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.

Current Advances in Graft-versus-host Disease After Intestinal Transplantation.

Graft-versus-host disease (GvHD) remains a potentially fatal complication following intestinal transplant (ITx). Over the past decade, advances in the understanding of the pathophysiology of this complex immunological phenomenon have led to the reassessment of the host systemic immune response and have created a gateway for novel preventive and therapeutic strategies. Although sufficient evidence dictates the use of corticosteroids as a first-line option, the treatment for refractory disease remains contentious and lacks a standardized therapeutic approach. Timely diagnosis remains crucial, and the advent of chimerism detection and immunological biomarkers have transformed the identification, prognostication, and potential for survival after GvHD in ITx. The objectives of the following review aim to discuss the clinical and diagnostic features, pathophysiology, advances in immune biomarkers, as well as therapeutic opportunities in the prevention and treatment of GvHD in ITx.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Oral N-acetylcysteine decreases IFN-γ production and ameliorates ischemia-reperfusion injury in steatotic livers.

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

Type 1 Innate Lymphoid Cells Are Proinflammatory Effector Cells in Ischemia-Reperfusion Injury of Steatotic Livers.

Innate lymphoid cells (ILCs), the most recently described family of lymphoid cells, play fundamental roles in tissue homeostasis through the production of key cytokine. Group 1 ILCs, comprised of conventional natural killer cells (cNKs) and type 1 ILCs (ILC1s), have been implicated in regulating immune-mediated inflammatory diseases. However, the role of ILC1s in nonalcoholic fatty liver disease (NAFLD) and ischemia-reperfusion injury (IRI) is unclear. Here, we investigated the role of ILC1 and cNK cells in a high-fat diet (HFD) murine model of partial warm IRI. We demonstrated that hepatic steatosis results in more severe IRI compared to non-steatotic livers. We further elicited that HFD-IRI mice show a significant increase in the ILC1 population, whereas the cNK population was unchanged. Since ILC1 and cNK are major sources of IFN-γ and TNF-α, we measured the level of ex vivo cytokine expression in normal diet (ND)-IRI and HFD-IRI conditions. We found that ILC1s in HFD-IRI mice produce significantly more IFN-γ and TNF-α when compared to ND-IRI. To further assess whether ILC1s are key proinflammatory effector cells in hepatic IRI of fatty livers, we studied both Rag1-/- mice, which possess cNK cells, and a substantial population of ILC1s versus the newly generated Rag1-/-Tbx21-/- double knockout (Rag1-Tbet DKO) mice, which lack type 1 ILCs, under HFD IRI conditions. Importantly, HFD Rag1-Tbet DKO mice showed significant protection from hepatic injury upon IRI when compared to Rag1-/- mice, suggesting that T-bet-expressing ILC1s play a role, at least in part, as proinflammatory effector cells in hepatic IRI under steatotic conditions.

Long-term outcomes of endoscopic mucosal resection for early-stage esophageal adenocarcinoma.

BACKGROUND: With growing application of endoscopic therapy for early-stage esophageal cancer, we sought to review our experience of endoscopic mucosal resections (EMRs). The aim of our study was to understand the natural course of these patients, especially with positive margins. METHODS: A prospectively maintained database of all patients undergoing endoscopic therapies at Georgetown University Hospital for esophageal cancer was used for the analysis between 2010 and 2020. RESULTS: Of 80 patients in the EMR database, 35 were performed as index cases for esophageal adenocarcinoma. Majority (74.3%) had a pre-treatment ultrasound confirming absence of regional adenopathy. There were no post-EMR bleeding or perforation events requiring re-intervention. Complete R0 resection was achieved in 22/35 (62.9%) after initial EMR. Thirteen patients had positive margins. Of these 13 patients, only 7 patients underwent repeat endoscopic resection, 2 underwent subsequent esophagectomy, 2 received definitive radiation given poor surgical candidacy, and 2 were lost to follow-up. Overall and 5-year survival of all patients undergoing EMR was 67.9 months and 85%, respectively. Subset analysis of the 13 patients with R1 resection demonstrated an overall survival of 49.2 months and 60% 5-year survival vs overall survival of 78.9 months and 93% 5-year survival for R0 resection. At a median follow-up of 60.5 months, cancer recurrence occurred in 3 patients. All of them were successfully managed with repeat EMR. CONCLUSIONS: Endoscopic resections represent a safe and effective treatment for early-stage esophageal cancer. Patients with high-risk features should be counseled to undergo an esophagectomy if they are operable candidates.

Pancreaticoduodenectomy hospital resource utilization in octogenarians.

BACKGROUND: Although pancreaticoduodenectomy (PD) is feasible in patients greater than or equal to 80 years, little is known about the potential strain on resource utilization. METHODS: Outcomes and inpatient charges were compared across age cohorts (I: ≤70, II: 71 to 79, III: ≥80 years) in 99 patients who underwent PD (2005 to 2013) at our institution. The generalized linear modeling approach was used to estimate the impact of age. RESULTS: Perioperative complications were equivalent among cohorts. Increasing age was associated with intensive care unit use, increased length of stay (LOS), and the likelihood of discharge to a skilled facility. After controlling for covariates, hospital charges were significantly higher in Cohort III (P = .006) and Cohort II (P = .035) when compared with Cohort I. However, hospital charges between Cohorts II and III were equivalent (P = .374). Complications (P = .005) and LOS (P < .001) were associated with higher hospital charges. CONCLUSIONS: Increasing age was associated with increased intensive care unit, LOS, and discharge to skilled facilities. However, octogenarians had equivalent PD charges and outcome measures when compared with septuagenarians and future studies should validate these findings in larger national studies.

Laparoscopic-assisted versus open pancreaticoduodenectomy: early favorable physical quality-of-life measures.

BACKGROUND: We compared outcomes and postpancreatectomy quality of life (QOL) in paired cohorts of patients undergoing conventional open pancreaticoduodenectomy (OPD) or laparoscopic-assisted pancreaticoduodenectomy (LAPD). METHODS: Comparative analysis of QOL was performed in a matched cohort of 53 patients after OPD or LAPD between 2010 and 2013. The Medical Outcomes Study Short Form-36 Health Survey and the Karnofsky score were used. RESULTS: Physical component score, mental component score, and Karnofsky scores were calculated at multiple time points for OPD (n = 25) and LAPD (n = 28). Operative times, complications, and readmission rates were equivalent. Time to starting adjuvant therapy trended toward clinical importance in LAPD (61 vs 110 days, P = .0878). Duration of stay was less in LAPD (7.10 vs 9.44 days, P = .02). LAPD had a superior QOL centered on functional status compared with OPD (physical component score 49.09 vs 38.4, P = .04; Karnofsky 92.22 vs 66.92%, P = .003). These statistical differences were not observed beyond 6 months. CONCLUSION: LAPD provided a more favorable QOL within the first 6 months and shorter length of stay compared with conventional OPD. LAPD may serve as an alternative operative therapy to potentially minimize delays in receipt of and enhance tolerability of adjuvant therapies.