Amount of co-transplanted donor-derived leukocytes determines in-vivo microchimerism and mixed lymphocyte culture changes post-liver transplantation.

OBJECTIVE: To evaluate the impact of passenger leukocytes in liver grafts on the rate of microchimerism induction after liver transplantation and to evaluate immunological changes thereafter based on serial donor-specific MLC's in these patients. METHODS: 26 orthotopic liver transplant recipients were prospectively evaluated for immunological changes based on the co-transplantation of donor-derived leukocytes. Intraoperatively harvested liver biopsies and peripheral blood-lymphocytes of liver transplant recipients were sampled at various time points. Donor spleen cells were obtained during organ procurement. RESULTS: HLA-PCR analysis demonstrated a stable pattern of microchimerism in 15 out of 26 patients. Microchimerism was detectable by PCR up to a mean of 7 weeks after transplantation, when chimerism in the peripheral blood became negative. Passenger donor leukocytes were present in all biopsies obtained during backtable preparation of the liver graft. For the 15 patients presenting microchimerism the rate of passenger leukocytes in the liver graft biopsies showed a mean of 155.8 leukocytes per mm 2 liver tissue (SD +/- 23.2 cells/mm2, range 121 to 217 cells per mm2 tissue). Otherwise patients without chimerism showed a mean of 90.4 passenger leukocytes per mm2 tissue (SD +/- 14.5 cells/mm2, range: 52 to 99 cells/mm2). Lymphocyte proliferation, determined by donor-specific "multiple" single-way- mixed-lymphocyte-cultures (dsmMLC) was reduced to a mean of 62.2 % of preoperative values (SD +/- 14.5 %, range 33 % to 88 %) in the 15 patients with stable microchimerism. Otherwise in the 11 patients without microchimerism dsmMLC results stayed at continuously higher levels with a mean of 106 % (SD +/- 13.4, range 92 % to 134 %). CONCLUSIONS: The results from these studies of microchimerism and lymphocyte reactivity after liver transplantation suggest that the co-transplantation of donor leukocytes plays an important and active role in the modulation of the host-immune system.

[State of hepatocyte transplantation: a risk or a chance?]. / Stand der Hepatozytentransplantation: Risiko oder Chance?

Over the past few years, hepatocyte transplantation has been considered as an alternative method for orthotopic liver transplantation for the treatment of various liver diseases. Beside curative approach for genetic metabolic deficiencies (familial hypercholesterolemia, hemophilia, etc.), it could be a useful tool for bridging the waiting period until an appropriate donor organ is obtained. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally or into the spleen settle down in the diseased liver. This enables genetic modification to correct inborn metabolic deficiencies and improves survival in acute liver failure. In 1992, the first clinical transplantation of isolated hepatocytes in 10 patients was performed. In 1998, Fox and coworkers described the successful transplantation of allogeneic liver cells in a child with Crigler-Najjar syndrome. Accomplished studies of Strom et al. resp. Bilir et al. of the same year proved the effectiveness of liver cell transplantation for transient treatment of acute liver failure. Prerequisite of this cell-based therapeutic strategy is a sufficient amount of highly differentiated hepatocytes, hence, a well established in-vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes and to preserve the physiological cell function. This review discusses the different experimental approaches regarding the cultivation of human hepatocytes and also the use of alternative cell sources (like animal hepatocytes, immortalized cells of human origin, progenitor cells from fetal human liver/liver stem cells) for hepatocyte transplantation.

[Total endoscopic Nissen fundoplication with the robotic device "da Vinci" in children. Hemodynamics, gas exchange, and anesthetic management]. / Roboterassistierte, endoskopische Fundoplikatio nach Nissen bei Kindern. Hämodynamik, Gasaustausch und anästhesiologisches Management.

The robot device "da Vinci" represents the latest stage in laparoendoscopic surgery. We report the first two cases worldwide of endoscopic Nissen fundoplication with a telemanipulatory robot system in two children, aged 10 and 12 years. In addition to standard monitoring, we used invasive blood pressure monitoring during the 300 min periods of general anesthesia. Arterial blood gas samples were analyzed in short intervals. During surgery, which included 177 and 180 min periods of intraperitoneal insufflation of carbon dioxide, no significant changes of pH, PaO2, PaCO2, etCO2, heart rate, and mean arterial pressure were observed. Body temperature was maintained with an external warming blanket. Extubation was achieved immediately after the end of the operation, and both patients were discharged home on postoperative day 6. Robot-assisted techniques may possibly add significant progress and improvement to laparoendoscopic surgery. Nonetheless, we conclude that, despite our first encouraging results, potential risks of robot-assisted surgery have not yet been definitively defined. Therefore, patients are in need for intensive and even invasive monitoring, unless a larger number of patients has been studied.

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

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.

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.

Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy.

Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity.

Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen. PicoGreen has been shown to detect as little as 0.5 ng pure DNA or 10(2) cells (interassay SD < 10%, intraassay SD < 5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen, cells were digested with papain for 20 h at 60 degrees C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.

Role of HLA antigens in liver transplantation with special reference to cellular immune reactions.

