Liver-based Metabolic Diseases Research Articles

Microbial Ecosystem in Diabetes Mellitus: Consideration of the Gastrointestinal System.

Intestinal microbiota is established to be a crucial element in the control of human health, and keeping the symbiotic relationship between the human body and intestinal microbes will have paramount importance. A number of investigations illustrated that many chronic diseases are associated with intestinal micro-ecological disorders implying intestinal floras as an important component among the environmental factors, and perturbations in their composition are correlated with metabolic disorders, including obesity and diabetes mellitus (DM). Increased evidence suggests that alterations in the gut microbial ecosystem have been involved in part in the pathogenesis of both type 1 and type 2 DM. Short chain fatty acids (SCFAs), derived from microbiota, have been studied for their potential action in modulating CNS, gut barrier axis, and the immune system as a promising mechanism for the observed protective effects on diabetes pathogenesis. Besides, the role of bile acid (BA) stimulated receptors to have a significant role in liver metabolism, and pathophysiology of liver-based metabolic diseases has also been investigated. In the current review, we will try to summarize the correlation between intestinal microbiota and diabetes considering the existing current evidence revealing the role of gut microbiota in onset and disease progression.

Liver transplantation during infancy: No increased rate of neurological complications.

pLT is a highly standardized therapy for children with end-stage liver disease and liver-based metabolic diseases. However, NCs after transplantation occur and especially younger children are considered as more vulnerable and susceptible to NCs. Up to now, detailed data particularly for the very young age group do not exist. We therefore retrospectively studied NCs in children after pLT under age of 24months. Forty children aged between 19days and 22months were evaluated according to type of NC and potential risk factors. NCs occurred in 8/40 patients (20%). All experienced new-onset seizures and in 1/6 surviving patients, seizures evolved into epilepsy. Other NCs were intracerebral abscess (1/8 patients) and subdural hemorrhage (1/8 patients). The overall 3-year mortality rate was 10% (4/40 patients). Significant risk factors for NCs and therefore seizures were HAT (P=0.020), total surgery time (P=0.009), retransplantation (P<0.001), period of catecholamine therapy (P=0.024), period of mechanical ventilation (P=0.014), and period of sedation (P=0.010). Our study is the first to provide detailed information on NCs after pLT in children under 24months of age. The incidence of NCs in this particular group of very young patients was not increased compared to previously published data of children of all ages. Main NC was new-onset seizure. In the surviving infants, prognosis of seizure was excellent and the risk of developing epilepsy was low. Even more, the occurrence of NCs did not significantly affect mortality or survival in this particular age group.

Instant Blood-Mediated Inflammatory Reaction in Hepatocyte Transplantation: Current Status and Future Perspectives.

Hepatocyte transplantation (HT) is emerging as a promising alternative to orthotopic liver transplantation (OLT) in patients with certain liver-based metabolic disease and acute liver failure. Hepatocytes are generally infused into the portal venous system, from which they migrate into the liver cell plates of the native organ. One of the major hurdles to the sustained success of this therapy is early cell loss, with up to 70% of hepatocytes lost immediately following infusion. This is largely thought to be due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in the activation of complement and coagulation pathways. Transplanted hepatocytes produce and release tissue factor (TF), which activates the coagulation pathway, leading to the formation of thrombin and fibrin clots. Thrombin can further activate a number of complement proteins, leading to the activation of the membrane attack complex (MAC) and subsequent hepatocyte cell death. Inflammatory cells including granulocytes, monocytes, Kupffer cells, and natural killer (NK) cells have been shown to cluster around transplanted hepatocytes, leading to their rapid clearance shortly after transplantation. Current research aims to improve cell engraftment and prevent early cell loss. This has been proven successful in vitro using pharmacological interventions such as melagatran, low-molecular-weight dextran sulphate, and N-acetylcysteine (NAC). Effective inhibition of IBMIR would significantly improve hepatocyte engraftment, proliferation, and function, providing successful treatment for patients with liver-based metabolic diseases.

Adeno-associated virus vector-based gene therapy for monogenetic metabolic diseases of the liver.

