ABSTRACT
Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.
Subject(s)
Epstein-Barr Virus Infections , Cell Line , Epstein-Barr Virus Infections/metabolism , Herpesvirus 4, Human , Humans , Immunotherapy, Adoptive/methods , Interleukins , Killer Cells, NaturalABSTRACT
Argonaute 2 (Ago2) is the main component of the RNA-induced silencing complex. We recently showed that liver-specific Ago2-deficiency in mice (L-Ago2 knockout [KO] mice) enhances mitochondrial oxidation and alleviates obesity-associated pathophysiology. However, the precise mechanisms behind the role of hepatic Ago2 in regulating the mitochondrial oxidation associated with glucose metabolism are still unclear. Here, we show that hepatic Ago2 regulates the function of peroxisome proliferator-activated receptor α (PPARα) for oxidative metabolism. In both genetically and diet-induced severe obese conditions, L-Ago2 KO mice developed obesity and hepatic steatosis but exhibited improved glucose metabolism accompanied by lowered expression levels of pathologic microRNAs (miRNAs), including miR-802, miR-103/107, and miR-152, and enhanced expression of PPARα and its target genes regulating oxidative metabolism in the liver. We then investigated the role of hepatic Ago2 in the outcomes of vertical sleeve gastrectomy (VSG) in which PPARα plays a crucial role in a drastic transcription reprogram associated with improved glycemia post VSG. Whereas VSG reduced body weight and improved fatty liver in wild-type mice, these effects were not observed in hepatic Ago2-deficient mice. Conversely, glucose metabolism was improved in a hepatic Ago2-dependent manner post VSG. Treating Ago2-deficient primary hepatocytes with WY-14643, a PPARα agonist, showed that Ago2-deficiency enhances sensitivity to WY-14643 and increases expression of PPARα target genes and mitochondrial oxidation. Our findings suggest that hepatic Ago2 function is intrinsically associated with PPARα that links Ago2-mediated RNA silencing with mitochondrial functions for oxidation and obesity-associated pathophysiology.
Subject(s)
Argonaute Proteins/deficiency , Liver/metabolism , Obesity/metabolism , Obesity/surgery , PPAR alpha/metabolism , Animals , Argonaute Proteins/genetics , Bariatric Surgery , Glucose/metabolism , Glucose Tolerance Test , Glycemic Control , Hepatocytes/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/drug therapy , Obesity/genetics , Oxidative Stress , PPAR alpha/genetics , Pyrimidines/administration & dosageABSTRACT
We report the successful use of peripheral nerve blocks for provision of surgical anesthesia for knee surgery in a patient who had end-stage heart failure, who was supported by a HeartMate II left ventricular assist device, and who was anticoagulated. We discuss the anesthetic implications involved in the care of patients being anticoagulated and on left ventricular assist device.