B. H. Markus

Cytomegalovirus- and interferon-related effects on human endothelial cells : cytomegalovirus infection reduces upregulation of HLA class II antigen expression after treatment with interferon-γ

Cultured human umbilical vein endothelial cells (HUVEs) were infected with human cytomegalovirus (HCMV) strain AD169. Up to 50% HUVEs proved to be positive for HCMV early nuclear antigens 24 hours after inoculation with virus. Following infection kinetics of surface expression of HLA class I and II, intercellular adhesion molecule (ICAM-1) and endothelial lymphocyte adhesion molecule (ELAM-1) on HUVEs were investigated by means of flow cytometry. A slight increase in HLA class I expression was observed, whereas expression of HLA class II (DR, DP, DQ) antigens was not induced by infection with HCMV. Furthermore, when compared with uninfected cells treated with interferon-gamma (IFN-gamma), reduced enhancement of HLA-DR expression was conspicuous in HCMV-infected cells treated with IFN-gamma. There is evidence that only a portion of HUVE is affected in its ability to upregulate HLA class II antigens. While expression of ICAM-1 was found to be enhanced between 8 and 20 hours after infection with a maximum at 12 hours after infection, no modulation of ELAM-1 was seen.

A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation

Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.

Roboterassistierte laparoskopische Cholecystektomie und Fundoplicatio – erste Erfahrungen mit dem Da-Vinci-System

Abstract. We report on our first five robot-assisted laparoscopic cholecystectomies and one fundoplication (Da Vinci system). No postoperative complications were observed. For the cholecystectomies (three elective and two acute cases) mean operation time was 1 h 35 min, and mean hospital stay was 5 days; for fundoplication the operation time was 2 h 15 min. The main advantages seem to be improved visualization by using a stereo camera und ease of precise dissection by micromechanical instruments directed by masterslaves from a distant console. The main disadvantage is the high cost. To fully evaluate the benefit for the patient, prospective clinical trials are warranted.Zusammenfassung. Wir berichten über unsere ersten Erfahrungen mit der roboterassistierten laparoskopischen Cholecystektomie (n = 5) und Fundoplicatio (n = 1). Stereooptik und Instrumente, die von einer Konsole aus über Masterarme vom Operateur gesteuert werden, erlauben eine präzise und sichere Operation. Subjektiv ist die Kombination eines dreidimensionalen Sehens, gepaart mit der dimensionalen Erweiterung des Instrumenteneinsatzes, im Vergleich zur herkömmlichen laparoskopischen Technik der größte Vorteil. Da zur Zeit der Einsatz des Da-Vinci-Systems noch mit erheblichen Kosten verbunden ist, dürften der Verbreitung dieser Technik enge Grenzen gesetzt sein. Es ist aber absehbar, daß Entwicklungen der Computertechnik, der Mikro/Nanomechanik und haptischen Resonanz dazu führen werden, Robotiktechnologie auch im Bereich der Visceralchirurgie präsent zu machen.

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.

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.

Roboterassistierte, endoskopische Fundoplikatio nach Nissen bei Kindern: Hämodynamik, Gasaustausch und anästhesiologisches Management

ZusammenfassungIn der laparoskopischen Chirurgie stellt der Operationsroboter “da Vinci” die neueste Entwicklung dar. Wir berichten über die weltweit ersten 2 Mädchen im Alter von 10 und 12 Jahren, die sich einer endoskopischen Fundoplikatio nach Nissen mit diesem Robotersystem unterzogen haben. Während der gesamten Dauer der jeweils knapp 300-minütigen Allgemeinanästhesie erfolgte ein erweitertes hämodynamisches Monitoring, das aus invasiver Blutdruckmessung sowie arteriellen Blutgasanalysen in kurzen Abständen bestand. Wir konnten während der Operation – ein Pneumoperitoneum bestand bei beiden Kindern für 177 bzw. 180 min – keine relevanten Veränderungen von pH, paO2, paCO2, etCO2, Herzfrequenz und arteriellem Mitteldruck beobachten. Die Körpertemperatur wurde mit einer Wärmedecke aufrechterhalten. Unmittelbar nach Operationsende konnten beide Kinder extubiert und am 6. postoperativen Tag nach Hause entlassen werden. Ungeachtet der Tatsache, dass roboterassistierte Techniken die endoskopische Chirurgie entscheidend verbessern könnten, müssen trotz dieser ermutigenden Ergebnisse aufgrund der bislang niedrigen Patientenzahlen mögliche unerwünschte Auswirkungen der Roboterchirurgie als gegenwärtig nicht hinreichend geklärt betrachtet werden. Die Patienten bedürfen daher noch besonders intensiver – und somit auch invasiver –Überwachung und Aufmerksamkeit.AbstractThe 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 monitioring, unless a larger number of patients has been studied.