Liver-based metabolic diseases account for a substantial burden of childhood diseases. In most patients, treatment is often limited to supportive measures and liver transplantation is ultimately required. Even despite the excellent long-term outcome of liver transplantation, the procedure is associated with a significant morbidity and mortality. Gene therapy, in contrast, has great potential to save lives, improve the quality of life, and offer few risks and adverse effects compared with present therapies including liver transplantation. The most promising results to date in liver gene transfer have been achieved with adeno-associated virus. Although safety issues, such as immunogenicity of vector and/or transgene product, remain an important concern, gene therapy is ready for clinical trials in adults and adolescents. Developing and testing safe approaches for efficient and long-term stable applications in newborns and small children, such as targeted integration and gene correction, is one of the remaining future challenges.

Intrasplenic Transplanted Adult Rat Isolated Hepatocyte Fraction But Not Cholangiocytes Forms Bile Canaliculi

BackgroundHepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation in countries in which ethical regulations do not allow organ transplantation. One of the problems remains that substances normally secreted by the surviving hepatocytes to bile cannot be removed because of lack of bile canaliculi. We found that ligation of the recipient's common bile duct in hepatocyte transplantation recipients is followed by formation of bile canaliculi. The question arose as to whether the signal released from the obstructed bile vasculature activated the transplanted hepatocytes (HC) or cholangiocyte (CH) to form bile canaliculi. MethodsWe transplanted separately isolated autologous HC and CH to spleens and observed the structural organization of the grafted cells. ResultsHC formed glycogen-rich clusters but not cords usually not attached to the CH of the new bile canaliculi. Separate clusters of bile canaliculi with keratin 7 and 19–positive and gamma-glutamyl transpeptidase–positive cells were observed. Transplanted CH remained keratin 7 and 19–positive and gamma-glutamyl transpeptidase positive but did not form canaliculi. ConclusionsThe transplanted HC fraction may contain hepatic progenitor cells for cholangiocytes, but they become activated only under the condition of bile stasis by an as-yet undefined factor.

Effects of edaravone, a radical scavenger, on hepatocyte transplantation.

Hepatocyte transplantation (HTx) has yielded significant improvements in liver function and survival in experimentally induced acute liver failure and liver-based metabolic disease. However, transplantation is inefficient, and it is thought that transplanted hepatocytes have a shortened lifespan because of inflammation involving excess nitric oxide (NO). The present study aimed to clarify whether edaravone, a free radical scavenger used to treat ischemic stroke, could reduce ischemic changes in hepatocyte-transplanted livers. Edaravone (3 mg/kg) was administered intravenously 24 h before HTx to Nagase analbuminemic rats (NARs). Hepatocytes were isolated, and 30 × 10(6) cells were injected in a 1.0-ml volume directly into the spleens of NARs. All experimental groups studied received FK506 to control rejection. Animals in Group A received medium-only; Group B received HTx only; and Group C received HTx and edaravone. Forty-eight hours after transplantation, the hepatocytes from animals were isolated and analyzed for staining with propidium iodide- and annexin-V using flow cytometry. Liver sections were also studied by immunostaining for albumin, and TUNEL. Peripheral blood serum albumin levels were measured on post-transplant days 0, 3, 5, 7, 10 and 14 using ELISA. The edaravone-treated animals demonstrated an increased number of engrafted donor hepatocytes in the liver. The edaravone-treated liver sections also contained fewer TUNEL-positive cells and animals that received edaravone had higher serum albumin levels post-transplantation. Hepatocytes were also found to have increased in numbers 2 weeks following treatment with edaravone. Edaravone administration during HTx can suppress apoptosis near the transplanted cells, increasing engraftment. These studies indicate its potential usefulness for future clinical application.

