Liver Transplant Laboratory Research
Laboratory research is also a vital component of our strategy at Lurie Children’s to improve the care opportunities and successes of our patients. Ongoing laboratory research in the study of liver diseases includes the following.
Childhood obesity is epidemic in the country, and along with it, so are the associated conditions of type-2 diabetes, hypertension, hyperlipidemia and fatty liver disease. It is estimated that 1.6 million American children have non-alcoholic fatty liver disease (NAFLD), making it the most prevalent liver disease affecting children. A significant proportion of children with NAFLD develop non-alcoholic steatohepatitis (NASH), which consists of inflammation (hepatitis) in addition to fatty liver. NASH often leads to scarring and even cirrhosis.
It is estimated that within 10 years NASH-related cirrhosis will be the leading indication for all liver transplants — pediatric and adult alike. The mechanisms involved in the progression of NASH are unknown, though weight reduction can reverse the disease.
Diets Deficient in Methionine & Choline
We have developed an animal model which produces NASH by feeding a diet that is deficient in methionine and choline. This model was used to show that osteopontin is a key cytokine in the progression of NASH. Using this model, we also showed that an anti-inflammatory drug can reduce the progression of NASH. These findings led to publications in the American Journal of Physiology – Gastrointestinal and Liver Physiology and in the Journal of Hepatology.
Critical Relationship Between Leptin & Osteopontin
We have developed a unique mouse model of NASH in conjunction with type-2 diabetes and obesity in which we have shown a critical relationship between leptin and osteopontin in the progression of NASH. Findings in this model led to publication in the American Journal of Physiology – Gastrointestinal and Liver Physiology.
We have also developed a unique cell culture model of NASH in which hepatocytes accrue fat and make inflammatory cytokines including osteopontin when grown in culture medium free of methionine and choline. This model has allowed us to dissect the molecular signaling involved in the development of NASH. A paper from this work was published in the American Journal of Physiology – Gastrointestinal and Liver Physiology.
Reactive Oxygen Species Generated by Mitochondria
Using this cell model we have examined the role of reactive oxygen species generated by mitochondria in response to nutritional perturbation acting as an initial signal in the cascade leading to fatty liver. This work showed that hepatocytes have a “nutrient sensing” function and that hydrogen peroxide is the signal molecule produced by mitochondria in response to a change in nutrition. This work was published in the Journal of Biological Chemistry.
Our current work is looking at the nutrient signaling of liver in a mouse model. It employs mice that have portal-systemic shunts to test whether portal blood flow is an essential conduit for the nutrient signal to reach the liver for sensing to occur.
Neonatal hemochromatosis (NH) is a rare gestational condition in which iron accumulates in the liver and extrahepatic sites of the fetus in a distribution similar to that seen in hereditary hemochromatosis. It is usually lethal to the fetus or neonate. The risk of recurrence in subsequent offspring of a woman after the index case is greater than 80%. Dr. Whitington has hypothesized that recurrent NH is an alloimmune gestational disorder.
Intravenous Immunoglobulin Derived from Pooled Serum of Multiple Donors
Dr. Whitington designed a treatment to prevent recurrent lethal NH, wherein women with a history of having had their last pregnancy ending in documented NH were treated with intravenous immunoglobulin derived from pooled serum of multiple donors. To date, over 50 treatments have been performed. The outcomes of treated gestations have been improved relative to untreated gestations in the same women (p < 0.0001). These results strongly indicate that treatment with high-dose IV-Ig during gestation modified recurrent NH so that it was not lethal to the fetus or newborn. Early results were published in the Lancet and a second comprehensive paper is in press in Pediatrics.
Serum Collected From Affected Women as a Source Of Antibody
We are seeking to identify the target of the immune attack responsible for NH. We are using serum collected from affected women as a source of antibody to detect common fetal antigens that may be the antigen in question. The team is currently attempting to clone the protein using a couple of approaches. We have tentatively identified the target using immunoprecipitation, 2D-PAGE and AMLTI-TOF mass spectroscopy. The identity has permitted us to express the protein in a cell system and determine if it is recognized by NH sera.
We are also cloning directly from a human fetal liver cDNA library using the sera to identify on immunoblot clones of interest. If successful, cloning the fetal liver protein target of NH will permit us to develop an assay for detecting and quantifying antibody in the serum of women at risk. Being able to measure antibody will permit refinement of the treatment protocol. Early results of this work have been published in the Journal of Pediatric Gastroenterology and Nutrition, Pediatric Transplantation, Hepatology and Seminars in Liver Disease.
Determining the Pathophysiology of NH
Our group has reproduced NH in mice by injecting serum from affected women into pregnant mice producing severe liver injury and substantial mortality in the pups. The mechanism of injury appears to be direct binding of IgG to fetal hepatocytes with binding and activation of complement and formation of membrane attack complexes to kill hepatocytes. This work has extended into cell culture experiments using fetal hepatocytes and into an advanced mouse model employing C3 and C5 knockout mice. The overall goal is to determine the pathophysiology of NH so that better treatments can be designed.
Progressive familial intrahepatic cholestasis (PFIC) and Alagille syndrome (AGS) are genetically determined diseases that result in cholestasis.
Understanding Five Genetically Determined Pediatric Liver Diseases
Lurie Children’s is a clinical center in the NIH-funded Rare Liver Diseases Research Consortium (Cholestatic Liver Disease Consortium or CLiC) with Dr. Whitington serving as the principal investigator on the grant. This consortium’s goals are to understand the mechanisms of disease and improve treatments of five genetically determined pediatric liver diseases, including PFIC and AGS.
Understanding the Development of Liver Cancer (Hepatocellular Carcinoma)
We participated in a world-wide study that identified a select group of children with genetic cholestasis, namely PFIC-2 (BSEP deficient) patients, who appear to have a high risk of developing liver cancer (hepatocellular carcinoma). This work was recently published in Hepatology.
Identifying the Particular Aspects of PFIC-2 Patients that Make them Cancer Susceptible
Following up on that work, our lab in collaboration with Dr. Bento Soares from the Research Center are studying the gene expression profiles of livers from BSEP patients and several control livers with a goal of identifying the particular aspects of PFIC-2 patients that make them cancer susceptible. This work will mainly involve the use of gene chip array technology.