Modulation of adhesion molecule expression on endothelial cells by verapamil and other Ca++ channel blockers

Cytokine-induced expression of adhesion molecules on leukocytes and endothelial cells (EC) is a crucial point in the process of organ transplant rejection. It has been shown that protein kinase C (PKC) is involved in this activation process. Verapamil and other calcium channel blockers seem to possess immunosuppressive qualities in vivo and in vitro; some authors suggested that this is due to PKC- or calmodulin-antagonism. Thus our objectives were to further investigate the second-messenger systems involved in the stimulation of EC and to analyze whether the beneficial influence of calcium channel blockers on the outcome of transplantation is due to impaired expression of adhesion molecules on EC. Our results, obtained in an in vitro model using human umbilical vein EC, show that IL-1-induced expression of intercellular adhesion molecule-1 (ICAM-1) is in part mediated by PKC and that parallel activation of calmodulin is required. Expression of ICAM-1 was reduced to 38.5% by PKC-inhibitor H7 and to 77.2% by calmodulin-inhibitor W7. In addition, data on the intracellular events in TNF-alpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1) is presented, showing that both PKC and, to a higher extent, calmodulin, are involved in this process. Expression of VCAM-1 was reduced to 63.7% by H7 and to 27.7% by W7. IL-1-induced expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) is PKC-dependent but insensitive to blocking of calmodulin. Though activation of adhesion molecule expression utilizes PKC and/or calmodulin as second-messenger pathways the investigated calcium channel blockers verapamil (R- and S-enantiomers), diltiazem and Ro 40-5967 failed to inhibit adhesion molecule expression. Surprisingly, higher concentrations of verapamil (> 12.5 micrograms/ml) or Ro 40-5967 (5 micrograms/ml) significantly enhanced IL-1-induced expression of ELAM-1. ICAM-1-expression was also enhanced by verapamil, but not by Ro 40-5967 or diltiazem. This enhancement was only seen if verapamil was added maximally one hour after the cytokine stimulus indicating that transcriptional modulation is responsible for the observed effects. Our findings indicate that calcium channel blockers have an immunomodulating effect independent of adhesion molecule expression.

ROLE OF HLA ANTIGENS IN LIVER TRANSPLANTATION WITH SPECIAL REFERENCE TO CELLULAR IMMUNE REACTIONS

Abstract 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.

Lymphocytes induce enhanced expression of HLA class I antigens on cytomegalovirus-infected syngeneic human endothelial cells

Human cytomegalovirus (HCMV) infection has been associated with enhanced expression of HLA antigens on the endothelium and with cellular infiltrates within the graft following human organ transplantation. We investigated the interactions between human cytomegalovirus-infected cultured endothelial cells and cocultured syngeneic as well as allogeneic lymphocytes. Our objective was to find out whether cocultured lymphocytes elicit HCMV-mediated immune responses. In this report we focus on the modified expression of HLA antigens on the surface membrane of human umbilical vein endothelial cells (HUVECs). Endothelial expression of HLA class I and II antigens was measured by means of flow cytometry. Cocultures of HCMV-infected HUVECs with unprimed autologous PBLs led to virus-specific lymphocyte response, resulting in enhanced expression of HLA class I on HUVECs. This effect was only observed when lymphocytes were added to HUVECs during the very early phase after virus inoculation and was due to the stimulation of the CD8+ T-cell subpopulation. The modification of endothelial HLA expression was not observed in transwell cocultures, indicating the importance of cellular contact between endothelial cells and lymphocytes to elicit this effect. We conclude that HCMV-infected endothelial cells may induce virus-specific responses of unprimed syngeneic lymphocytes that lead to upregulated HLA class I expression on the endothelium. This pathway might be of important relevance for graft rejection crises after transplantation.

Cultured human biliary epithelial cells induce allogeneic lymphocyte activation in vitro: possible relevance in liver transplant rejection.

The biliary epithelium is a major target for allograft-directed immune responses during rejection crises after liver transplantation. This paper deals with in vitro studies on the immunogenetic potential of cultured biliary epithelial cells (BECs) to elicit an allogeneic cellular immune response. Therefore, BECs were cocultured with syngeneic and allogeneic lymphocytes in order to study lymphocyte activation. The respective lymphocytes [3H]thymidine incorporation was a measure for the proliferative activity. While syngeneic peripheral blood lymphocytes (PBL) never exhibited BEC-induced proliferation allogeneic PBL were significantly (P < 0.05) activated in all experiments (n = 6). In experiments with purified subpopulations CD8+ cells but not CD4+ cells proved to be activated by BECs. In time kinetics (n = 5) the maximum of the BEC-induced proliferation was on day 9 while the endothelial cell-induced proliferation was found to be 2 days earlier on day 7 after onset of the experiments (P < 0.05). BEC-induced proliferation was accompanied by induced IL-2 secretion (> 300 pg/ml) by activated lymphocytes as determined by ELISA. Stimulation of BECs with 500 U/ml interferon-gamma, 1000 U/ml interferon-alpha or blocking expression of HLA molecules on the surface membrane of BECs by monoclonal antibodies did not alter BEC-induced allogeneic lymphocyte proliferation. Monoclonal antibodies against CD8+ but not CD4+ suppressed proliferative activity of PBL and CD8+ cells by 40 and 45%, respectively. Overall, these results provide evidence that BECs may induce CD8+ lymphocyte activation in vivo and therefore might play a crucial role in triggering immune responses related to liver transplant rejection episodes.