Joseph B. Lillegard

Artificial and bioartificial liver support.

Acute liver failure (ALF) is a widespread problem with an unfavorable prognosis. Currently, liver transplantation is the only direct means of treatment for patients in ALF. Due to the scarcity of donor organs, liver support technologies are being developed and clinically tested with the intent of supporting a patient in ALF until the patient regains native liver function or until a donor organ becomes available. Two major categories of devices are currently being tested. Artificial liver support is purely mechanical, including albumin dialysis. Bioartificial devices contain cellular material. No single system has reproducibly demonstrated improvement in patient mortality. However, with the advent of new technology and cell acquisition techniques, further randomized controlled trials will be necessary to determine the role of artificial and bioartificial liver support devices in the treatment of patients with ALF.

Portal hypertension correlates with splenic stiffness as measured with MR elastography

To investigate the correlation between MR elastography (MRE) assessed spleen stiffness and direct portal vein pressure gradient (D‐HVPG) measurements in a large animal model of portal hypertension.

Cell therapies for liver diseases

Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure, acute‐on‐chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential to be widely applied to other liver diseases, including noninherited liver diseases and liver cancer, and to improve the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases. Liver Transpl 18:9–21, 2012.

Role of Kupffer cells and toll-like receptor 4 in acetaminophen-induced acute liver failure.

BACKGROUND Significant morbidity associated with acute liver failure (ALF) is from the systemic inflammatory response syndrome (SIRS). Toll-like receptor 4 (TLR4) has been shown to play an integral role in the modulation of SIRS. However, little is known about the mechanistic role of TLR4 in ALF. Also, no cell type has been identified as the key mediator of the TLR4 pathway in ALF. This study examines the role of TLR4 and Kupffer cells (KCs) in the development of the SIRS following acetaminophen (APAP)-induced ALF. MATERIALS AND METHODS Five groups of mice were established: untreated wild-type, E5564-treated (a TLR4 antagonist), gadolinium chloride -treated (KC-depleted), clodronate-treated (KC-depleted), and TLR4-mutant. Following APAP administration, 72-h survival, biochemical and histologic liver injury, extent of lung injury and edema, and proinflammatory gene expression were studied. Additionally, TLR4 expression was determined in livers of wild-type and KC-depleted mice. RESULTS Following APAP administration, wild-type, TLR4-mutant, E5564-treated, and KC-depleted mice had significant liver injury. However, wild-type mice had markedly worse survival compared with the other four treatment groups. TLR4-mutant, E5564-treated, and KC-depleted mice had less lung inflammation and edema than wild-type mice. Selected proinflammatory gene expression (interleukin 1β, interleukin 6, tumor necrosis factor) in TLR4-mutant, E5564-treated, and KC-depleted mice was significantly lower compared with wild-type mice after acute liver injury. CONCLUSION This study demonstrates that survival in APAP-induced ALF potentially correlates with the level of proinflammatory gene expression. This study points to a link between TLR4 and KCs in the APAP model of ALF and, more importantly, demonstrates benefits of TLR4 antagonism in ALF.

Serum-Free Medium and Mesenchymal Stromal Cells Enhance Functionality and Stabilize Integrity of Rat Hepatocyte Spheroids

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.

Aldosteronomas—State of the Art

Primary aldosteronism (PA) is the most common cause of secondary hypertension in nonsmokers. Widespread screening of unselected hypertensives has identified PA in as many as 15% of patients. With such screening efforts using the PAC/PRA ratio and PAC, the widespread prevalence of the disease has become apparent while the relative percentage of APA has decreased. PA is confirmed by demonstrating lack of aldosterone suppressibility with sodium loading. Subtype evaluation is best achieved with high resolution CT scanning and AVS in the appropriate setting. In patients with PA and a unilateral source of aldosterone excess, laparoscopic adrenalectomy is the treatment of choice with excellent outcomes and low morbidity as compared with older open approaches. Patients with IHA, or those not amenable or agreeable to surgery, are best managed with a MR antagonist.

Hepatic Resection for the Carcinoid Syndrome in Patients with Severe Carcinoid Heart Disease: Does Valve Replacement Permit Safe Hepatic Resection?

