Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Reliability of histologic assessment for NAFLD and development of an expanded NAFLD activity score.

BACKGROUND AND AIMS: The NASH Clinical Research Network histologic scoring system, the gold-standard NASH histology assessment for clinical trials, has demonstrated intrarater and interrater variability. An expert panel in a previous systematic Research and Development/University of California Los Angeles (RAND/UCLA) study determined that existing histologic scoring systems do not fully capture NASH disease activity and fibrosis, and standardized definitions of histologic features are needed. We evaluated the reliability of existing and alternate histologic measures and their correlations with a disease activity visual analog scale to propose optimal components for an expanded NAFLD activity score (NAS). APPROACH AND RESULTS: Four liver pathologists who were involved in the prior RAND/UCLA study underwent standardized training and multiple discussions with the goal of improving agreement. They were blinded to clinical information and scored histologic measures twice, ≥2 weeks apart, for 40 liver biopsies representing the full spectrum of NAFLD. Index intraclass correlation coefficient (ICC) estimates demonstrated intrarater (0.80-0.85) and interrater (0.60-0.72) reliability. Hepatocyte ballooning items had similar interrater ICCs (0.68-0.79), including those extending scores from 0-2 to 0-4. Steatosis measures (interrater ICCs, 0.72-0.80) correlated poorly with disease activity. Correlations with disease activity were largest for hepatocyte ballooning and Mallory-Denk bodies (MDBs), with both used to develop the expanded NAS (intrarater ICC, 0.90; interrater ICC, 0.80). Fibrosis measures had ICCs of 0.70-0.87. CONCLUSIONS: After extensive preparation among a group of experienced pathologists, we demonstrated improved reliability of multiple existing histologic NAFLD indices and fibrosis staging systems. Hepatocyte ballooning and MDBs most strongly correlated with disease activity and were used for the expanded NAS. Further validation including evaluation of responsiveness is required.

Immunohistochemistry and immunofluorescence utilization audit by subspecialty in an academic setting: A step toward stewardship.

There are immunohistochemistry (IHC) and immunofluorescence (IF) panels described in the literature and established by personal and institutional experiences that are in common use by pathologists in their daily practice. Stewardship is a difficult discussion because IHC utilization is influenced by many factors including the pathologist's experience, background, practice setting, personal bias, and medicolegal culture. We developed the methodology to audit the IHC/IF utilization in our academic subspecialty practice. We aim to share this methodology and to provide our data that can be used for consideration by other subspecialized academic practices. This analysis included a total of 63,157 specimens that were accessioned during 2022, representing 38,612 cases. The likelihood of ordering IHC/IF ranged from 1 % (in genitourinary pathology) to 59 % (in renal pathology). The average percentage of specimens with IHC/IF was 21 % for the entire practice. In cases where IHC/IF was ordered, the number of stained slides averaged 4.9 per specimen for the entire practice. The number of IHC/IF slides per specimen ranged from 1.9 (in gastrointestinal pathology) to 12.2 (in renal pathology). The highest number of antibodies ordered for a single specimen by subspecialty ranged from 11 (in cardiac pathology) to 63 (in dermatopathology). Renal pathology was the only subspecialty that had an average number of IHC/IF slides that was statistically significantly different from all other subspecialties. We described the various patterns of utilization by subspecialty and rationalized their subtle differences. We also analyzed the types of cases that exceeded the reimbursement limits set by the Centers for Medicare and Medicaid Services (CMS).

Evaluation of a gene expression biomarker to identify operationally tolerant liver transplant recipients: the LITMUS trial.

