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BackgroundLoss of intestinal barrier integrity plays a fundamental role in the pathogenesis of various gastrointestinal diseases and is implicated in the onset of sepsis and multiple organ failure. An array of methods to assess different aspects of intestinal barrier function suffers from lack of sensitivity, prolonged periods of specimen collection, or high expense. We have developed a technique to measure the concentration of the food dye FD&C Blue #1 from blood and sought to assess its utility in measuring intestinal barrier function in humans.Materials and methodsFour healthy volunteers and 10 critically ill subjects in the intensive care unit were recruited in accordance with an institutional review board approved protocol. Subjects were given 0.5 mg/kg Blue #1 enterally as an aqueous solution of diluted food coloring. Five blood specimens were drawn per subject: 0 h (before dose), 1, 2, 4, and 8 h. After plasma isolation, organic extracts were analyzed by high-performance liquid chromatography/mass spectrometry detecting the presence of unmodified dye.ResultsWe found no baseline detectable absorption in healthy volunteers. After including the subjects in the intensive care unit, we compared dye absorption in the six subjects who met criteria for septic shock with the eight who did not. Septic patients demonstrated significantly greater absorption of Blue #1 after 2 h.ConclusionsWe have developed a novel, easy-to-use method to measure intestinal barrier integrity using a food grade dye detectable by mass spectrometry analysis of patient blood following oral administration.

Drug-induced liver failure is a significant indication for a liver transplant, and unexpected liver toxicity is a major reason that otherwise effective therapies are removed from the market. Various methods exist for monitoring liver injury but are often inadequate to predict liver failure. New diagnostic tools are needed. Methods: We evaluate in a preclinical model whether 18F-2-deoxy-2-fluoroarabinose (18F-DFA), a PET radiotracer that measures the ribose salvage pathway, can be used to monitor acetaminophen-induced liver injury and failure. Mice treated with vehicle, 100, 300, or 500 mg/kg acetaminophen for 7 or 21 h were imaged with 18F-FDG and 18F-DFA PET. Hepatic radiotracer accumulation was correlated to survival and percentage of nonnecrotic tissue in the liver. Mice treated with acetaminophen and vehicle or N-acetylcysteine were imaged with 18F-DFA PET. 18F-DFA accumulation was evaluated in human hepatocytes engrafted into the mouse liver. Results: We show that hepatic 18F-DFA accumulation is 49%-52% lower in mice treated with high-dose acetaminophen than in mice treated with low-dose acetaminophen or vehicle. Under these same conditions, hepatic 18F-FDG accumulation was unaffected. At 21 h after acetaminophen treatment, hepatic 18F-DFA accumulation can distinguish mice that will succumb to the liver injury from those that will survive it (6.2 vs. 9.7 signal to background, respectively). Hepatic 18F-DFA accumulation in this model provides a tomographic representation of hepatocyte density in the liver, with a R2 between hepatic 18F-DFA accumulation and percentage of nonnecrotic tissue of 0.70. PET imaging with 18F-DFA can be used to distinguish effective from ineffective resolution of acetaminophen-induced liver injury with N-acetylcysteine (15.6 vs. 6.2 signal to background, respectively). Human hepatocytes, in culture or engrafted into a mouse liver, have levels of ribose salvage activity similar to those of mouse hepatocytes. Conclusion: Our findings suggest that PET imaging with 18F-DFA can be used to visualize and quantify drug-induced acute liver injury and may provide information on the progression from liver injury to hepatic failure.

Drug-induced liver failure is a significant indication for a liver transplant, and unexpected liver toxicity is a major reason that otherwise effective therapies are removed from the market. Various methods exist for monitoring liver injury but are often inadequate to predict liver failure. New diagnostic tools are needed. Methods: We evaluate in a preclinical model whether 18F-2-deoxy-2-fluoroarabinose (18F-DFA), a PET radiotracer that measures the ribose salvage pathway, can be used to monitor acetaminophen-induced liver injury and failure. Mice treated with vehicle, 100, 300, or 500 mg/kg acetaminophen for 7 or 21 h were imaged with 18F-FDG and 18F-DFA PET. Hepatic radiotracer accumulation was correlated to survival and percentage of nonnecrotic tissue in the liver. Mice treated with acetaminophen and vehicle or N-acetylcysteine were imaged with 18F-DFA PET. 18F-DFA accumulation was evaluated in human hepatocytes engrafted into the mouse liver. Results: We show that hepatic 18F-DFA accumulation is 49%-52% lower in mice treated with high-dose acetaminophen than in mice treated with low-dose acetaminophen or vehicle. Under these same conditions, hepatic 18F-FDG accumulation was unaffected. At 21 h after acetaminophen treatment, hepatic 18F-DFA accumulation can distinguish mice that will succumb to the liver injury from those that will survive it (6.2 vs. 9.7 signal to background, respectively). Hepatic 18F-DFA accumulation in this model provides a tomographic representation of hepatocyte density in the liver, with a R2 between hepatic 18F-DFA accumulation and percentage of nonnecrotic tissue of 0.70. PET imaging with 18F-DFA can be used to distinguish effective from ineffective resolution of acetaminophen-induced liver injury with N-acetylcysteine (15.6 vs. 6.2 signal to background, respectively). Human hepatocytes, in culture or engrafted into a mouse liver, have levels of ribose salvage activity similar to those of mouse hepatocytes. Conclusion: Our findings suggest that PET imaging with 18F-DFA can be used to visualize and quantify drug-induced acute liver injury and may provide information on the progression from liver injury to hepatic failure.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.