Recurrence of autoimmune liver diseases after liver transplantation: Review and expert opinion statement.

Autoimmune liver diseases (AILD) constitute the fourth most common indication for liver transplantation (LT) across the world. In general, the outcomes after LT are acceptable, however, disease recurrence post-LT is common for all AILD which can negatively affect graft and overall survival. Several questions persist, including the risk factors associated with recurrent disease, optimal anti-rejection medications, strategies to reduce the risk of recurrence, and how to best incorporate these strategies into clinical practice. For that reason, we assembled an international group of experts to review evidence to address these outstanding questions regarding liver transplantation for AILD. Survival rates post-LT are approximately 90 and 70% at 1- and 5-years and recurrent disease occurs in 10 to 50% of patients with AILD. In patients with disease recurrence, graft survival decreased by 18% and 28% and overall survival by 8% and 12% at 5 and -10 years after LT, respectively. Recurrent AIH is associated with high aminotransferases and immunoglobulin G (IgG) before LT, lymphoplasmacytic infiltrates in the explants, and may be associated with the absence of steroids after LT. However, the efficiency and safety of triple immunosuppressive maintenance therapy is still debatable. Younger age at diagnosis with PBC or at LT are associated with PBC recurrence. Preventive use of ursodeoxycholic acid reduces the risk of recurrence and has a benefit in graft and patient survival. Episodes of systemic inflammation including T-cell mediated rejection, active ulcerative colitis and episodes of cholangitis are associated with recurrent PSC. Conclusions: Recurrent disease for AILD is associated with worse graft and patient survival. AIH patients could be considered for long-term low-dose predniso(lo)ne, whereas PBC patients should be placed on preventive UDCA after LT. There are no specific treatments for PSC recurrence; however, adequate control of inflammatory bowel disease and optimal immunosuppression to avoid T-cell-mediated rejection should be encouraged.

Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells.

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

Impact of Platelet Count on Perioperative Bleeding in Patients With Cirrhosis Undergoing Surgical Treatments of Liver Cancer.

In patients with cirrhosis with severe thrombocytopenia (platelet count [PC] <50 × 109 /L) and undergoing invasive procedures, it is common clinical practice to increase the PC with platelet transfusions or thrombopoietin receptor agonists to reduce the risk of major periprocedural bleeding. The aim of our study was to investigate the association between native PC and perioperative bleeding in patients with cirrhosis undergoing surgical procedures for the treatment of hepatocellular carcinoma (HCC). We retrospectively evaluated 996 patients with cirrhosis between 1996 and 2018 who underwent surgical treatments of HCC by liver resection (LR) or radiofrequency ablation (RFA) without prophylactic platelet transfusions. Patients were allocated to the following three groups based on PC: high (>100 × 109 /L), intermediate (51-100 × 109 /L), and low (≤50 × 109 /L). PC was also analyzed as a continuous covariate on multivariable analysis. The primary endpoint was major perioperative bleeding. The overall event rate of major perioperative bleeding was 8.9% and was not found to differ significantly between the high, intermediate, and low platelet groups (8.1% vs. 10.2% vs. 10.8%, P = 0.48). On multivariable analysis, greater age, aspartate aminotransferase, lower hemoglobin, and treatment with LR (vs. RFA) were found to be significant independent predictors of major perioperative bleeding, with associations with disease etiology and year of surgery also observed. After adjusting for these factors, the association between PC and major perioperative bleeding remained nonsignificant. Conclusion: Major perioperative bleeding was not significantly associated with PC in patients with cirrhosis undergoing surgical treatment of HCC, even when their PC was <50 × 109 /L. With the limit of a retrospective analysis, our data do not support the recommendation of increasing PC in patients with severe thrombocytopenia in order to decrease their perioperative bleeding risk.

The Sample Size Matters: To What Extent the Participant Reduction Affects the Outcomes of a Neuroscientific Research. A Case-Study in Neuromarketing Field.