LITMUS was a single-centre, Phase 2a study designed to investigate whether the gene biomarker FGL2/IFNG previously reported for the identification of tolerance in murine models could identify operationally tolerant liver transplant recipients. Multiplex RT-PCR was used to amplify eight immunoregulatory genes in peripheral blood mononuclear cells (PBMC) from 69 adult liver transplant recipients. Patients with PBMC FGL2/IFNG ≥ 1 and a normal liver biopsy underwent immunosuppression (IS) withdrawal. The primary end point was the development of operational tolerance. Secondary end points included correlation of tolerance with allograft gene expression and immune cell markers. Twenty-eight of 69 patients (38%) were positive for the PBMC tolerance biomarker and 23 proceeded to IS withdrawal. Nine of the 23 patients had abnormal baseline liver biopsies and were excluded. Of the 14 patients with normal biopsies, eight (57%) have achieved operational tolerance and are off IS (range 12-57 months). Additional studies revealed that all of the tolerant patients and only one non-tolerant patient had a liver gene ratio of FOXP3/IFNG ≥ 1 prior to IS withdrawal. Increased CD4+ T regulatory T cells were detected both in PBMC and livers of tolerant patients following IS withdrawal. Higher expression of SELE (gene for E-selectin) and lower expression of genes associated with inflammatory responses (GZMB, CIITA, UBD, LSP1, and CXCL9) were observed in the pre-withdrawal liver biopsies of tolerant patients by RNA sequencing. These results suggest that measurement of PBMC FGL2/IFNG may enrich for the identification of operationally tolerant liver transplant patients, especially when combined with intragraft measurement of FOXP3/IFNG. Clinical Trial Registration: ClinicalTrials.gov (LITMUS: NCT02541916).

Microbiota-Driven Activation of Intrahepatic B Cells Aggravates NASH Through Innate and Adaptive Signaling.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis is rapidly becoming the leading cause of liver failure and indication for liver transplantation. Hepatic inflammation is a key feature of NASH but the immune pathways involved in this process are poorly understood. B lymphocytes are cells of the adaptive immune system that are critical regulators of immune responses. However, the role of B cells in the pathogenesis of NASH and the potential mechanisms leading to their activation in the liver are unclear. APPROACH AND RESULTS: In this study, we report that NASH livers accumulate B cells with elevated pro-inflammatory cytokine secretion and antigen-presentation ability. Single-cell and bulk RNA sequencing of intrahepatic B cells from mice with NASH unveiled a transcriptional landscape that reflects their pro-inflammatory function. Accordingly, B-cell deficiency ameliorated NASH progression, and adoptively transferring B cells from NASH livers recapitulates the disease. Mechanistically, B-cell activation during NASH involves signaling through the innate adaptor myeloid differentiation primary response protein 88 (MyD88) as B cell-specific deletion of MyD88 reduced hepatic T cell-mediated inflammation and fibrosis, but not steatosis. In addition, activation of intrahepatic B cells implicates B cell-receptor signaling, delineating a synergy between innate and adaptive mechanisms of antigen recognition. Furthermore, fecal microbiota transplantation of human NAFLD gut microbiotas into recipient mice promoted the progression of NASH by increasing the accumulation and activation of intrahepatic B cells, suggesting that gut microbial factors drive the pathogenic function of B cells during NASH. CONCLUSION: Our findings reveal that a gut microbiota-driven activation of intrahepatic B cells leads to hepatic inflammation and fibrosis during the progression of NASH through innate and adaptive immune mechanisms.

A Stepwise Algorithmic Approach and External Validation Study for Noninvasive Prediction of Advanced Fibrosis in Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Advanced F3-4 fibrosis predicts liver-related mortality in nonalcoholic fatty liver disease (NAFLD). Noninvasive tests, designed to rule in/out advanced fibrosis, are limited by indeterminates, necessitating biopsy. We aimed to determine whether stepwise combinations of noninvasive serum-based tests and elastography (VCTE) could predict F3-4, reduce indeterminates, and decrease liver biopsies. METHODS AND RESULTS: Five hundred forty-one biopsy-proven NAFLD cases were identified between 2010 and 2018 from two Canadian centers. Characteristics of training (n = 407)/validation (n = 134) cohorts included: males 54%/59%; mean age 48.5/52.5 years; mean body mass index 32.3/33.6 kg/m2; diabetes mellitus 30%/34%; and F3-4 48%/43%. For training/validation cohorts, area under the receiver operating curve (AUROC) for FIB-4, AST-platelet ratio index (APRI), NAFLD fibrosis score (NFS), BARD score, and AST/ALT ratio ranged from 0.70 to 0.83/0.68 to 0.81, with indeterminates 25-39%/34-45%, for F3-4. In the training cohort, parallel FIB-4 + NFS had good accuracy (AUROC = 0.81) but was limited by 38% indeterminates and 16% misclassified. Sequential FIB-4 → NFS reduced indeterminates to 10%, and FIB-4 → VCTE to 0%, misclassified 20-22%, while maintaining high specificity (0.88-0.92) and accuracy (AUROC 0.75-0.78) for combined cohorts. Liver biopsy could have been avoided in 27-29% of patients using sequential algorithms. CONCLUSIONS: Sequential FIB-4 âž” NFS/VCTE predicts F3-4 with high specificity and good accuracy, while reducing indeterminates and need for biopsy. Parallel algorithms are limited by high indeterminates. Sequential FIB-4 âž” NFS had similar accuracy to VCTE-containing algorithms. Validation in low-prevalence cohorts may allow for potential use in community or resource-limited areas for risk stratification.

