Normothermic regional perfusion in controlled DCD liver procurement: Outcomes of the Swedish national implementation protocol.

Liver transplantation (LTX) using donors after controlled circulatory death (cDCD) is associated with poorer graft survival and increased incidence of nonanastomotic biliary strictures (NASs) compared to livers procured from brain-dead donors (DBD). The use of normothermic regional perfusion (NRP) during cDCD procurement may improve posttransplant outcomes and reduce the incidence of NAS. In Sweden, cDCD LTX was introduced through a national pilot protocol with mandatory NRP. This study aims to evaluate the outcome of cDCD LTX during the pilot period. Donor and recipient data were collected on all cDCD liver transplants during the pilot period between January 2020 to December 2022. Outcome on NAS, patient and graft survival, early allograft dysfunction, acute kidney injury, and comprehensive complication index was compared to a matched cohort of 28 patients transplanted with a DBD liver between 2018 and 2022. Eighteen patients were transplanted with a liver from a cDCD donor after using NRP. The mean functional warm ischemia time was 29 ± 6 minutes. The mean lactate reduction during NRP was 8.7 ± 2.4 mmol/L, and the end NRP perfusate alanine aminotransferase was 1.4 ± 1 µkat/L. When comparing recipients of cDCD liver transplant to DBD, no significant differences were observed in the incidence of NAS, patient and graft survival, comprehensive complication index, early allograft dysfunction, or acute kidney injury. Study protocol magnetic resonance cholangiopancreatography in cDCD patients showed no signs of subclinical biliary strictures. Evaluation of the Swedish national pilot of cDCD LTX with mandatory NRP shows comparable outcomes to a matched DBD cohort with 94.4% 1-year patient and graft survival and no incidence of NAS within the first year.

Potential of neonatal organ donation and outcome after transplantation.

Organ transplantation is limited by access to suitable organs. Infant recipient waitlist mortality is increased due to the scarcity of size-matched organs. Neonatal organ donors have been proposed as an underutilized source of donor organs. However, the literature on the actual prevalence and outcome of neonatal organ donation and transplantation is fragmented and not well analyzed. This literature review aims to summarize the available literature on the potential of neonatal organ donation and to analyze published cases of neonatal organ transplantation. A systematic search of the Medline and Cochrane databases yielded 2964 articles, which were screened for eligibility. In total, 86 articles were considered eligible, of which 34 were included in the literature review: 8 articles describing the potential of neonatal organ donation programs, and 26 articles describing clinical transplantation. Current evidence suggests there is a large pool of potential neonatal organ donors. In contrast, the literature on neonatal organ donor utilization is sparse. However, case series of successful kidney, heart, liver, hepatocyte, and multivisceral transplantation using organs from neonatal donors are summarized. Although good posttransplant organ function was achieved, the use of neonatal organs is associated with increased risk of thrombosis in both kidney and liver transplantation. Neonatal organ donation is a promising alternative for expanding the current donor pool. Experience is limited, but reported patient and graft survival are acceptable and more research on the subject is warranted.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Liver transplantation in patients with post-hepatectomy liver failure - A Northern European multicenter cohort study.

BACKGROUND: Liver transplantation (LTX) has been described as a rescue treatment option in severe, intractable post-hepatectomy liver failure (PHLF), but is not considered to be indicated for this condition by many hepatobiliary and transplant surgeons. In this article we describe the clinical experience of five northern European tertiary centers in using LTX to treat selected patients with severe PHLF. METHODS: All patients subjected to LTX due to PHLF at the participating centers were identified from prospective clinical databases. Preoperative variables, surgical outcome (both resection surgery and LTX) and follow-up data were assessed. RESULTS: A total of 10 patients treated with LTX due to severe PHLF from September 2008 to May 2020 were identified and included in the study. All patients but one were male and the median age was 70 years (range 49-72). In all patients the indication for liver resection was suspected malignancy, but in six patients post-resection pathology revealed benign or pre-malignant disease. There was no 90-day mortality after LTX. Patients were followed for a median of 49 months (13-153) and eight patients were alive without recurrence at last follow-up. DISCUSSION: In selected patients with PHLF LTX can be a life-saving procedure with low short-term risk.

