Hepatocyte-induced CD4+ T cell alloresponse is associated with major histocompatibility complex class II up-regulation on hepatocytes and suppressible by regulatory T cells.

Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune-mediated loss of transplanted cells is observed, and longterm acceptance has not been achieved yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Tregs), which therefore also might enable reduction of posttransplant cell loss and enhance longterm allograft acceptance. We hence characterized hepatocyte-induced immune reactions and evaluated the immunomodulatory potential of Tregs applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4+ CD25high CD127low Tregs were added to cocultures in single-/trans-well setups with/without supplementation of anti-interferon γ (IFNγ) antibodies. Hepatocyte-induced alloresponses were then analyzed by multicolor flow cytometry. Measurements indicated that T cell response upon stimulation was associated with IFNγ-induced major histocompatibility complex (MHC) class II up-regulation on hepatocytes and mediated by CD4+ T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8+ T cells showed early up-regulation of CD69 despite lack of cell proliferation in the course of coculture. Supplementation of Tregs effectively abrogated hepatocyte-induced alloresponses and was primarily cell contact dependent. In conclusion, human hepatocytes induce a CD4+ T cell alloresponse in vitro, which is associated with MHC class II up-regulation on hepatocytes and is susceptible to suppression by Tregs. Liver Transplantation 24 407-419 2018 AASLD.

Antibiotic-Resistant Bacteria Colonizing the Bile Duct Are Associated with Increased Morbidity and Mortality after Resection of Extrahepatic Cholangiocarcinoma.

Background: Patients with extrahepatic cholangiocarcinoma (CCA) face considerable morbidity including septic complications after surgery. The aim of this study was to characterize the bacterial spectrum of the common hepatic duct (CHD) and its clinical relevance regarding morbidity and mortality after resection of extrahepatic CCA. Methods: We retrospectively analyzed data from 205 patients undergoing surgery for extrahepatic CCA in our department between January 2000 and March 2015. Patients were reviewed for pre-operative medical conditions, biliary bacterial flora obtained from intra-operative swabs, different septic complications, and post-operative outcome. Results: Bacterial colonization of the CHD was observed in 84.9% of the patients, with Enterococcus faecalis being detected most frequently (28.3%). Wound infections occurred in 30.7% of patients. Bacterial flora of the CHD and of the post-operatively colonized wounds coincided in 51.5% and of intra-abdominal swabs obtained during surgical revisions in 40.0%. Ciprofloxacin-resistant bacteria in the CHD were identified as independent risk factor for wound infections (odds ratio [OR], 3.330; 95% confidence interval [CI], 1.771-6.263; p < 0.001) and for complications requiring surgical revision (OR, 2.417; 95% CI, 1.288-4.539; p = 0.006). Most important independent risk factors for intra-hospital mortality were ampicillin-sulbactam-resistant bacteria in the CHD (OR, 3.969; 95% CI, 1.515-10.399; p = 0.005) and American Society of Anesthesiologists (ASA) grading >2 (OR, 2.936; 95% CI, 1.337-6.451; p = 0.007). Conclusions: Antibiotic-resistant bacteria from the CHD are associated with increased morbidity and mortality in patients undergoing resection for extrahepatic CCA.

Longitudinal imaging and femtosecond laser manipulation of the liver: How to generate and trace single-cell-resolved micro-damage in vivo.

The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imaging in vivo on single cell level including re-localisation of ablated regions in follow-up measurements after 2-7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3-6.6 µJ led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5-15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopy in vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed.

Establishment of a guided, in vivo, multi-channel, abdominal, tissue imaging approach.

Novel tools in humane animal research should benefit the animal as well as the experimentally obtained data. Imaging technologies have proven to be versatile and also in accordance with the demands of the 3 R principle. However, most imaging technologies are either limited by the target organs, number of repetitive imaging sessions, or the maximal resolution. We present a technique-, which enables multicolor abdominal imaging on a tissue level. It is based on a small imaging fiber endoscope, which is guided by a second commercial endoscope. The imaging fiber endoscope allows the distinction of four different fluorescence channels. It has a size of less than 1 mm and can approximately resolve single cells. The imaging fiber was successfully tested on cells in vitro, excised organ tissue, and in mice in vivo. Combined with neural networks for image restauration, high quality images from various abdominal organs of interest were realized. The second endoscope ensured a precise placement of the imaging fiber in vivo. Our approach of guided tissue imaging in vivo, combined with neuronal networks for image restauration, permits the acquisition of fluorescence-microscope like images with minimal invasive surgery in vivo. Therefore, it is possible to extend our approach to repetitive imaging sessions. The cost below 30 thousand euros allows an establishment of this approach in various scenarios.

Alloresponses of Mixed Lymphocyte Hepatocyte Culture to Immunosuppressive Drugs as an In-Vitro Model of Hepatocyte Transplantation.

BACKGROUND Hepatocyte transplantation (HCTx) has the potential for the treatment of end-stage liver disease. However, failure of engraftment and the long-term acceptance of cellular allografts remain significant challenges for its clinical application. The aim of this study was to investigate the efficacy of the immunosuppressive agents, Cyclosporine, Everolimus, and Belatacept to suppress the alloresponse of primary human hepatocytes in a mixed lymphocyte-hepatocyte culture (MLHC) and their potential hepatotoxicity in vitro. MATERIAL AND METHODS Primary human hepatocytes were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an MLHC. Proliferative alloresponses were determined by flow cytometry, and cytokine secretion was measured using Luminex-based multiplex technology. Using an MLHC, the alloresponses of primary human hepatocytes were compared in the presence and absence of Cyclosporine, Everolimus, and Belatacept. Cultured primary human hepatocytes were assessed for the production of albumin, urea, aspartate transaminase (AST) and DNA content. Metabolic activity was determined with the MTT assay. RESULTS Immune responses induced by primary human hepatocytes were effectively suppressed by Cyclosporine, Everolimus, and Belatacept. Everolimus significantly reduced the metabolic activity of primary human hepatocytes in vitro, suggesting impairment of cell viability. However, further functional analysis showed no significant differences between treated and untreated controls. CONCLUSIONS Cyclosporine, Everolimus, and Belatacept suppressed the alloresponse of primary human hepatocytes in an MLHC without significant cytotoxicity or functional cell impairment.