The sample size is a crucial concern in scientific research and even more in behavioural neurosciences, where besides the best practice it is not always possible to reach large experimental samples. In this study we investigated how the outcomes of research change in response to sample size reduction. Three indices computed during a task involving the observations of four videos were considered in the analysis, two related to the brain electroencephalographic (EEG) activity and one to autonomic physiological measures, i.e., heart rate and skin conductance. The modifications of these indices were investigated considering five subgroups of sample size (32, 28, 24, 20, 16), each subgroup consisting of 630 different combinations made by bootstrapping n (n = sample size) out of 36 subjects, with respect to the total population (i.e., 36 subjects). The correlation analysis, the mean squared error (MSE), and the standard deviation (STD) of the indexes were studied at the participant reduction and three factors of influence were considered in the analysis: the type of index, the task, and its duration (time length). The findings showed a significant decrease of the correlation associated to the participant reduction as well as a significant increase of MSE and STD (p < 0.05). A threshold of subjects for which the outcomes remained significant and comparable was pointed out. The effects were to some extents sensitive to all the investigated variables, but the main effect was due to the task length. Therefore, the minimum threshold of subjects for which the outcomes were comparable increased at the reduction of the spot duration.

Ex situ Normothermic Split Liver Machine Perfusion: Protocol for Robust Comparative Controls in Liver Function Assessment Suitable for Evaluation of Novel Therapeutic Interventions in the Pre-clinical Setting.

Background: Ex situ donor liver machine perfusion is a promising tool to assess organ viability prior to transplantation and platform to investigate novel therapeutic interventions. However, the wide variability in donor and graft characteristics between individual donor livers limits the comparability of results. We investigated the hypothesis that the development of a split liver ex situ machine perfusion protocol provides the ideal comparative controls in the investigation of machine perfusion techniques and therapeutic interventions, thus leading to more comparable results. Methods: Four discarded human donor livers were surgically split following identification and separation of right and left inflow and outflow vessels. Each lobe, on separate perfusion machines, was subjected to normothermic perfusion using an artificial hemoglobin-based oxygen carrier solution for 6 h. Metabolic parameters as well as hepatic artery and portal vein perfusion parameters monitored. Results: Trends in hepatic artery and portal vein flows showed a general increase in both lobes throughout each perfusion experiment, even when normalized for tissue weight. Progressive decreases in perfusate lactate and glucose levels exhibited comparable trends in between lobes. Conclusion: Our results demonstrate comparability between right and left lobes when simultaneously subjected to normothermic machine perfusion. In the pre-clinical setting, this model provides the ideal comparative controls in the investigation of therapeutic interventions.

Accuracy of Transient Elastography in Assessing Fibrosis at Diagnosis in Naïve Patients With Primary Biliary Cholangitis: A Dual Cut-Off Approach.

BACKGROUND AND AIMS: Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration-controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC. APPROACH AND RESULTS: We collected data from 167 consecutive treatment-naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry. CONCLUSIONS: In a multicenter study of treatment-naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

Organ Restoration With Normothermic Machine Perfusion and Immune Reaction.