Comparison of cystatin C, creatinine, and iohexol clearance in pediatric liver transplantation-a retrospective cohort study.

Impaired renal function after pediatric (LT) is a recognized problem. Accurate monitoring of (GFR) is imperative to detect declining renal function. GFR can be estimated via s-creatinine and/or p-cystatin C or measured by inulin and or/iohexol clearances. We retrospectively compared eGFRcrea and eGFRcyst, to mGFRiohex after LT. Data from 91 children with 312 concomitant measurements of s-creatinine, p-cystatin C, and iohexol clearance, obtained between 2007 and 2015, were analyzed. eGFR was calculated by using the p-cystatin C-based CAPA and CKD-EPI formulas, and the s-creatinine-based Schwartz-LYON, FAS, revised Schwartz and MDRD formulas. Also, the arithmetic means of cystatin C-based and creatinine-based equations were used. Every calculated eGFR was compared to mGFRiohex in statistical correlation, accuracy, precision, bias, and misclassifications. Among the different equations, p-cystatin C-based formulas (CAPA and CKD-EPI) as well as the s-creatinine-based Schwartz-LYON formula showed the most correct estimates regarding accuracy (84-87.5%), bias (0.19-4.0 ml/min/1.73 m2 ), and misclassification rate (24.7-25%). In patients with renal function <75 ml/min/1.73 m2 , cystatin C-based formulas were significantly more accurate and less biased than creatinine-based formulas. In conclusion, S-creatinine could be used in a clinical setting on a regular basis in liver transplanted pediatric patients, with reliable results, if eGFR is calculated by the Schwartz-LYON formula. When suspected renal dysfunction, cystatin C-based eGFR should be calculated, since it gives more accurate and less biased estimates than creatinine-based eGFR, and should be confirmed by mGFR (iohexol).

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Liver Transplantation for Liver Metastasis of a Pseudopapillary Pancreatic Neoplasm in a Male Patient.

BACKGROUND Solid pseudopapillary neoplasm (SPN) of the pancreas, which predominantly affects young women, is an uncommon condition with low malignant potential. It is often asymptomatic. This tumor has a low metastatic rate and a good prognosis in contrast to other pancreatic tumors. Approximately 14% of SPNs develop liver metastasis, but for SPNs with malignant features liver metastasis has been reported to occur in over 55% of cases. Complete surgical resection is the treatment of choice for increasing the survival rate in metastatic recurrent disease. When surgical resection is impossible, liver transplantation has shown promising results in a few cases. The purpose of this article is to present the first case of a male patient who underwent liver transplantation for this indication. CASE REPORT We present the case of a 60-year-old male patient who previously had pancreas surgery, numerous liver resections, and chemotherapy for SPN, but nevertheless developed recurrence of multiple liver metastases. His metastatic liver disease was regarded as unresectable. The lymphatic structure was also affected. The patient underwent orthotopic liver transplantation with a deceased donor graft after multidisciplinary evaluation. Resection of involved lymphatic structures was also performed. At 2-year follow-up, the patient was alive and recurrence free. CONCLUSIONS This is the first published report of a male patient who underwent liver transplantation due to SPN metastasis. Our case demonstrates that liver transplantation should be further investigated for selected cases of SPN of the pancreas with liver metastatic disease when surgical resection is deemed unattainable.

Oxygen-charged HTK-F6H8 emulsion reduces ischemia-reperfusion injury in kidneys from brain-dead pigs.

