Jaime M. Glorioso

Pivotal preclinical trial of the spheroid reservoir bioartificial liver

BACKGROUND & AIMS The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF). METHODS Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST+No-cell device, ST+SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90h. RESULTS At 48h, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90h after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83%, n=6) compared to ST alone (0%, n=6, p=0.003) and No-cell device therapy (17%, n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment. CONCLUSIONS The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.

Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH(+/-) pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH(-/-) pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, the animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopathy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH(-/-) pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH(-/-) pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of the efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.

Distinguishing between Hepatic Inflammation and Fibrosis with MR Elastography

Purpose To investigate the utility of magnetic resonance (MR) elastography-derived mechanical properties in the discrimination of hepatic inflammation and fibrosis in the early stages of chronic liver diseases. Materials and Methods All studies were approved by the institutional animal care and use committee. A total of 187 animals were studied, including 182 mice and five pigs. These animals represented five different liver diseases with a varying combination and extent of hepatic inflammation, fibrosis, congestion, and portal hypertension. Multifrequency three-dimensional MR elastography was performed, and shear stiffness, storage modulus, shear loss modulus, and damping ratio were calculated for all animals. Necroinflammation, fibrosis, and portal pressure were either histologically scored or biochemically and physically quantified in all animals. Two-sided Welch t tests were used to evaluate mean differences between disease and control groups. Spearman correlation analyses were used to evaluate the relationships between mechanical parameters and quantitative fibrosis extent (hydroxyproline concentration) and portal pressure. Results Liver stiffness and storage modulus increased with progressively developed fibrosis and portal hypertension (mean stiffness at 80 Hz and 48-week feeding, 0.51 kPa ± 0.12 in the steatohepatitis group vs 0.29 kPa ± 0.01 in the control group; P = .02). Damping ratio and shear loss modulus can be used to distinguish inflammation from fibrosis at early stages of disease, even before the development of histologically detectable necroinflammation and fibrosis (mean damping ratio at 80 Hz and 20-week feeding, 0.044 ± 0.012 in the steatohepatitis group vs 0.014 ± 0.008 in the control group; P < .001). Damping ratio and liver stiffness vary differently with respect to cause of portal hypertension (ie, congestion- or cirrhosis-induced hypertension). These differentiation abilities have frequency-dependent variations. Conclusion Liver stiffness and damping ratio measurements can extend hepatic MR elastography to potentially enable assessment of necroinflammatory, congestive, and fibrotic processes of chronic liver diseases.

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.

Preservation of the deep muscular fascia and locoregional control in melanoma

BACKGROUND Locoregional recurrence occurs in approximately 20% of patients with melanoma and is associated with a significantly worse prognosis. Standards are well established for peripheral margins; however, there is insufficient evidence regarding depth of resection. METHODS Retrospective review of 964 patients undergoing excision of trunk or extremity melanoma ≥1 mm thick during a 29-year period at a tertiary academic center. Multivariate analysis and hazard ratios were used to determine the effect of excision of the deep muscular fascia on locoregional recurrence. RESULTS A total of 278 (29%) patients underwent resection of the muscular fascia. Of these patients, 18 (6%) developed local, 33 (12%) developed in-transit, and 68 (24%) developed nodal recurrence within 5 years. A total of 686 (71%) patients underwent excision of their primary melanoma with preservation of the muscular fascia. Of these patients, 40 (6%) developed local, 30 (4%) developed in-transit, and 84 (12%) developed nodal recurrence at 5 years. In multivariate analysis excision of the deep muscular fascia was an independent predictor of locoregional recurrence in patients treated with sentinel lymph node biopsy. Specifically, fascia resection was associated with a 2.5-fold increased risk of nodal recurrence but not associated with local recurrence or overall survival. CONCLUSION On the basis of no demonstrated advantage for resection of the deep muscular fascia, but potential for increased risk of intralymphatic recurrences, we recommend preservation of the deep muscular fascia during resection of primary cutaneous melanoma.

Noninvasive 3-dimensional imaging of liver regeneration in a mouse model of hereditary tyrosinemia type 1 using the sodium iodide symporter gene

Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild‐type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine‐binding globulin promoter. NIS‐transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS‐transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS‐transduced HCs were readily imaged in vivo by single‐photon emission computed tomography, and this demonstrated for the first time noninvasive 3‐dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models. Liver Transpl 21:442–453, 2015.

Characterization of a porcine model for associating liver partition and portal vein ligation for a staged hepatectomy