Liver transplantation is the only recognized effective treatment for end-stage liver disease. However, organ shortages have become the main challenge for patients and physicians within the transplant community. Waiting list mortality remains an issue with around 10% of patients dying whilst waiting for an available organ. The post-transplantation period is also associated with an adverse complication rate for these specific cohorts of high-risk patients, particularly regarding patient and graft survival. Ischaemia reperfusion injury (IRI) has been highlighted as the mechanism of injury that increases parenchymal damage, which eventually lead to significant graft dysfunction and other poor outcome indicators. The consequences of IRI in clinical practice such as reperfusion syndrome, primary non-function of graft, allograft dysfunction, ischaemic biliary damage and early biliary complications can be life-threatening. IRI dictates the development of a significant inflammatory response that drives the pathway to eventual cell death. The main mechanisms of IRI are mitochondrial damage due to low oxygen tension within the hepatic micro-environment and severe adenosine triphosphate (ATP) depletion during the ischaemic period. After the restoration of normal blood flow, this damage is further enhanced by reoxygenation as the mitochondria respond to reperfusion by releasing reactive oxygen species (ROS), which in turn activate Kupffer cells within the hepatic micro-environment, leading to a pro-inflammatory response and eventual parenchymal cell apoptosis and associated tissue degradation. Machine perfusion (MP) is one emergent strategy considered to be one of the most important advances in organ preservation, restoration and transplantation. Indeed, MP has the potential to rescue frequently discarded organs and has been shown to limit the extent of IRI, leading to suppression of the deleterious pro-inflammatory response. This immunomodulation reduces the prevalence of allograft rejection, the use of immunosuppression therapy and minimizes post-transplant complications. This review aims to update the current knowledge of MP with a focus on normothermic machine liver perfusion (NMLP) and its potential role in immune response pathways.

Stress Assessment by Combining Neurophysiological Signals and Radio Communications of Air Traffic Controllers.

Air Traffic Control (ATC) has been classified as the fourth most stressful job. In this regard, sixteen controllers were asked to perform ecological ATC simulation during which behavioral (Radio Communications with pilots - RCs), subjective (stress perception) and neurophysiological signals (brain activity and skin conductance - SC) were collected. All the considered parameters reported significant changes under high stress conditions. In particular, the theta, alpha, and beta brain rhythms increased significantly (all p<0.05) all over the brain areas, and both the SC components exhibited higher values (p<0.01). Additionally, the number of speech under high stress decreased significantly (p<10-4) while both the mean and median value of the F0 component of the RC increased (p<0.01). The results can be employed to objectively measure and track the controller's stress level while dealing with ATC activities to better tailoring the workshift and maintaining high safety levels.

The Immunological Basis of Liver Allograft Rejection.

Liver allograft rejection remains a significant cause of morbidity and graft failure in liver transplant recipients. Rejection is caused by the recognition of non-self donor alloantigens by recipient T-cells. Antigen recognition results in proliferation and activation of T-cells in lymphoid tissue before migration to the allograft. Activated T-cells have a variety of effector mechanisms including direct T-cell mediated damage to bile ducts, endothelium and hepatocytes and indirect effects through cytokine production and recruitment of tissue-destructive inflammatory cells. These effects explain the histological appearances of typical acute T-cell mediated rejection. In addition, donor specific antibodies, most typically against HLA antigens, may give rise to antibody-mediated rejection causing damage to the allograft primarily through endothelial injury. However, as an immune-privileged site there are several mechanisms in the liver capable of overcoming rejection and promoting tolerance to the graft, particularly in the context of recruitment of regulatory T-cells and promotors of an immunosuppressive environment. Indeed, around 20% of transplant recipients can be successfully weaned from immunosuppression. Hence, the host immunological response to the liver allograft is best regarded as a balance between rejection-promoting and tolerance-promoting factors. Understanding this balance provides insight into potential mechanisms for novel anti-rejection therapies.

The Human Immune Response to Cadaveric and Living Donor Liver Allografts.

