Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

The First Collective Examination of Immunosuppressive Practices Among American Intestinal Transplant Centers.

Background: Unlike other solid organs, no standardized treatment algorithms exist for intestinal transplantation (ITx). We established a consortium of American ITx centers to evaluate current practices. Methods: All American centers performing ITx during the past 3 y were invited to participate. As a consortium, we generated questions to evaluate and collect data from each institution. The data were compiled and analyzed. Results: Ten centers participated, performing 211 ITx during the past 3 y (range, 3-46; mean 21.1). Induction regimens varied widely. Thymoglobulin was the most common, used in the plurality of patients (85/211; 40.3%), but there was no consensus regimen. Similarly, regimens for the treatment of acute cellular rejection, antibody-mediated rejection, and graft-versus-host disease varied significantly between centers. We also evaluated differences in maintenance immunosuppression protocols, desensitization regimens, mammalian target of rapamycin use, antimetabolite use, and posttransplantation surveillance practices. Maintenance tacrolimus levels, stoma presence, and scoping frequency were not associated with differences in rejection events. Definitive association between treatments and outcomes, including graft and patient survival, was not the intention of this initial collaboration and is prevented by the lack of patient-level data and the presence of confounders. However, we identified trends regarding rejection episodes after various induction strategies that require further investigation in our subsequent collaborations. Conclusions: This initial collaboration reveals the extreme heterogeneity of practices among American ITx centers. Future collaboration will explore patient-level data, stratified by age and transplant type (isolated intestine versus multivisceral), to explore the association between treatment regimens and outcomes.

Factors Associated With 5- and 10-Year Survival After Intestinal Transplantation in Infants and Children.

OBJECTIVES: Intestinal transplantation is an option for permanent intestinal failure with parenteral nutrition intolerance. We sought to determine long-term intestinal graft survival in pediatric patients at our center and to identify factors influencing survival. METHODS: Retrospective chart review of 86 patients transplanted between 2003 and 2013, targeting potential explanatory variables related to demographics, perioperative factors, and postoperative complications. RESULTS: Intestinal graft survival was 71% and 65% after 5 and 10 years, respectively. Five-year graft survival was attained in 79% of patients with a history of anatomic intestinal failure compared with 45% with functional intestinal failure (P = 0.0055). Compared with nonsurvival, 5-year graft survival was also associated with reduced incidences of graft-versus-host disease (2% vs 16%, P = 0.0237), post-transplant lymphoproliferative disorder (3% vs 24%, P = 0.0067), and de novo donor-specific antibodies (19% vs 57%, P = 0.0451) plus a lower donor-recipient weight ratio (median 0.727 vs 0.923, P = 0.0316). Factors not associated with 5-year intestinal graft survival included graft rejection of any severity and inclusion of a liver graft. Factors associated with graft survival at 10 years were similar to those at 5 years. CONCLUSIONS: In our experience, outcomes in pediatric intestinal transplantation have improved substantially for anatomic but not functional intestinal failure. Graft survival depends on avoidance of severe infectious and immunological complications including GVHD, whereas inclusion of a liver graft provides no obvious survival benefit. Reduced success with functional intestinal failure may reflect inherently increased susceptibility to complications in this group.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Optimized adeno-associated virus 8 produces hepatocyte-specific Cre-mediated recombination without toxicity or affecting liver regeneration.

UNLABELLED: Engineering viral vectors to produce liver-specific protein expression may help advance understanding of hepatic regeneration and disease states. In addition to introducing genes of interest to the liver, these vectors can be adapted for gene deletion when designed to express Cre recombinase. The ability to use this system requires high, liver-restricted expression, low toxicity, and no effect on the process of interest. We developed an adeno-associated virus 8 (AAV8) with a codon-optimized Cre recombinase under a hepatocyte-specific major urinary protein (MUP) promoter (MUP-iCre-AAV8) that fulfills these requirements. A single intravenous injection of ROSA26R reporter mice, which express lacZ after Cre-mediated recombination, demonstrated homogeneous beta-galactosidase expression limited to hepatocytes after only 7 days. Cre protein expression remained strong for at least 31 days. Serum liver function tests and histology demonstrated minimal liver toxicity. The presence of MUP-iCre-AAV8 did not affect hepatocyte proliferation after partial hepatectomy as measured by Ki67 staining. CONCLUSION: AAV8 with the MUP promoter, by virtue of its lack of hepatic toxicity or effect on liver regeneration, may be an efficient alternative to complex transgenic methodologies for studies of the mouse liver.