Experience of Combined Liver-Kidney Transplantation for Acute-on-Chronic Liver Failure Patients With Renal Dysfunction

BackgroundThe aim of this study was to evaluate the outcomes of orthotopic liver transplantation (OLT) or combined liver-kidney transplantation (CLKT) for acute-on-chronic liver failure (ACLF) patients with renal dysfunction. MethodsFrom January 2001 to December 2009, 133 patients underwent OLT for ACLF at our center. Among them, 30 had both ACLF and renal dysfunction. Of the 30 patients, 12 underwent CLKT for end-stage renal disease (ESRD), and the other 18 with hepatorenal syndrome type 1 (HRS1) underwent OLT alone. ACLF was defined according to the Asian Pacific Association for the Study of Liver Consensus Meeting. Clinical data were reviewed for survival outcomes. ResultsThe median model for end-stage liver disease score (MELD) of patients with ACLF was 28. Among the 133 patients, all of whom received deceased donor liver grafts, 12 also got the kidney grafts from the same deceased donor. The hospital mortality rate was 21.8% for all patients with ACLF. The 5-year survival rates were 72.8% for patients without renal dysfunction and 70% for patients with renal dysfunction. The results of patients with ESRD who underwent CLKT were better than those of subjects without renal dysfunction or patients with HRS1 who underwent OLT alone. ConclusionsOLT alone improved renal function in most patients with HRS1, including those requiring short-term hemodialysis. Simultaneous liver-kidney transplantation was an excellent strategy for patients with both ACLF and ESRD. It provided protection to the kidney allograft for liver-based metabolic diseases affecting the kidney. The rate of acute rejection episodes in kidneys was low.

Placental stem cell correction of murine intermediate maple syrup urine disease

There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme-proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latter's safety and ethical concerns and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases.

Current Status of Hepatocyte Transplantation

Hepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation. The results are encouraging in genetic liver conditions where HT can replace the missing enzyme or protein. However, there are limitations to the technique, which need to be overcome. Unused donor livers to isolate hepatocytes are in short supply and are often steatotic, although addition of N-acetylcysteine improves the quality of the cells obtained. Hepatocytes are cryopreserved for later use and this is detrimental to metabolic function on thawing. There are improved cryopreservation protocols, but these need further refinement. Hepatocytes are usually infused into the hepatic portal vein with many cells rapidly cleared by the innate immune system, which needs to be prevented. It is difficult to detect engraftment of donor cells in the liver, and methods to track cells labeled with iron oxide magnetic resonance imaging contrast agents are being developed. Methods to increase cell engraftment based on portal embolization or irradiation of the liver are being assessed for clinical application. Encapsulation of hepatocytes allows cells to be transplanted intraperitoneally in acute liver failure with the advantage of avoiding immunosuppression. Alternative sources of hepatocytes, which could be derived from stem cells, are needed. Mesenchymal stem cells are currently being investigated particularly for their hepatotropic effects. Other sources of cells may be better if the potential for tumor formation can be avoided. With a greater supply of hepatocytes, wider use of HT and evaluation in different liver conditions should be possible.

Simultaneous combined liver and kidney transplantation: a single center experience

Renal dysfunction is common in patients awaiting liver transplantation (LT) and affects outcome following LT. Combined liver and kidney transplantation (CLKT) has been proposed as effective treatment for patients with chronic diseases of both organs, some with hepatorenal syndrome and for liver-based metabolic diseases affecting kidney. This study is undertaken to analyze results of CLKT at a single center. Of 2690 LTs performed between 1992 and 2007, there were 39 CLKTs; most common indications were metabolic, cirrhosis and polycystic disease. With follow-up of up to 170 months, 11 died (overall survival 71.8%); one-, five-, and 10-yr patient and liver graft survival is 77%, 73.7%, and 73.7%, respectively, and kidney graft survival is 77%, 70%, and 70%, respectively. Survival among metabolic group (78.6%) appeared to be better than non-metabolic group (68%); however, this difference was not significant (p = 0.39). Fifteen surviving patients (53.6%) have mild/moderate renal impairment (creatinine > or = 125 micromol/L). None has severe renal failure (serum creatinine > or = 250 micromol/L) or end-stage renal disease requiring hemodialysis. CLKT has good results in selected groups of patients. It provides protection to kidney allograft in liver-based metabolic diseases affecting kidney. The rate of acute rejection episodes of kidney is low. Significant proportion develops long-term mild/moderate renal dysfunction. Careful attention to immunosuppression to minimize nephrotoxicity may help.