The liver is an important contributor to the human immune system and it plays a pivotal role in the creation of both immunoreactive and tolerogenic conditions. Liver transplantation provides the best chance of survival for both children and adults with liver failure or cancer. With current demand exceeding the number of transplantable livers from donors following brain death, improved knowledge, technical advances and the desire to prevent avoidable deaths has led to the transplantation of organs from living, ABO incompatible (ABOi), cardiac death donors and machine based organ preservation with acceptable results. The liver graft is the most well-tolerated, from an immunological perspective, of all solid organ transplants. Evidence suggests successful cessation of immunosuppression is possible in ~20-40% of liver transplant recipients without immune mediated graft injury, a state known as "operational tolerance." An immunosuppression free future following liver transplantation is an ambitious but perhaps not unachievable goal. The initial immune response following transplantation is a sterile inflammatory process mediated by the innate system and the mechanisms relate to the preservation-reperfusion process. The severity of this injury is influenced by graft factors and can have significant consequences. There are minimal experimental studies that delineate the differences in the adaptive immune response to the various forms of liver allograft. Apart from ABOi transplants, antibody mediated hyperacute rejection is rare following liver transplant. T-cell mediated rejection is common following liver transplantation and its incidence does not differ between living or deceased donor grafts. Transplantation in the first year of life results in a higher rate of operational tolerance, possibly due to a bias toward Th2 cytokines (IL4, IL10) during this period. This review further describes the current understanding of the immunological response toward liver allografts and highlight the areas of this topic yet to be fully understood.

Primary Sclerosing Cholangitis: Burden of Disease and Mortality Using Data from the National Rare Diseases Registry in Italy.

Introduction: Studies on the epidemiology of primary sclerosing cholangitis (PSC) are mainly based on tertiary referral centers; and are retrospective case series susceptible to selection bias. The aim of this study was to estimate incidence; survival and cause of mortality of PSC in Italy; using population-based data. Methods: Data collected from the National Rare Diseases Registry (RNMR) and the National Mortality Database (NMD) were integrated and analyzed. Results: We identified 502 PSC incident cases. The crude incidence rate between 2012 and 2014 was 0.10 per 100,000 individuals. Sixty percent were male; mean age at disease onset and at diagnosis were 33 and 37 years; respectively; highlighting a mean diagnostic delay of 4 years. The rate of interregional mobility was 12%. Ten-year survival was 92%. In 32% of cases the cause of death was biliary-related; 12% died of biliary or gallbladder cancer. Conclusions: For rare diseases such as PSC; population-based cohort's studies are of paramount importance. Incidence rates of PSC in Italy are markedly lower and survival much longer than the ones reported from tertiary; single-centre series. Moreover; the diagnostic delay and the patient interregional mobility highlights the need for increasing awareness on the disease and for resource reallocation among Italian regions within the National Health Service.

Immune system and cholangiocytes: A puzzling affair in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic liver disease characterized by the destruction of the small and medium bile ducts. Its pathogenesis is still unknown. Despite the genome wide association study findings, the therapies targeting the cytokines pathway, tested so far, have failed. The concept of the biliary epithelium as a key player of the PBC pathogenesis has emerged over the last few years. It is now well accepted that the biliary epithelial cells (BECs) actively participate to the genesis of the damage. The chronic stimulation of BECs via microbes and bile changes the cell phenotype toward an active state, which, across the production of proinflammatory mediators, can recruit, retain, and activate immune cells. The consequent immune system activation can in turn damage BECs. Thus, the crosstalk between both innate and adaptive immune cells and the biliary epithelium creates a paracrine loop responsible for the disease progression. In this review, we summarize the evidence provided in literature about the role of BECs and the immune system in the pathogenesis of PBC. We also dissect the relationship between the immune system and the BECs, focusing on the unanswered questions and the future potential directions of the translational research and the cellular therapy in this area.

Prognostic models in primary biliary cholangitis.