Nonalcoholic Steatohepatitis: A Review.

Importance: Nonalcoholic steatohepatitis (NASH) is the inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) and is associated with disease progression, development of cirrhosis, and need for liver transplant. Despite its importance, NASH is underrecognized in clinical practice. Observations: NASH affects an estimated 3% to 6% of the US population and the prevalence is increasing. NASH is strongly associated with obesity, dyslipidemia, type 2 diabetes, and metabolic syndrome. Although a number of noninvasive tests and scoring systems exist to characterize NAFLD and NASH, liver biopsy is the only accepted method for diagnosis of NASH. Currently, no NASH-specific therapies are approved by the US Food and Drug Administration. Lifestyle modification is the mainstay of treatment, including dietary changes and exercise, with the primary goal being weight loss. Substantial improvement in histologic outcomes, including fibrosis, is directly correlated with increasing weight loss. In some cases, bariatric surgery may be indicated to achieve and maintain the necessary degree of weight loss required for therapeutic effect. An estimated 20% of patients with NASH will develop cirrhosis, and NASH is predicted to become the leading indication for liver transplants in the US. The mortality rate among patients with NASH is substantially higher than the general population or patients without this inflammatory subtype of NAFLD, with annual all-cause mortality rate of 25.56 per 1000 person-years and a liver-specific mortality rate of 11.77 per 1000 person-years. Conclusions and Relevance: Nonalcoholic steatohepatitis affects 3% to 6% of the US population, is more prevalent in patients with metabolic disease and obesity, progresses to cirrhosis in approximately 20% of cases, and is associated with increased rates of liver-specific and overall mortality. Early identification and targeted treatment of patients with nonalcoholic steatohepatitis are needed to improve patient outcomes, including directing patients toward intensive lifestyle modification to promote weight loss and referral for bariatric surgery as indicated for management of obesity and metabolic disease.

Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts.

Normothermic ex situ liver perfusion (NEsLP) offers the opportunity to assess biomarkers of graft function and injury. We investigated NEsLP parameters (biomarkers and markers) for the assessment of liver viability in a porcine transplantation model. Grafts from heart-beating donors (HBD), and from donors with 30 minutes (donation after cardiac death [DCD]30'), 70 minutes (DCD70'), and 120 minutes (DCD120') of warm ischemia were studied. The HBD, DCD30', and DCD70'-groups had 100% survival. In contrast, 70% developed primary nonfunction (PNF) and died in the DCD120'-group. Hepatocellular function during NEsLP showed low lactate (≤1.1 mmol/L) in all the groups except the DCD120'-group (>2 mmol/L) at 4 hours of perfusion (P = .04). The fold-urea increase was significantly lower in the DCD120'-group (≤0.4) compared to the other groups (≥0.65) (P = .01). As for cholangiocyte function, bile/perfusate glucose ratio was significantly lower (<0.6) in all the groups except the DCD120'-group (≥0.9) after 3 hours of perfusion (<0.01). Bile/perfusate Na+ ratio was significantly higher (≥1.2) after 3 hours of perfusion in all the groups except for the DCD120'-group (≤1) (P < .01). Three hours after transplantation, the DCD120'-group had a significantly higher international normalized ratio (>5) compared to the rest of the groups (≤1.9) (P = .02). Rocuronium levels were higher at all the time-points in the animals that developed PNF during NEsLP and after transplantation. This study demonstrates that biomarkers and markers of hepatocellular and cholangiocyte function during NEsLP correlate with the degree of ischemic injury and posttransplant function.

