Bernd H. Markus

Transplantation for Primary Biliary Cirrhosis

Primary biliary cirrhosis is a frequent indication for liver transplantation. The purpose of this report is to present our experience with liver transplantation for primary biliary cirrhosis. Attention is given to the causes of hepatic dysfunction seen in allografts. In addition, we review the postoperative problems encountered and the quality of life at time of last follow-up in patients with transplants for primary biliary cirrhosis. A total of 97 orthotopic liver transplant procedures were performed in 76 patients with advanced primary biliary cirrhosis at the University of Pittsburgh from March 1980 through September 1985. The transplant operation was relatively easy to perform. The most common technical complications experienced were fragmentation and intramural dissection of the recipient hepatic artery, which required an arterial graft in 20% of the cases. Most of the postoperative mortality occurred in the first 6 mo after transplantation, with an essentially flat actuarial life survival curve from that time point to a projected 5-yr survival of 66%. Common causes of death included rejection and primary graft nonfunction. Thirteen of the 76 patients had some hepatic dysfunction at the time of the last follow-up, although none were jaundiced. Recurrence of primary biliary cirrhosis could not be demonstrated in any of the patients. Antimitochondrial antibody was detected in the serum of almost all of the patients studied postoperatively for it. Most important, almost all of the 52 surviving patients have been rehabilitated socially and vocationally.

Impact of oxidative stress on human cytomegalovirus replication and on cytokine-mediated stimulation of endothelial cells.

Transplantation-related pathogenic factors such as ischemia or allograft-directed inflammation are associated with oxidative changes that might lead to cellular oxidative stress. The aim of this study was to investigate the impact of oxidative stress on: (1) CMV replication in cultured human endothelial cells and (2) the stimulation of endothelial cells by proinfiammatory cytokines. Both pathomechanisms are known to contribute to graft rejection crises in vivo. Oxidative stress was induced in endothelial cell cultures with 10-200 microM buthionine sulfoximine. Western blotting showed a significant increase in the production of CMV-specific immediate early and late proteins in buthionine sulfoximine-treated cultures. Immunocytochemical staining suggested that this effect was caused by increased numbers of CMV antigen expressing cells (66% immediate early; 78%, late). Quantitative polymerase chain reaction for CMV-specific DNA and virus titration revealed that enhanced viral replication levels correlated with increased virion production. As a measure for the endothelial cell activation status, the surface expression of HLA-ABC and HLA-DR and adhesion molecules (ICAM-1, ELAM-1, VCAM-1) was quantified by fluorometric methods. Whereas oxidative stress alone did not modulate any surface molecule expression, the IFN-gamma-mediated expression of HLA-ABC and HLA-DR and the IL-1-mediated expression of ICAM-1, but not of ELAM-1 and VCAM-1 (IL-1 + TNF-alpha), was amplified. Interestingly, the amplification of HLA molecule expression was even higher in CMV-infected endothelial cells. This study provides evidence that oxidative stress contributes to the regulation of CMV replication, virus shedding, and the activation of endothelial cells by proinflammatory cytokines as it is observed in transplant recipients.

ISOLATION AND PRIMARY CULTURES OF HUMAN INTRAHEPATIC BILE DUCTULAR EPITHELIUM

SummaryA technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.

Preservation of the Synthetic and Metabolic Capacity of Isolated Human Hepatocytes by Coculture With Human Biliary Epithelial Cells

Bioartificial liver support systems have demonstrated limited efficacy in compensation of liver detoxification and substitution of liver‐derived factors. However, in these devices, the biological substitution of the complex liver function has been restricted to xenogeneic or transformed hepatocytes. Therefore, we have examined the long‐term effect of coculturing normal human hepatocytes (HCs) with allogeneic biliary epithelial cells (BECs). We applied functional in vitro assays to examine their metabolic potential by ammonia detoxification to urea, cytochrome P450‐dependent lignocaine conversion to mono‐ethyl‐glycine‐xylidide (MEGX), and protein expression and secretion. As the liver has a pivotal role in the synthesis of coagulation factors, we measured antithrombin III (AT III), factor VII, and albumin, comparing HCs plated on collagen or inside 3‐dimensional collagen gels. Over 30 days, expression and secretion of albumin and clotting factors by human HCs were augmented by culture inside collagen gel, but were also enhanced and better maintained by coculture with BECs. Higher proportions of BECs cocultured with HCs substantially increased the protein synthesis and urea production. Remarkably, the almost absent cytochrome P450 activity of HC alone after 1 week could be reversed and maintained over 3 weeks by coculture with BECs. The pattern of these effects differed from the extent of interleukin‐6 (IL‐6) production and HC viability under the compared conditions. In conclusion, coculture of human HCs with BECs impressively restores the synthetic and metabolic liver function in vitro. These results suggest mechanisms of improved liver epithelial differentiation supported by coculture conditions. This technique offers new perspectives in bioartificial liver support, hepatocyte transplantation, and stem cell differentiation. (Liver Transpl 2005;11:410–419.)

In vitro analysis of verapamil-induced immunosuppression: Potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells

BACKGROUND Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.