Risk prediction modelling is important to better understand the determinants of the course and outcome of PBC and to inform the risk across the disease continuum in PBC enabling risk-stratified follow-up care and personalised therapy. Current prognostic models in PBC are based on treatment response to ursodeoxycholic acid because of the well-established relationship between alkaline phosphatase on treatment and long-term outcome. In addition, serum alkaline phosphatase correlates with ductular reaction and biliary metaplasia, which are hallmark of biliary injury. Considering the waiting time for treatment failure in high-risk patients is not inconsequential, efforts are focused on bringing forward risk stratification at diagnosis by predicting treatment response at onset. There is a need for better prognostic variables that are central to the disease process. We should take an integrative approach that incorporates multiple layers of information including genetic and environmental influences, host characteristics, clinical data, and molecular alterations for risk assessments. Biomarker discovery has an accelerated pace taking advantage of the emergence of large-scale omics platforms (genomics, epigenomics, transcriptomics, proteomics, metabolomics, and others) and whole-genome sequencing. In the digital era, applications of artificial intelligence, such as machine learning, can support the computing power required to analyse the vast amount of data produced by omics. The information is then used for the development of personalised risk prediction models that through clinical trials and hopefully industry partnerships can guide risk management strategies. We are facing an unprecedented opportunity for the integration of molecular diagnostics into the clinic, which promotes progress toward the personalised management of patients with PBC.

Pretreatment prediction of response to ursodeoxycholic acid in primary biliary cholangitis: development and validation of the UDCA Response Score.

BACKGROUND: Treatment guidelines recommend a stepwise approach to primary biliary cholangitis: all patients begin treatment with ursodeoxycholic acid (UDCA) monotherapy and those with an inadequate biochemical response after 12 months are subsequently considered for second-line therapies. However, as a result, patients at the highest risk can wait the longest for effective treatment. We determined whether UDCA response can be accurately predicted using pretreatment clinical parameters. METHODS: We did logistic regression analysis of pretreatment variables in a discovery cohort of patients in the UK with primary biliary cholangitis to derive the best-fitting model of UDCA response, defined as alkaline phosphatase less than 1·67 times the upper limit of normal (ULN), measured after 12 months of treatment with UDCA. We validated the model in an external cohort of patients with primary biliary cholangitis and treated with UDCA in Italy. Additionally, we assessed correlations between model predictions and key histological features, such as biliary injury and fibrosis, on liver biopsy samples. FINDINGS: 2703 participants diagnosed with primary biliary cholangitis between Jan 1, 1998, and May 31, 2015, were included in the UK-PBC cohort for derivation of the model. The following pretreatment parameters were associated with lower probability of UDCA response: higher alkaline phosphatase concentration (p<0·0001), higher total bilirubin concentration (p=0·0003), lower aminotransferase concentration (p=0·0012), younger age (p<0·0001), longer interval from diagnosis to the start of UDCA treatment (treatment time lag, p<0·0001), and worsening of alkaline phosphatase concentration from diagnosis (p<0·0001). Based on these variables, we derived a predictive score of UDCA response. In the external validation cohort, 460 patients diagnosed with primary biliary cholangitis were treated with UDCA, with follow-up data until May 31, 2016. In this validation cohort, the area under the receiver operating characteristic curve for the score was 0·83 (95% CI 0·79-0·87). In 20 liver biopsy samples from patients with primary biliary cholangitis, the UDCA response score was associated with ductular reaction (r=-0·556, p=0·0130) and intermediate hepatocytes (probability of response was 0·90 if intermediate hepatocytes were absent vs 0·51 if present). INTERPRETATION: We have derived and externally validated a model based on pretreatment variables that accurately predicts UDCA response. Association with histological features provides face validity. This model provides a basis to explore alternative approaches to treatment stratification in patients with primary biliary cholangitis. FUNDING: UK Medical Research Council and University of Milan-Bicocca.

Toward precision medicine in primary biliary cholangitis.

Primary biliary cholangitis is a chronic, cholestatic liver disease characterized by a heterogeneous presentation, symptomatology, disease progression and response to therapy. In contrast, clinical management and treatment of PBC is homogeneous with a 'one size fits all' approach. The evolving research landscape, with the emergence of the -omics field and the availability of large patient cohorts are creating a unique opportunity of translational epidemiology. Furthermore, several novel disease and symptom-modifying agents for PBC are currently in development. The time is therefore ripe for precision medicine in PBC. In this manuscript we describe the concept of precision medicine; review current approaches to risk-stratification in PBC, and speculate how precision medicine in PBC might develop in the near future.