Predictors of De Novo Nonalcoholic Fatty Liver Disease After Liver Transplantation and Associated Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) can occur de novo in patients undergoing liver transplantation (LT) for indications other than NAFLD, and it has been increasingly recognized as a complication in the post-LT setting. This study aims to better characterize de novo NAFLD after LT by identifying risk factors for its development, describing incidence and extent of fibrosis, assessing the diagnostic utility of noninvasive serum fibrosis algorithms, and comparing survival to those without NAFLD. This was a retrospective single-center analysis of de novo NAFLD in a post-LT cohort. Those whose primary indication for LT was nonalcoholic steatohepatitis (NASH) were excluded. Risk factors were analyzed by univariate and multivariate analyses. De novo NAFLD and fibrosis were assessed on posttransplant liver biopsies, and noninvasive fibrosis scores were calculated from concomitant blood tests. After applying the exclusion criteria, 430 for-cause post-LT biopsies were evaluated; 33.3% (n = 143) had evidence of de novo steatosis and/or NASH at a median of 3.0 years after transplant. On multivariate analysis, body mass index (BMI; odds ratio [OR], 1.12; P < 0.001), diabetes mellitus (OR, 3.01; P = 0.002), hepatitis C virus (OR, 4.61; P < 0.001), weight gain (OR, 1.03; P = 0.007), and sirolimus use (OR, 3.11; P = 0.02) were predictive of de novo NAFLD after LT. Significant fibrosis (≥F2) was present in almost 40% of the cohort. Noninvasive serum fibrosis scores were not useful diagnostic tests. There was no significant difference in the short-term or longterm survival of patients who developed de novo NAFLD. In conclusion, diabetes, BMI, weight gain after LT, and sirolimus-based immunosuppression, in keeping with insulin resistance, were the only modifiable factors associated with development of de novo NAFLD. A significant proportion of patients with de novo NAFLD had fibrosis and given the limited utility of noninvasive serum fibrosis algorithms, alternative noninvasive tools are required to screen for fibrosis in this population. There was no significant difference in the short-term or longterm survival of patients who developed de novo NAFLD.

Inhibition of the Fibrinogen-Like Protein 2:FcγRIIB/RIII immunosuppressive pathway enhances antiviral T-cell and B-cell responses leading to clearance of lymphocytic choriomeningitis virus clone 13.

Persistent viruses evade immune detection by interfering with virus-specific innate and adaptive antiviral immune responses. Fibrinogen-like protein-2 (FGL2) is a potent effector molecule of CD4+  CD25+  FoxP3+ regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, FcγRIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone-13 (LCMV cl-13) was assessed in this study. Chronically infected fgl2+/+ mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC-II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2-/- mice or fgl2+/+ mice that had been pre-treated with antibodies to FGL2 and FcγRIIB/RIII and then infected with LCMV cl-13 developed a robust CD4+ and CD8+ antiviral T-cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and FcγRIIB/RIII was shown to rescue the number and functionality of virus-specific CD4+ and CD8+ T cells with reduced total and virus-specific T-cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.

Mechanism of hard-nanomaterial clearance by the liver.

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

Subnormothermic ex vivo liver perfusion is a safe alternative to cold static storage for preserving standard criteria grafts.