Model for end-stage liver disease (MELD) exception for unusual metabolic liver diseases

Metabolic liver disease is the underlying diagnosis in only a small proportion of patients who undergo liver transplantation (LT), but for these patients, LT is lifesaving. Patients with metabolic liver disease often do not present with typical findings of end-stage liver disease and require special consideration and scrutiny concerning the appropriateness and timing of LT. Liverbased metabolic disease is classified into 3 types: (1) disease that causes structural liver damage with liver failure or cirrhosis, (2) metabolic disease without structural liver damage that affects other organs (especially the central nervous system), and (3) metabolic disease with systemic deficiencies that are partially represented in the liver. There may be overlap in presentation, with some disease forms presenting either with or without structural liver disease. General considerations that affect review board decisions may include the relative contraindication of the use of living-related donor organs and the unpredictable metabolic course that may cause severe central nervous system complications in several of these disease states. Also, although many of these diseases present mostly in children, adolescents and adults previously managed medically are increasingly presenting for LT consideration when medical management becomes more difficult or complex as they mature.

Liver repopulation after cell transplantation in mice treated with retrorsine and carbon tetrachloride.

Efficiency of engraftment after liver cell transplantation is less than 1% under conventional conditions. Our aim was to develop a high-efficiency, nonsurgical, no-genetic-advantage mouse model of liver repopulation with transplanted cells. Mice were conditioned with nonlethal doses of a cell cycle inhibitor, retrorsine, 70 mg/kg, to irreversibly block proliferation of native hepatocytes. After the drug was eliminated, 2 million freshly isolated beta-galactosidase-labeled liver cells were transplanted into the spleens of C57BL/6J recipient mice. To stimulate donor cell proliferation, three doses of carbon tetrachloride (CCl4), 0.5 ml/kg, were given. Several control groups were studied to evaluate the contribution of each treatment to liver repopulation. Repopulation, as measured by cell isolation from recipient livers 1-7 months after transplantation, was on average 20%. Repopulation was 10% if CCl4 was given only once, between 0.5% and 1% if only retrorsine or CCl4 were used, and 0.05% if no conditioning was used. Phenotypically, whole livers turned blue on exposure to X-gal staining, whereas negative (control) livers remained pale brown. More than 55% of liver repopulation resulted from clusters containing 21 or more cells, some of which contained more than 200 cells, suggesting seven or more rounds of cell division in a subset of transplanted cells. This murine study demonstrates high levels of repopulation after liver cell transplantation into nongenetically modified livers, using a cell cycle inhibitor and chemical liver injury to provide transplanted cells a proliferative advantage. Liver repopulation was effected mostly by a small fraction of transplanted cells. Analogous nonsurgical liver cell transplantation strategies, but with clinically applicable drugs, could be devised for the treatment of liver-based metabolic diseases.

Liver cell transplantation: The road to clinical application

J Lab Clin Med 2001;138:298-312.

A reversibly immortalized human hepatocyte cell line as a source of hepatocyte‐based biological support

The application of hepatocyte transplantation (HTX) is increasingly envisioned for temporary metabolic support during acute liver failure and provision of specific liver functions in inherited liver-based metabolic diseases. Compared with whole liver transplantation, HTX is a technically simple procedure and hepatocytes can be cryopreserved for future use. A major limitation of this form of therapy in humans is the worldwide shortage of human livers for isolating an adequate number of transplantable human hepatocyes when needed. Furthermore, the numbers of donor livers available for hepatocyte isolation is limited by competition for their use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for immediate preparation of consistent and functional cells, it is unlikely that human hepatocytes can be obtained on such a scale to treat a large number of patients with falling liver functions. The utilization of xenogenic hepatocytes will result in additional concerns regarding transmission of infectious pathogens and immunological and physiological incompatibilities between animals and humans. An attractive alternative to primary human hepatocytes is the use of tightly regulated human hepatocyte cell lines. Such cell lines can provide the advantages of unlimited availability, sterility and uniformity. We describe here methods for creating transplantable human hepatocyte cell lines using currently available cell cultures and gene transfer technology.

Successful Retroviral Gene Transfer of Simian Virus 40 T Antigen and Herpes Simplex Virus-Thymidine Kinase into Human Hepatocytes 1.