Effects Of Proinflammatory Cytokines On Cultivated Primary Human Hepatocytes: Fluorometric Measurement of Intercellular Adhesion Molecule-1 and Human Leukocyte Antigen-a, -b, -c, and -dr Expression

Expression of adhesion molecules and human leukocyte antigens on the surface of hepatocytes (HC) may play an important role in the immune reaction in different types of infectious and noninfectious hepatitis, liver graft rejection, and autoimmune liver diseases. The aim of this study was to evaluate the influence of the proinflammatory cytokines IFN-alpha, IFN-gamma, and IL-1 alpha on the expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-A, -B, -C, and -DR on highly purified primary human HC in cell culture. Expression was assessed by semiquantitative measurement of HC in cell culture by means of computer-aided fluorometry after immunofluorescent labeling. Avidin-biotin-immunoperoxidase staining was applied on parallel cultures to evaluate cell purity (> 99%) and to confirm the results obtained by fluorometry. ICAM-1 was expressed constitutively on untreated HC in vitro. Stimulation of HC with IFN-gamma and IL-1 alpha for 24 hr resulted in an increase of ICAM-1 expression. Cultured HC were moderately HLA-A, -B, and -C positive, but HLA-DR negative. Stimulation of HC with 500 U/ml IFN-gamma for 72 hr resulted in an increase of HLA-A, -B, -C, and -DR expression, whereas stimulation with 10 U/ml IL-1 alpha for 72 hr had no influence. By using 5000 U/ml IFN-alpha for 72 hr, we achieved an increase of HLA-A, -B, and -C expression; effects on the other tested antigens were not significant. In contrast to endothelial cells and transformed human hepatocytic cell lines, ICAM-1 on HC was changed more intensively by IFN-gamma than by IL-1 alpha. Furthermore, the results reveal differences in HLA and ICAM-1 expression between HC in vivo and in vitro.

Effects of desferrioxamine on human cytomegalovirus replication and expression of HLA antigens and adhesion molecules in human vascular endothelial cells

Abstract Desferrioxamine (DFO), commonly used in therapy as a chelator of ferric ion in disorders of iron overload, is a potent inhibitor of human cytomegalovirus (HCMV) replication in cultured fibroblast cells. Moreover, DFO has immunomodulatory activity both in vitro and in vivo. We studied DFO effects on HCMV replication in cultured human endothelial cells and on the expression of several cell surface molecules, which mediate interactions of endothelial cells with other cell types in the immune system. The concentrations of DFO required for 50% reduction in the number of endothelial cells expressing HCMV late antigen, ranged for several HCMV strains from 5.2 to 8.8 μM. DFO concentrations ranging from 5 to 40 μM inhibited cellular DNA synthesis in a dose-dependent manner without any significant effects on the cell viability. DFO at 10 μM concentration suppressed expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1), while it had no significant effect on the expression of vascular cell adhesion molecule-1 (VCAM-1). Expression of HLA class I and class II was not influenced by DFO treatment. The results showed that DFO is both effective in inhibition of HCMV replication and expression of ICAM-1 and ELAM-1 in endothelial cells, a combination that warrants attention to its potential use to prevent HCMV-induced allograft rejection in transplant recipients.

Immunomodulatory properties of the metal chelators desferrioxamine and diethylenetriamine penta-acetic acid in vitro

In this study, we investigated the effects of the intracellular metal chelator desferrioxamine (DFO) and the extracellular metal chelator diethylenetriamine penta-acetic acid (DTPA), which were previously shown to have strong anticytomegalovirus potencies, on their ability to elicit immunomodulatory effects in vitro[fcn,3]. The results showed that nontoxic and in vivo attainable concentrations of both DFO and DTPA inhibited mitogen- and allogen-induced proliferation of peripheral blood lymphocytes. The immunomodulatory effects of DFO/DTPA seem to be due to the impaired expression of interleukin-2 receptor and the reduced secretion of interleukin-2. However, metal chelators were more effective than cyclosporine or tacrolimus (FK506) in our in vitro experiments. Moreover, cytotoxicity mediated by lymphokine-activated killer cells and natural killer cells and the expression of HLA and adhesion molecules on cytokine-stimulated endothelial cells were differentially impaired by DFO/DTPA. These results warrant further study of the immunological effects of metal chelators in vivo.

Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogeneic T cells or cytokines

BACKGROUND Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. METHODS Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. RESULTS Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. CONCLUSION The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.

Alloreactive Lymphocyte Adherence to Human Arterial Endothelial Cell Monolayers

The early events of the cellular infiltration of allografts involve interactions between lymphocytes and vascular endothelium. To investigate the role of HLA antigens in the recognition and adherence of recipient lymphocytes to donor epithelium, an in vitro adherence assay has been developed. Donor-primed lymphocytes (DPL) exhibited a nearly two-fold increase in adherence during coculture with donor human arterial endothelial cells (HAEC) as compared to third-party primed lymphocytes (TPL). The degree of DPL adherence correlated well with the extent of donor HLA antigen expression on HAEC. DPL adherence to HAEC expressing class I HLA antigens was blocked by class I-specific monoclonal antibodies (mAbs). Following treatment with gamma-interferon (Y-IFN) HAEC express class I and class II HLA antigens, and DPL adherence is blocked by class I or class II-specific mAbs. In contrast, the adherence of TPL to HAEC was unaffected by these mAbs. These studies suggest that, in the allograft situation, allorecognition may play a role in the adherence of recipient lymphocytes to the allograft endothelium.