User Guide-Ultrafree®-MC and -CL Centrifugal Filter Devices
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Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.
- The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that beta(2)-glycoprotein I (beta(2)GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize beta(2)GPI when bound to anionic surfaces and not in solution. We showed that beta(2)GPI can exist in at least 2 different conformations: a circular plasma conformation and an "activated" open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of beta(2)GPI. By changing pH and salt concentration, we were able to convert the conformation of beta(2)GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-beta(2)GPI complex. We also demonstrate that the open conformation of beta(2)GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-beta(2)GPI antibodies. The conformational change of beta(2)GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.
- Document Type:
- Reference
- Product Catalog Number:
- UFC30GV0S
- Product Catalog Name:
- Ultrafree-MC Centrifugal Filter
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β₂-glycoprotein I: a novel component of innate immunity.
- Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the host's immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that β₂-glycoprotein I (β₂GPI) is a scavenger of LPS. In vitro, β₂GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of β₂GPI to LPS caused a conformational change in β₂GPI that led to binding of the β₂GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of β₂GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that β₂GPI is involved in the neutralization and clearance of LPS and identify β₂GPI as a component of innate immunity.
- Document Type:
- Reference
- Product Catalog Number:
- UFC30GV0S
- Product Catalog Name:
- Ultrafree-MC Centrifugal Filter
-
Delivery of human acetylcholinesterase by adeno-associated virus to the acetylcholinesterase knockout mouse.
- The purpose of this work was to develop a gene delivery system that expressed acetylcholinesterase (AChE) for prolonged periods. An adeno-associated virus (AAV) expressing human AChE was constructed by co-transfecting three plasmids into HEK 293T cells. The purified vector expressed 0.17 microg AChE per 1 million viral particles in culture medium in 23 h, or 0.8 U/ml. The AAV/hAChE was injected into muscle of adult AChE knockout mice and into the brains of 3-6 week old AChE knockout mice. Intramuscular injection yielded plasma AChE levels approaching 50% of the AChE activity of wild-type mouse plasma. The highest AChE activity was found on day 3 post-injection. AChE activity declined thereafter to a constant 7% of normal. The decreased level was accompanied by the appearance of anti-human AChE antibodies, suggesting partial clearance of AChE from plasma by antibodies. Intrastriatal injection resulted in AChE expression in the striatum. No antibodies were detected in animals treated intrastriatally. Motor coordination was improved and the lifespan of intrastriatally-treated AChE knockout mice was prolonged. Human AChE was expressed in mouse brain for up to 7 months after intrastriatal injection of an AAV/hAChE construct. Gene-therapy to supply AChE to the striatum improved motor coordination and prolonged the life of mice genetically deficient in AChE, probably by reducing their susceptibility to spontaneous seizures. This supports the hypothesis that their seizures are induced by excess acetylcholine.
- Document Type:
- Reference
- Product Catalog Number:
- MAB304