positive relationship between plasma and synovial fluid glypican-3 levels in knee OA patients (r = 0.515, P < 0.001). Additionally, overexpression of glypican-3 mRNA was observed in inflamed synovium of OA patients (P = 0.021). CONCLUSIONS: The present study revealed that plasma and synovial glypican-3 levels were negatively associated with radiographic severity of knee OA. Glypican-3 could emerge as a potential biomarker for reflecting the severity of knee OA and might play a plausible role in the pathophysiology of degenerative joint disease.

Increased serum glypican-3 is associated with liver stiffness and hepatic dysfunction in children with biliary atresia.

AIM OF THE STUDY: Biliary atresia (BA) is an uncommon disorder of the liver and bile ducts affecting infants and is characterized by progressive fibrosclerosing obstruction of the extrahepatic biliary tree leading to end-stage liver failure. The purpose of this study was to determine serum glypican-3 (GPC3) levels and liver stiffness in children with BA and the correlation of glypican-3 with clinical parameters. MATERIAL AND METHODS: Seventy-five post-Kasai BA patients and 28 healthy age-matched controls were registered. Serum GPC3 levels were examined by enzyme-linked immunosorbent assay. Liver stiffness measurement was analyzed by transient elastography. RESULTS: BA patients had significantly greater serum GPC3 and liver stiffness values than controls (p < 0.001). Serum GPC3 and liver stiffness values were significantly higher in jaundiced BA patients than in non-jaundiced BA patients (p < 0.001). Additionally, serum glypican-3 was associated with liver stiffness and serum total bilirubin (p < 0.001, respectively). CONCLUSIONS: Elevated serum GPC3 levels were associated with hepatic dysfunction and the severity of BA. As a result, serum GPC3 and liver stiffness might serve as biomarkers reflecting the deterioration of hepatic function and the outcome in post-Kasai BA.