BACKGROUND Hepatic resection of metastatic carcinoid cancer can prolong survival and control symptomatic endocrinopathy. Decompensated carcinoid heart disease (CHD) can develop in some patients with metastatic carcinoid cancers, which can preclude operation for resectable hepatic metastases. We hypothesized that outcomes after hepatic resection for patients with the carcinoid syndrome after valve replacement for CHD would be similar to carcinoid patients without CHD. STUDY DESIGN We compared the survival and symptom control after hepatic resection for patients undergoing valve replacement for CHD to carcinoid patients without CHD matched for age, sex, and extent of hepatectomy. RESULTS Fourteen patients with earlier valve replacement for CHD were compared with 28 carcinoid patients without CHD. All patients had hepatic resection for metastatic carcinoid disease and carcinoid syndrome. Mean age, sex distribution, and extent of hepatectomy (major hepatectomy, 78%) was similar between groups. Mean interval from valve replacement to hepatectomy was 101 days. There was no operative mortality. Major operative morbidity, inclusive of operative blood loss and cardiorespiratory events, occurred in 28.5% and 14.2% for CHD and non-CHD groups, respectively (p = 0.16). Symptom-free survival for CHD and non-CHD groups was 69% and 81% at 1 year (p = 0.22) and 61% and 44% (p = 0.17) at 5 years, respectively. Octreotide-free survival after hepatectomy 69% and 84% (p = 0.15) at 1 year and 62% and 52% (p = 0.29) 5 years, respectively. Overall survival CHD and non-CHD groups 100% at 1 year and 100% and 70% (p = 0.002) 5 years. CONCLUSIONS Valve replacement for severe CHD is safe and hepatic resection is associated with similar outcomes as patients without CHD undergoing hepatic resection for carcinoid syndrome. Identifying resectable hepatic metastases from carcinoids in patients with severe CHD should prompt valve replacement and interval hepatic resection.

Normal atmospheric oxygen tension and the use of antioxidants improve hepatocyte spheroid viability and function

Hepatocyte spheroids have been proposed for drug metabolism studies and in bioartificial liver devices. However, the optimal conditions required to meet the aerobic demands of mitochondria‐rich hepatocyte spheroids is not well studied. We hypothesized that an optimal concentration of oxygen could be identified and that the health of hepatocyte spheroids might be further improved by antioxidant therapy. Rat hepatocyte spheroids were maintained in suspension culture for 7 days under a mixture of 5% CO2 plus O2:N2 to achieve fractional oxygen contents of 6% (C1), 21% (C2), 58% (C3), and 95% (C4). Spheroid health was assessed under each condition by vital staining, TEM, oxygen consumption, and mitochondrial counts. Hepatocyte differentiation was assessed by expression of 10 liver‐related genes (HNF4a, HNF6, Cyp1A1, albumin, Nags, Cps1, Otc, Ass, Asl, Arg1). Functional markers (albumin and urea) were measured. The influence of oxygen tension and antioxidant treatment on the production of reactive oxygen species (ROS) was assessed by confocal microscopy. We observed that the hepatocyte spheroids were healthiest under normal atmospheric (C2) conditions with antioxidants ascorbic acid and L‐carnitine. Cell death and reduced functionality of hepatocyte spheroids correlated with the formation of ROS. Normal atmospheric conditions provided the optimal oxygen tension for suspension culture of hepatocyte spheroids. The formation and deleterious effects of ROS were further reduced by adding antioxidants to the culture medium. These findings have direct application to development of the spheroid reservoir bioartificial liver and the use of hepatocyte spheroids in drug metabolism studies. J. Cell. Physiol. 226: 2987–2996, 2011.

In utero transplanted human hepatocytes allow postnatal engraftment of human hepatocytes in pigs

In utero cell transplantation (IUCT) can lead to the postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCT have involved hematopoietic stem cells. It is unknown whether human hepatocytes used for IUCT in fetal pigs will lead to the engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1 × 107 human hepatocytes in utero and were delivered by cesarean section at term. The piglets received a second direct liver injection of 5 × 107 human hepatocytes 1 week after birth. The serum was analyzed for human albumin 2, 4, and 6 weeks after engraftment. Piglet livers were harvested 6 weeks after transplantation and were examined by immunohistochemistry, polymerase chain reaction, and fluorescence in situ hybridization for human‐specific sequences. Piglets undergoing IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all postengraftment time points. Human albumin gene expression, the presence of human hepatocytes, and the presence of human beta‐2 microglobulin were all confirmed 6 weeks after engraftment. IUCT in fetal pigs with human hepatocytes early in gestation allowed the engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large‐scale expansion of human hepatocytes in genetically engineered pigs. Liver Transpl 19:328–335, 2013.

Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type 1.

Transplantation of gene-corrected autologous hepatocytes can cure metabolic disease in a preclinical pig model of hereditary tyrosinemia type 1. Skipping the waiting list The only cure for hereditary tyrosinemia type 1 (HT1)—an inherited metabolic disease—is a liver transplant. However, owing to the shortage of liver donors, Hickey et al. turned to gene therapy as a way to cure HT1. The authors took liver cells from pigs that have HT (through a defect in the gene Fah), transduced them with the correct Fah, and then put the cells back into the same animals. The ex vivo gene therapy approach prevented liver failure and fibrosis and also restored metabolic function, which is deteriorated in HT1 disease. Having demonstrated in large animals the use of materials that are safe for use in people, the technology is now poised to move into patients, to regenerate their own livers and spare them the long wait times on the liver transplant list. We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase–deficient (Fah−/−) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah−/− pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH+ cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah−/− liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah−/− pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.