In previous statistical analyses we have demonstrated the importance of the dualistic effect of HLA on liver transplant survival: HLA compatibility decreases cellular rejection but also increases other immunologically mediated, HLA-restricted mechanisms of allograft injury. More recently these results have been confirmed by other researchers, and several studies have shown higher recurrence rates of infectious diseases such as hepatitis B and C and CMV hepatitis for HLA-compatible liver transplants. Although current practice does not consider HLA in liver transplantation, cellular in vitro studies show a significant role of HLA antigens in clinically relevant phenomena. While increasing the amount of infection-related immune damage, HLA compatibility also decreases the alloproliferative response of the recipient to the donor tissue. Further studies must examine whether non-HLA antigens such as tissue-specific antigens and heat-shock-proteins participate in this process, and how target cells can present different peptides such as soluble HLA antigens or viral proteins to the recipient.

Dedifferentiation of human hepatocytes by extracellular matrix proteins in vitro: quantitative and qualitative investigation of cytokeratin 7, 8, 18, 19 and vimentin filaments.

BACKGROUND/AIMS: Liver cirrhosis and carcinogenesis are accompanied by an alteration in extracellular matrix material. Histological studies reveal upregulation of the intermediate filaments cytokeratins 8 and 18 and de novo synthesis of vimentin, and cytokeratin 7 or 19 in hepatocytes. The aim of this study was to investigate how these two processes are linked. METHODS: Human hepatocytes were seeded: (i) on the matrix components collagen I, IV, laminin, or fibronectin; (ii) on stoichiometrically different complete matrices, derived from human placenta (matrix I) or the Englebreth-Holm-Swarm tumor (matrix II), and (iii) inside a three-dimensional collagen I sandwich. Filament expression and assembly were measured by cytofluor analysis or confocal laserscan microscopy. RESULTS: The matrix components or complete matrices triggered enhancement of cytokeratins 8 and 18 and de novo synthesis of cytokeratins 7, 19 and vimentin in a characteristic way. Confocal images demonstrated a dense and uniform network of cytokeratin 18 in freshly isolated cells, which was "replaced" by a few, thick protein bundles within 20 days. Interestingly, newly synthesized cytokeratin 19 structurally resembled the cytokeratin 19 organization in biliary epithelial cells. Marked cytokeratin alterations could be partially prevented when hepatocytes were grown in a three-dimensional collagen sandwich. CONCLUSIONS: Pathological alterations to the chemical composition, molecular structure, or spatial arrangement of the liver matrix lead to specific changes in the intermediate filament pattern in human hepatocytes. We assume that degradation of the matrix results in pathological alterations to the hepatocyte-receptor matrix-ligand ratio, followed by a switch from physiological to pathological cell-activation.

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Expression of human leukocyte antigens class I and class II on cultured biliary epithelial cells after cytomegalovirus infection.

The influence of cytomegalovirus (CMV) infection on the HLA expression on cultured biliary epithelial cells (BEC) was investigated. CMV-infection augmented expression of HLA class I but not of HLA class II. CMV reduced the IFN-gamma-mediated induction of the de novo expression of HLA class II while the stimulated expression of HLA class I was not impaired. Autologous but not allogeneic PBL responded to CMV-infected BEC. This response resulted in upregulation of HLA class I on BEC which was significantly higher compared with the expression on infected BEC alone or on uninfected BEC cocultured with autologous PBL. The results suggest that CMV modulates the immunogenic potential of BEC, which is important for the HLA and CMV-mediated pathomechanisms in vivo.

The immunological role of biliary epithelial cells in human liver transplant rejection.

From histopathological analyses after liver transplantation it is evident that the biliary epithilium is an important target for leucocytes of the graft recipient. Besides clinical and histopathological investigations undertaken by several authors it was also endeavoured to determine the immunological impact of the biliary epithelial cells (BEC) in vitro. As for the intrahepatic BEC, in vitro studies proved to be restricted owing to difficult isolation procedures and the limited number of cells yielded from transplanted organs. Therefore, studies on cultured extrahepatic BEC served as a model for the immunological features of the biliary epithelium in transplantation. Herein, in vivo and in vitro studies dealing with BEC and immunologically mediated hepatic disorders are reviewed in order to understand better the pathogenesis after liver transplantation. Furthermore, possible underlying mechanisms of BEC-directed immunity with regard to BEC-leucocyte interactions are discussed.

[Liver transplantation and splenectomy in idiopathic portal hypertension]. / Lebertransplantation und Splenektomie bei idiopathischer portaler Hypertension.

Idiopathic portal hypertension (IPH) was diagnosed in a 30-year-old man. Clinical signs were splenomegaly, leucothrombocytopenia, and esophageal varices of fourth degree. The histology of the liver biopsy showed portal fibrosis with no evidence of cirrhosis. No causing agent or known disease could be found for the histopathological and clinical features. Due to a severe deterioration of general condition and a decline of synthetic liver function, liver transplantation and splenectomy were performed. The histological examination of the explanted liver revealed features of IPH, demonstrating portal fibrosis and dilated vessels adjacent to portal tracts; no cirrhosis was found. The postoperative recovery was without any severe complications. The duration of hospitalization was 28 days. Following liver transplantation, the esophageal varices disappeared and leucocytes, platelets as well as parameters of hepatic synthesis reached normal values. Initially, the immunosuppression was composed of prednisolon, tacrolimus, and antibodies against IL-2 receptors (BT 563) and was later continued with prednisolon and tacrolimus. Within the follow-up observation of 26 months, there was no evidence for graft rejection, severe infection, or occurrence of portal hypertension. Up till now the patient is in good condition with normal graft function. Liver transplantation may be a curative therapy for patients with advanced disease of IPH but the long-term follow-up after transplantation has to show whether IPH can reoccur.