Current clinical reports have indicated that hepatocyte transplantation (HTX) could be used in patients with liver failure and in children with liver-based metabolic diseases. One of the major limiting factors of HTX is a serious shortage of donor livers for hepatocyte isolation. To address this issue, we immortalized adult human hepatocytes with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase simultaneously. One of the resulting clones, NKNT-3, grew steadily in chemically defined serum-free medium without any obvious crisis and showed the gene expression of differentiated liver functions. Under the administration of 5 μM ganciclovir, NKNT-3 cells stopped proliferation and died in in vitro experiments. We have established a tightly regulated immortal human hepatocyte cell line. The cells could allow the need for immediate availability of consistent and functionally uniform cells in sufficient quantity and adequate quality.

Complete neurological recovery of an adult patient with type II citrullinemia after living related partial liver transplantation.

Type II citrullinemia is an adult-onset hepatocerebral disease caused by a deficiency of argininosuccinate synthetase in liver. A 25-year-old Japanese man suddenly developed encephalopathy, showing disorientation and flapping tremor. Plasma concentrations of ammonia and citrulline were extremely high, and hepatic argininosuccinate synthetase activity was deficient. The patient's condition deteriorated rapidly in spite of intensive medications. Therefore, we performed a partial liver transplantation using a graft obtained from his healthy 61-year-old father. After surgery, his neurological symptoms soon disappeared and plasma levels of ammonia and citrulline were normalized within 3 months after operation. Type II citrullinemia is one fulminant form of various liver-based metabolic diseases, and immediate liver transplantation is necessary to rescue patients with this disease. As liver transplantation from cadaveric donor is still not possible in Japan, it seems justifiable to use living related partial liver transplantation for our patient.

Orthotopic liver transplantation for hepatic-based metabolic disorders

Abstract Between January 1989 and June 1993, a total of 470 liver transplantations were performed at King's College Hospital. Thirty-seven transplantations were performed in 34 patients with liver-based metabolic disease. There were 16 females and 18 males with a median age of 19 years (range 1 month to 62 years). There were 14 patients under 16 years of age. The indications for liver transplantation were Wilson's disease (n= 16), alpha 1-antitrypsin deficiency (n= 10), tyrosinaemia (n= 2), primary hyperoxaluria type 1 (PH1; n= 2), congenital haemochromatosis (n= 1), familial amyloidotic polyneuropathy (FAP; n= 1, familial hypercholesterolaemia) (n= 1) and Crigler-Najjar syndrome type I (CNS1; n= 1). These included two patients who received combined heart-liver grafts for familial hypercholesterolaemia and FAP, respectively. Two patients received combined liver-kidney transplants for PH1. There were four deaths: from sepsis (n= 2), acute hepatic vein obstruction in a left lateral segment graft (n= 1) and portal vein thrombosis with liver necrosis (n= 1). Three patients were re-transplanted, one for chronic rejection and two for hepatic artery thrombosis, giving an overall graft survival of 81% and patient survival of 88% (30/34), at a mean follow-up of 34 months (range 10–64 months).

Orthotopic liver transplantation for hepatic-based metabolic disorders

Between January 1989 and June 1993, a total of 470 liver transplantations were performed at King's College Hospital. Thirty-seven transplantations were performed in 34 patients with liver-based metabolic disease. There were 16 females and 18 males with a median age of 19 years (range 1 month to 62 years). There were 14 patients under 16 years of age. The indications for liver transplantation were Wilson's disease (n = 16), alpha 1-antitrypsin deficiency (n = 10), tyrosinaemia (n = 2), primary hyperoxaluria type 1 (PH1; n = 2), congenital haemochromatosis (n = 1), familial amyloidotic polyneuropathy (FAP; n = 1, familial hypercholesterolaemia) (n = 1) and Crigler-Najjar syndrome type I (CNS1; n = 1). These included two patients who received combined heart-liver grafts for familial hypercholesterolaemia and FAP, respectively. Two patients received combined liver-kidney transplants for PH1. There were four deaths: from sepsis (n = 2), acute hepatic vein obstruction in a left lateral segment graft (n = 1) and portal vein thrombosis with liver necrosis (n = 1). Three patients were retransplanted, one for chronic rejection and two for hepatic artery thrombosis, giving an overall graft survival of 81% and patient survival of 88% (30/34), at a mean follow-up of 34 months (range 10-64 months).