The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.

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The pathogenesis and treatment of nonalcoholic steatohepatitis (NASH) are not well established. Feeding a diet deficient in both methionine and choline (MCD) is one of the most common models of NASH, which is characterized by steatosis, mitochondrial dysfunction, hepatocellular injury, oxidative stress, inflammation, and fibrosis. However, the individual contribution of the lack of methionine and choline in liver steatosis, advanced pathology and impact on mitochondrial S-adenosyl-Lmethionine (SAM) and glutathione (GSH), known regulators of disease progression, has not been specifically addressed. Here, we examined the regulation of mitochondrial SAM and GSH and signs of disease in mice fed a MCD, methionine-deficient (MD), or choline-deficient (CD) diet. The MD diet reproduced most of the deleterious effects of MCD feeding, including weight loss, hepatocellular injury, oxidative stress, inflammation, and fibrosis, whereas CD feeding was mainly responsible for steatosis, characterized by triglycerides and free fatty acids accumulation. These findings were preceded by MCD-or MD-mediated SAM and GSH depletion in mitochondria due to decreased mitochondrial membrane fluidity associated with a lower phosphatidylcholine/phosphatidylethanolamine ratio. MCD and MD but not CD feeding resulted in increased ceramide levels by acid sphingomyelinase. Moreover, GSH ethyl ester or SAM therapy restored mitochondrial GSH and ameliorated hepatocellular injury in mice fed a MCD or MD diet. Thus, the depletion of SAM and GSH in mitochondria is an early event in the MCD model of NASH, which is determined by the lack of methionine. Moreover, therapy using permeable GSH prodrugs may be of relevance in NASH.

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The terms extended donor or expanded donor mean changes in donor acceptability criteria. In almost all cases, the negative connotations of these terms cannot be justified. Factors considered to affect donor or organ acceptability have changed with time, after showing that they did not negatively affect graft or patient survival per se or when the adequate measures had been adopted. There is no age limit to be an organ donor. Kidney and liver transplantation from donors older than 65 years can have excellent graft and patient actuarial survival and graft function. Using these donors can be from an epidemiological point of view the most important factor to esablish the final number of cadaveric liver and kidney transplantations. Organs with broad structural parenchyma lesion with preserved functional reserve and organs with reversible functional impairment can be safely transplanted. Bacterial and fungal donor infection with the adequate antibiotic treatment of donor and/or recipient prevents infection in the latter. The organs, including the liver, from donors with infection by the hepatitis B and C viruses can be safely transplanted to recipients with infection by the same viruses, respectively. Poisoned donors and non-heart-beating donors, grafts from transplant recipients, reuse of grafts, domino transplant and splitting of one liver for two recipients can be an important and safe source of organs for transplantation.

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Running title: Cholesterol and hepatic ischemia-reperfusion injury.Key words: Hepatic steatosis; ischemia/reperfusion; liver transplantation; triglycerides; cholesterol; statins; squalene synthase.

Our study demonstrates that patients with brain death caused by bacterial meningitis due to meningococci, pneumococci, or E coli may be suitable organ donors. Transplantation of organs from such donors does not increase the risk of infection transmission to the recipient, provided that both donor and recipient had received adequate antibiotic therapy.