This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.
The Stabilin receptors are a two-member family in the type H class of scavenger receptors. These dynamic receptors bind and internalize multiple ligands from the cell surface for the purpose of clearing extracellular material including some synthetic drugs and for sensing the external environment of the cell. Stabilin-1 was the first receptor to be cloned, though the biological activity of Hyaluronic Acid Receptor for Endocytosis (HARE)/Stabilin-2 was observed about 10 years prior to the cloning of Stabilin-1. Stabilin-1 has a more diverse expression profile among the tissues than HARE/Stabilin-2. This review will focus on HARE/Stabilin-2 and its interactions with hyaluronan, heparin, and phosphorothioate antisense oligonucleotides and what is known about how this receptor participates in signaling upon ligand binding.
IntroductionParaneoplastic necrotizing myopathy is a rare disorder, described as a proximal, symmetrical, and rapidly progressing myopathy that is manifested as a paraneoplastic syndrome. Diagnosis is established via histological examination of the muscle biopsy.Case presentationWe present the case of a 53-year-old woman, born in Guinea-Bissau, with a history of locally advanced breast cancer, diagnosed ten months previously. The patient had experienced a progressively proximal muscle weakness of the lower extremities, which led to a total inability to walk. Upon neurological examination, the patient showed muscle weakness and atrophy in both proximal lower extremities without myalgia. Muscle strength was graded according to the Medical Research Council Scale as 2 out of 5 in the bilateral iliopsoas muscle, and 4 out of 5 in the bilateral quadriceps femoris. The deep-tendon reflexes were hypoactive. The laboratory examination showed increased values of serum creatinine kinase and myoglobin. An electromyogram showed an incomplete interference pattern during voluntary contraction in the iliopsoas and quadriceps femoris. The motor nerve conduction was 44.1 m/s and 44.3 m/s in the right and left tibial nerves, respectively, and 46.5 m/s and 46.1 m/s in the right and left peroneal nerves, respectively. The sensory motor nerve conductions and the compound motor action potential amplitudes were normal. These findings, despite not being specific, suggested a myopathy. Consequently, a muscle biopsy was performed. A biopsy specimen showed myopathic changes that were characteristic of a necrotizing myopathy.ConclusionTreatment for this syndrome consists of controlling the tumor, and providing corticoid therapy. This led to the partial remission of the neurological manifestations.
Summary Lipopolysaccharides (LPSs) cause lethal endotoxemia if not rapidly cleared from blood circulation. Liver sinusoidal endothelial cells (LSEC) systemically clear LPS by unknown mechanisms. We discovered that LPS clearance through LSEC involves endocytosis and lysosomal inactivation via Stabilin-1 and 2 (Stab1 and Stab2) but does not involve TLR4. Cytokine production was inversely related to clearance/endocytosis of LPS by LSEC. When exposed to LPS, Stabilin double knockout mice (Stab DK) and Stab1 KO, but not Stab2 KO, showed significantly enhanced systemic inflammatory cytokine production and early death compared with WT mice. Stab1 KO is not significantly different from Stab DK in circulatory LPS clearance, LPS uptake and endocytosis by LSEC, and cytokine production. These data indicate that (1) Stab1 receptor primarily facilitates the proactive clearance of LPS and limits TLR4-mediated inflammation and (2) TLR4 and Stab1 are functionally opposing LPS receptors. These findings suggest that endotoxemia can be controlled by optimizing LPS clearance by Stab1.
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