We developed a novel technique of subnormothermic ex vivo liver perfusion (SNEVLP) for the storage of liver grafts before transplantation. To test the safety of SNEVLP for the nonextended criteria grafts (standard grafts), we compared it to a control group with minimal cold static storage (CS) time. Heart-beating pig liver retrieval was performed. Grafts were either stored in cold unmodified University of Wisconsin solution (CS-1), in cold University of Wisconsin solution with ex vivo perfusion additives (CS-2), or preserved with a sequence of 3 hours CS and 3 hours SNEVLP (33°C), followed by orthotopic liver transplantation. Liver function tests and histology were investigated. Aspartate aminotransferase (AST) levels during SNEVLP remained stable (54.3 ± 12.6 U/L at 1 hour to 47.0 ± 31.9 U/L at 3 hours). Posttransplantation, SNEVLP versus CS-1 livers had decreased AST levels (peak at day 1, 1081.9 ± 788.5 versus 1546.7 ± 509.3 U/L; P = 0.14; at day 2, 316.7 ± 188.1 versus 948.2 ± 740.9 U/L; P = 0.04) and alkaline phosphatase levels (peak at day 1, 150.4 ± 19.3 versus 203.7 ± 33.6 U/L; P = 0.003). Bilirubin levels were constantly within the physiological range in the SNEVLP group, whereas the CS-1 group presented a large standard deviation, including pathologically increased values. Hyaluronic acid as a marker of endothelial cell (EC) function was markedly improved by SNEVLP during the early posttransplant phase (5 hours posttransplant, 1172.75 ± 598.5 versus 5540.5 ± 2755.4 ng/mL). Peak international normalized ratio was similar between SNEVLP and CS-1 groups after transplantation. Immunohistochemistry for cleaved caspase 3 demonstrated more apoptotic sinusoidal cells in the CS-1 group when compared to SNEVLP grafts 2 hours after reperfusion (19.4 ± 19.5 versus 133.2 ± 48.8 cells/high-power field; P = 0.002). Adding normothermic CS-2 had no impact on liver injury or function after transplantation when compared to CS-1. In conclusion, SNEVLP is safe to use for standard donor grafts and is associated with improved EC and bile duct injury even in grafts with minimal CS time.

Anti-inflammatory signaling during ex vivo liver perfusion improves the preservation of pig liver grafts before transplantation.

Normothermic ex vivo liver perfusion (NEVLP) improves graft preservation by avoiding cold ischemia injury. We investigated whether the protective effects of NEVLP can be further improved by applying strategies targeted on reducing the activation of proinflammatory cytokines during perfusion. Livers retrieved under heart-beating conditions were perfused for 4 hours. Following the preservation period, a pig liver transplantation was performed. In group 1 (n = 5), anti-inflammatory strategies (alprostadil, n-acetylcysteine, carbon monoxide, sevoflurane, and subnormothermic temperature [33°C]) were applied. This was compared with a perfused control group (group 2) where livers (n = 5) were perfused at 37°C without anti-inflammatory agents, similar to the setup used in current European clinical trials, and to a control group preserved with static cold storage (group 3). During 3-day follow-up, markers of reperfusion injury, bile duct injury, and liver function were examined. Aspartate aminotransferase (AST) levels during perfusion were significantly lower in the study versus control group at 1 hour (52 ± 6 versus 162 ± 86 U/L; P = 0.01), 2 hours (43 ± 5 versus 191 ± 111 U/L; P = 0.008), and 3 hours (24 ± 16 versus 218 ± 121 U/L; P = 0.009). During perfusion, group 1 versus group 2 had reduced interleukin (IL) 6, tumor necrosis factor α, and galactosidase levels and increased IL10 levels. After transplantation, group 1 had lower AST peak levels compared with group 2 and group 3 (1400 ± 653 versus 2097 ± 1071 versus 1747 ± 842 U/L; P = 0.47) without reaching significance. Bilirubin levels were significantly lower in group 1 versus group 2 at day 1 (3.6 ± 1.5 versus 6.60 ± 1.5 µmol/L; P = 0.02) and 3 (2 ± 1.1 versus 9.7 ± 7.6 µmol/L; P = 0.01). A trend toward decreased hyaluronic acid, as a marker of improved endothelial cell function, was observed at 1, 3, and 5 hours after reperfusion in group 1 versus group 2. Only 1 early death occurred in each group (80% survival). In conclusion, addition of anti-inflammatory strategies further improves warm perfused preservation. Liver Transplantation 22 1573-1583 2016 AASLD.