BACKGROUND: Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media. METHODS: Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane. After 18 h of cold ischemia the kidneys were transplanted into allogeneic recipients and assessed for adenosine triphosphate content, morphology, and expression of genes related to hypoxia, environmental stress, inflammation, and apoptosis. RESULTS: Compared with HTK-flushed kidneys, organs preserved using oxygen-precharged HTK-perfluorohexyloctane emulsion had increased elevated adenosine triphosphate content and a significantly lower gene expression of hypoxia inducible factor-1α, vascular endothelial growth factor, interleukin-1α, tumor necrosis factor-α, interferon-α, JNK-1, p38, cytochrome-c, Bax, caspase-8, and caspase-3 at all time points assessed. In contrast, the mRNA expression of Bcl-2 was significantly increased. CONCLUSIONS: The present study has demonstrated that in brain-dead pigs the perfusion of kidneys with oxygen-precharged HTK-perfluorohexyloctane emulsion results in significantly reduced inflammation, hypoxic injury, and apoptosis and cellular integrity and energy content are well maintained. Histologic examination revealed less tubular, vascular, and glomerular changes in the emulsion-perfused tissue compared with the HTK-perfused counterparts. The concept of perfusing organs with oxygen-precharged emulsion based on organ preservation media represents an efficient alternative for improved organ preservation.

SOULMATE: the Swedish study of liver transplantation for isolated colorectal cancer liver metastases not suitable for operation or ablation, compared to best established treatment-a randomized controlled multicenter trial.

BACKGROUND: Around one fourth of patients with colorectal cancer present themselves with distant metastases at the time of diagnosis, and one additional one fifth of the patients will develop distant metastases during the disease, most commonly in the liver. Surgical treatment such as liver resection or ablation, often combined with chemotherapy and targeted therapy, is the only treatment option with curative potential, but only about 20% of the patients with liver metastases are candidates for surgical intervention. Standard treatment for unresectable patients is palliative oncological therapy; however, less than 10% of these patients will achieve a 5-year survival. Non-randomized studies indicate that liver transplantation could be an option for selected patients with colorectal liver metastases (CRLM), which are not suitable for operation or ablation due to surgical technical reasons such as massive tumor burden and small future liver remnant, or oncological reasons, for example, early relapse after liver surgery. Since there is a shortage of donated liver grafts, it is important to select the patient group that benefit most from the treatment. Although some studies present positive results from liver transplantation of CRLM, the results must be validated in a randomized controlled trial before this new indication for liver transplantation can be introduced as a clinical routine. METHODS: The SOULMATE study is a randomized study evaluating if liver transplantation with liver grafts, primarily from extended criteria donors, increases overall survival in patients with CRLM, not suitable for resection or ablation, in comparison with best established treatment. Patients will be randomized to liver transplantation (LT)+ best established treatment (BET) or to best established treatment only. In the SOULMATE trial, we will evaluate the use of livers from extended criteria donors to decrease the risk of prolonging waiting time for patients on the waiting list for LT. DISCUSSION: The SOULMATE study has the possibility to confirm the positive results of previous studies in a randomized setting. The use of extended criteria donors will make the results transferable globally, as most countries are struggling with organ shortage. TRIAL REGISTRATION: Clinical Trial number: NCT04161092 registered 13 November 2019.

Prediction of survival in patients with colorectal liver metastases- development and validation of a prognostic score model.

BACKGROUND: Metastatic spread of colorectal cancer to the liver impacts prognosis. Advances in chemotherapy have resulted in increased resectability rates and thereby improved survival in patients with colorectal liver metastases (CRLM). However, criteria are needed to ensure that patients selected for hepatic resection benefit from the invasive therapy. The study aimed to construct a predictive model for overall survival (OS) in patients with CRLM, based on preoperatively available information. METHODS: The retrospective cohort study reviewed all patients with CRLM discussed at multidisciplinary team conference at Karolinska University Hospital, Stockholm, Sweden, 2013-2018. Independent prognostic factors for OS were identified, based on which a score model was generated. The model was validated on patients treated for CRLM at Hôpital Universitaire Paul Brousse, Villejuif, France, 2007-2018. Calibration and discrimination methods were used for internal and external validation. RESULTS: The Swedish development cohort included 1013 patients, the French validation cohort 391 patients. Poor OS was significantly associated with age>60years (hazard ratio (HR) 3.57 (95%CI 2.18-9.94)), number of CRLM (HR 4.59 (2.83-12.20)), diameter of largest CRLM>5 cm (HR 2.59 (1.74-5.03)), right-sided primary tumour (HR 2.98 (2.00-5.80)), extrahepatic disease (HR 4.14 (2.38-15.87)) and non-resectability (HR 0.77 (0.66-0.90)). The C-statistic for prediction of OS was .74, in the development cohort and 0.69 in the validation cohort. CONCLUSION: The presented predictive score model can adequately predict OS for patients at the initial diagnosis of CRLM. The prognostic model could be of clinical value in the management of all patients with CRLM, by predicting individualized survival and thereby facilitating treatment recommendations.

Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death.

Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015-2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80-210] minutes. Median viability was 86% (IQR: 71%-91%). Median yield was 6.9 (IQR: 3.4-12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.

The potential use of extended criteria donors and eligible recipients in liver transplantation for unresectable colorectal liver metastases in Central Sweden.

BACKGROUND: Unresectable colorectal liver metastases (CRLM) is a condition with poor prognosis. A recent treatment alternative improving survival in patients with unresectable CRLM, has emerged with the introduction of liver transplantation (LT), yet not uncontroversial with the current organ shortage. This study aimed to retrospectively investigate the potential of declined donors with acceptable risk as liver graft donors and patients with unresectable CRLM as potential recipients. METHODS: All declined donors in central Sweden and all patients with CRLM discussed at multidisciplinary team conference at Karolinska University Hospital, January 2013-October 2018, were identified. Donors were classified according to the European Committee Guide to the quality and safety of organs for transplantation and potential recipients were evaluated by selection criteria, based on studies on the Norwegian Secondary Cancer study database. RESULTS: Out of 1,462 evaluated potential donors, 62 (2.7 pmp) donors were identified, corresponding to 6-18% of the utilized donor pool. Out of 1,008 included patients with CRLM, 25 (2.1 pmp) potential recipients were recognized. Eligibility for LT and left-sided colon cancer were favorable prognostic factors. CONCLUSIONS: Today's donor pool could increase with the use of extended criteria donors, which is sufficient and display an acceptable risk-benefit ratio for patients with unresectable CRLM. With current selection criteria a small subset of patients with unresectable CRLM are eligible recipients. This subset of patients has a better survival compared to patients ineligible for LT.

CD5 Surface Expression Marks Intravascular Human Innate Lymphoid Cells That Have a Distinct Ontogeny and Migrate to the Lung.

Innate lymphoid cells (ILCs) contribute to immune defense, yet it is poorly understood how ILCs develop and are strategically positioned in the lung. This applies especially to human ILCs due to the difficulty of studying them in vivo. Here we investigated the ontogeny and migration of human ILCs in vivo with a humanized mouse model ("MISTRG") expressing human cytokines. In addition to known tissue-resident ILC subsets, we discovered CD5-expressing ILCs that predominantly resided within the lung vasculature and in the circulation. CD5+ ILCs contained IFNγ-producing mature ILC1s as well as immature ILCs that produced ILC effector cytokines under polarizing conditions in vitro. CD5+ ILCs had a distinct ontogeny compared to conventional CD5- ILCs because they first appeared in the thymus, spleen and liver rather than in the bone marrow after transplantation of MISTRG mice with human CD34+ hematopoietic stem and progenitor cells. Due to their strategic location, human CD5+ ILCs could serve as blood-borne sentinels, ready to be recruited into the lung to respond to environmental challenges. This work emphasizes the uniqueness of human CD5+ ILCs in terms of their anatomical localization and developmental origin compared to well-studied CD5- ILCs.

ABO Incompatible Liver Transplantation in Children: A 20 Year Experience from Centres in the TransplantChild European Reference Network.