BACKGROUND Publications using the ALPPS (associating liver partition and portal vein ligation for a staged hepatectomy) procedure have demonstrated a future liver remnant growth of 40-160% in only 6-9 days. The present study aimed to develop and describe the first large animal model of ALPPS that can be used for future studies. METHODS A total of 13 female domestic pigs underwent ALPPS stage 1 (portal vein division and parenchymal transection) followed by ALPPS stage 2 (completion left-extended hepatectomy) 7 days later. An abdominal computed tomography (CT) scan was performed immediately prior to ALPPS stage 1 surgery and again 7 days later to assess hypertrophy immediately prior to ALPPS stage 2 surgery. Blood samples, as well as tissue analysis for Ki-67, were performed. RESULTS On CT volumetric analysis, the mean size of the future liver remnant (FLR) prior to ALPPS stage 1 was 21 ± 2% and 40 ± 6% prior to ALPPS stage 2. The median degree of growth was 75% with a mean kinetic growth rate of 11% per day. Liver weights at autopsy correlated well with CT volumetric analysis (r = 0.87). There was no significant difference in mean lab values [asparate aminotransferase (AST), alanine aminotransferase (ALT), ammonia, International Normalized Ratio (INR) or bilirubin] from baseline until immediately prior to ALPPS stage 2. Post ALPPS stage 2 there was a significant increase in INR from baseline 1.1 to 1.6 (P = 0.012). No post-operative deaths secondary to liver failure were observed. CONCLUSION The present study describes the first reproducible large animal model of the ALPPS procedure. The degree of liver growth and the kinetic rate of growth were similar to that which has been demonstrated in human publications. This model will be valuable as future laboratory studies are performed.

Cold storage of rat hepatocyte spheroids.

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

Association between kidney intracapsular pressure and ultrasound elastography

BackgroundKidney congestion is a common pathophysiologic pathway of acute kidney injury (AKI) in sepsis and heart failure. There is no noninvasive tool to measure kidney intracapsular pressure (KIP) directly.MethodsWe evaluated the correlation of KIP with kidney elasticity measured by ultrasound surface wave elastography (USWE). We directly measured transcatheter KIP in three pigs at baseline and after bolus infusion of normal saline, norepinephrine, vasopressin, dopamine, and fenoldopam; infiltration of 2-L peritoneal dialysis solution in the intra-abdominal space; and venous, arterial, and ureteral clamping. KIP was compared with USWE wave speed.ResultsOnly intra-abdominal installation of peritoneal dialysis fluid was associated with significant change in KIP (mean (95% CI) increase, 3.7 (3.2–4.2)] mmHg; P < .001). Although intraperitoneal pressure and KIP did not differ under any experimental condition, bladder pressure was consistently and significantly greater than KIP under all circumstances (mean (95% CI) bladder pressure vs. KIP, 3.8 (2.9–4.) mmHg; P < .001). USWE wave speed significantly correlated with KIP (adjusted coefficient of determination, 0.71; P < .001). Estimate (95% CI) USWE speed for KIP prediction stayed significant after adjustment for KIP hypertension (−0.8 (− 1.4 to − 0.2) m/s; P = .008) whereas systolic and diastolic blood pressures were not significant predictors of KIP.ConclusionsIn a pilot study of the swine model, we found ultrasound surface wave elastography speed is significantly correlated with transcatheter measurement of kidney intracapsular and intra-abdominal pressures, while bladder pressure overestimated kidney intracapsular pressure.

Reply to: “Pivotal preclinical trial of the spheroid reservoir bioartificial liver”

To the Editor: We appreciate the kind words from Dr. Chamuleau and his respected colleagues at Academic Medical Centre, University of Amsterdam. Yes, studies in large animal models of ALF are challenging. Especially recovery studies requiring continuous care of animals over multiple days in succession. As a result, treatment time and membrane porosity were combined to limit our study to three treatment groups (SRBAL device, no cell device, no device) and two device configurations (400 kD membrane porosity, 6 h duration vs. 65 kD membrane porosity, 24 h duration) as detailed in our manuscript [1]. Is it possible that the converse device configurations of 400 kD membrane porosity, 24 h duration or 65 kD membrane porosity, 6 h duration may have offered greater benefit? That possibility is unlikely. The biochemical and hemodynamic profiles of sick ALF pigs were most improved using the 65 kD membrane when loaded with greater than 100 grams of porcine hepatocyte spheroids. This benefit would have been reduced by shortening the treatment duration from 24 h to 6 h. As stated in the Discussion, superior outcome of SRBAL therapy using a membrane of 65 kD vs. 400 kD was unexpected; improved outcome appeared to correlate with lower shifts in plasma protein levels with the tighter membrane. We agree, it is possible that some conclusions drawn from our study may be specific to the SRBAL configuration. In future clinical trials of the SRBAL, longer treatment duration and greater cell dose will be evaluated based on their beneficial effects observed in the pivotal preclinical trial. Dr. Chamuleau and colleagues mentioned the complete moratorium on xenotransplantation in the European Union, presumably due to potential risks of zoonosis from animal cells. Yes, extracorporeal therapies pose potential risks of infectious transmission. There are also potential risks of tumor spread when using a proliferative human hepatocyte cell source as proposed by Dr. Chamuleau and colleagues. With regards to the potential risk of zoonosis during SRBAL therapy, a careful review of blood samples from 160 patients exposed to living pig tissue has been reported [2]. This rigorous retrospective study included up to 12 years follow-up and was required by the U.S. FDA before resuming studies of human xeno-pig therapies in 1999. This pig exposure study, published in Science, included several cohorts of patients and all patients who had been treated by a porcine hepatocyte bioartificial liver to that date. The authors reported that no porcine endogenous retrovirus (PERV) viremia was detected in any patient. Other human clinical reports uphold this negative conclusion including a report in The Lancet [3] by the same team of investigators who first reported PERV transmission under in vitro conditions using a highly receptive human cell line HEK293 [4]. Yes, the potential of zoonosis needs to be addressed scientifically and responsibly. To date, the data indicates that cells obtained from pigs raised in approved barrier facilities are safe, and often