The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts.

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-ß and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

Infiltrative (sinusoidal) and hepatitic patterns of injury in acute cellular rejection in liver allograft with clinical implications.

Acute cellular rejection post liver transplant occurs most commonly but not exclusively in the first year. In this study, we report two patterns: sinusoidal infiltrative and hepatitic, which are not considered in the Banff system. We describe their presentation, response to Solu-Medrol, and compare these to the typical moderate-severe acute cellular rejection. Patients transplanted from 2007 to 2012 at University Health Network, who had biopsy-proven rejection in the first year, were studied. Baseline transaminases and bilirubin, time of acute cellular rejection, follow-up, and treatment responses were analyzed. A total of 407 biopsies were received, of which 77 had diagnosis of acute cellular rejection with rejection activity index 5 or above; 49 from viral hepatitis patients were excluded. Twenty-eight were included; 15/28 (54%) had typical acute cellular rejection (tACR) using Banff criteria. Six (21%) had hepatitic acute cellular rejection overlapping with typical features of acute cellular rejection; seven (25%) had infiltrative acute cellular rejection (iACR) overlapping with typical features. The iACR occurred later than the tACR (124 versus 50 days; P = 0.032) and had a higher rise in baseline aspartate aminotransferase (ΔAST) compared with tACR (289 U/l versus 109 U/l; P=0.046). Only one out of seven patients with iACR (14 versus 40% in tACR) failed Solu-Medrol boluses and required thymoglobulin. Patients with hepatitic acute cellular rejection (hACR) had similar ΔAST (P = 0.12) but higher bilirubinemia than typical acute cellular rejection (tACR) (160 µmol/l versus 35 mol/l; P = 0.039) and required thymoglobulin in four out of six (67% versus 40%) instances. Patients with iACR had higher ΔAST than tACR but better Solu-Medrol response compared with both tACR and hACR. hACR is different from plasma cell-rich late-occurring cellular rejection in its pattern but similar in its poor Solu-Medrol response.

Subnormothermic ex vivo liver perfusion reduces endothelial cell and bile duct injury after donation after cardiac death pig liver transplantation.

An ischemic-type biliary stricture (ITBS) is a common feature after liver transplantation using donation after cardiac death (DCD) grafts. We compared sequential subnormothermic ex vivo liver perfusion (SNEVLP; 33°C) with cold storage (CS) for the prevention of ITBS in DCD liver grafts in pig liver transplantation (n = 5 for each group). Liver grafts were stored for 10 hours at 4°C (CS) or preserved with combined 7-hour CS and 3-hour SNEVLP. Parameters of hepatocyte [aspartate aminotransferase (AST), international normalized ratio (INR), factor V, and caspase 3 immunohistochemistry], endothelial cell (EC; CD31 immunohistochemistry and hyaluronic acid), and biliary injury and function [alkaline phosphatase (ALP), total bilirubin, and bile lactate dehydrogenase (LDH)] were determined. Long-term survival (7 days) after transplantation was similar between the SNEVLP and CS groups (60% versus 40%, P = 0.13). No difference was observed between SNEVLP- and CS-treated animals with respect to the peak of serum INR, factor V, or AST levels within 24 hours. CD31 staining 8 hours after transplantation demonstrated intact EC lining in SNEVLP-treated livers (7.3 × 10(-4) ± 2.6 × 10(-4) cells/µm(2)) but not in CS-treated livers (3.7 × 10(-4) ± 1.3 × 10(-4) cells/µm(2) , P = 0.03). Posttransplant SNEVLP animals had decreased serum ALP and serum bilirubin levels in comparison with CS animals. In addition, LDH in bile fluid was lower in SNEVLP pigs versus CS pigs (14 ± 10 versus 60 ± 18 µmol/L, P = 0.02). Bile duct histology revealed severe bile duct necrosis in 3 of 5 animals in the CS group but none in the SNEVLP group (P = 0.03). Sequential SNEVLP preservation of DCD grafts reduces bile duct and EC injury after liver transplantation.