An increasing number of AB0-incompatible (AB0i) liver transplantations (LT) are being undertaken internationally in recent years due to organ shortages and the need for urgent transplantation. The aim of our study was establish the value of ABOi LT from available retrospective results of AB0i pediatric liver transplantations performed in European reference centers now belonging to the TransplantChild, European Reference Network (ERN). Data from medical records were analyzed, including demographic data, diagnosis, urgency of transplantation, time on the waiting list, PELD/MELD score, desensitization procedures, immunosuppression, selected post-transplant complications, and patient and graft survival. A total of 142 patients (pts) with transplants between 1986 and 2018 in 8 European transplant centers were included in the study. The indications for liver transplantation were: cholestatic diseases in 62 pts, acute liver failure in 42 pts, and other conditions in the remaining 38 pts. Sixty-six patients received grafts from living donors, and seventy-six received grafts from deceased donors. Both patient and graft survival were significantly affected by deceased donor type, urgent transplantation, and the development of vascular complications. In the multivariate analysis, vascular complications had a negative impact on patient and graft survival, while a longer time from the first AB0i LT in the study showed better results, suggesting an international learning experience. In conclusion, we believe that AB0i LT in children is now a safe procedure that may be adopted more readily in children.

Disorganization and degeneration of liver sympathetic innervations in nonalcoholic fatty liver disease revealed by 3D imaging.

Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.

Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection.

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcgammaRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16(+) CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.

Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes.

Bile acids (BAs) are detergents essential for intestinal absorption of lipids. Disruption of BA homeostasis can lead to severe liver damage. BA metabolism is therefore under strict regulation by sophisticated feedback mechanisms. The hormone-like protein Fibroblast growth factor 19 (FGF19) is essential for maintaining BA homeostasis by down regulating BA synthesis. Here, the impact of both FGF19 and chenodeoxycholic acid (CDCA) on primary human hepatocytes was investigated and a possible autocrine/paracrine function of FGF19 in regulation of BA synthesis evaluated. Primary human hepatocytes were treated with CDCA, recombinant FGF19 or conditioned medium containing endogenously produced FGF19. RNA sequencing revealed that treatment with CDCA causes deregulation of transcripts involved in BA metabolism, whereas treatment with FGF19 had minor effects. CDCA increased FGF19 mRNA expression within 1 h. We detected secretion of the resulting FGF19 protein into medium, mimicking in vivo observations. Furthermore, medium enriched with endogenously produced FGF19 reduced BA synthesis by down regulating CYP7A1 gene expression. However, following knockdown of FGF19, CDCA still independently decreased BA synthesis, presumably through the regulatory protein small heterodimer partner (SHP). In summary, we show that in primary human hepatocytes CDCA regulates BA synthesis in an FGF19-independent manner.

Blood Group Antigen Expression in Isolated Human Liver Cells in Preparation for Implementing Clinical ABO-Incompatible Hepatocyte Transplantation.

BACKGROUND: ABO blood group antigens in the liver are expressed mainly on endothelial cells or biliary epithelial cells but not on hepatocytes. This suggests that ABO-incompatible hepatocyte transplantation (ABOi-HTx) is theoretically feasible. However, the effects of stress on ABO blood group antigen expression caused by isolation and intraportal infusion require thorough investigation before ABOi-HTx can be implemented in clinical settings. METHODS: Human hepatocytes were isolated from liver tissue obtained from liver resection or deceased donor livers. The expression of blood group antigens on cryopreserved human liver tissues and isolated hepatocyte smear specimens were examined by immunofluorescent staining. The effect of proinflammatory cytokines on blood group antigen expression of hepatocytes was evaluated by flow cytometry. Instant blood-mediated inflammatory reaction after hepatocyte incubation with ABO-incompatible whole blood was examined using the tubing loop model. RESULTS: Blood group antigens were mainly expressed on vessels in the portal area. In hepatocyte smear specimens, isolated hepatocytes did not express blood group antigens. In contrast, a subset of cells in the smear specimens of nonparenchymal liver cells stained positive. In the flow cytometry analysis, isolated hepatocytes were negative for blood group antigens, even after 4-h incubation with cytokines. Platelet counts and complement activation were not significantly different in ABO-identical versus ABO-incompatible settings in the tubing loop model. CONCLUSION: Our study showed that blood group antigens were not expressed on hepatocytes, even after isolation procedures or subsequent incubation with cytokines. This finding is an important step toward removing the restriction of ABO matching in hepatocyte transplantation. Our results suggest that ABOi-HTx is a feasible therapeutic option, especially in patients who require urgent treatment with freshly isolated hepatocytes, such as those with acute liver failure.