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Berlin Brandenburg

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  • 1
    In: Transplantation, 2000, Vol.69(9), pp.1977-1981
    Description: 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 γ-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HUVEC 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, Ca 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.
    Keywords: Endothelium ; Cytokines ; Lymphocytes T ; Immunosuppression ; Transplantation ; Interleukin 1 ; ^G-Interferon ; Prostaglandin E2 ; Mycophenolate Mofetil ; Clinical ; Man ; Immunology ; Gamma -Interferon ; Immunology ; Man ; Mycophenolate Mofetil ; Prostaglandin E2;
    ISSN: 0041-1337
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  • 2
    In: Transplantation, 2000, Vol.69(4), pp.588-597
    Description: 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 Ca plays a critical role in cell-cell interaction, the Ca-channel blocker verapamil might be a good cany.didate for supporting CsA/tacrolimus-based therap 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 μM (CD4-adhesion) and 11 μM (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 μM; 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.
    Keywords: Immunosuppression ; Endothelium ; Lymphocytes T ; Immunosuppressive Agents ; Cell Motility ; Verapamil ; Calcium Channel Blockers ; Experimental ; Function ; Immunology ; Calcium Channel Blockers ; Cell Motility ; Immunology ; Verapamil;
    ISSN: 0041-1337
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  • 3
    Language: English
    In: Toxicology in Vitro, 2004, Vol.18(4), pp.527-532
    Description: In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three- dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c- met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two- dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.
    Keywords: Human Hepatocytes ; Drug Toxicity ; Cell Architecture ; Pharmacy, Therapeutics, & Pharmacology ; Chemistry ; Public Health
    ISSN: 0887-2333
    E-ISSN: 1879-3177
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  • 4
    Language: English
    In: International journal of molecular medicine, May 2008, Vol.21(5), pp.611-20
    Description: Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.
    Keywords: Autocrine Communication ; DNA Replication ; Cell Differentiation -- Physiology ; Hepatocytes -- Physiology
    ISSN: 1107-3756
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 5
    Language: English
    In: Transplant Immunology, 1998, Vol.6(4), pp.251-259
    Description: The novel immunosuppressive drug mycophenolate mofetil (CelICept registered , 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 super(+) and CD8 super(+) T-cell adhesion and penetration by MMF. The ID sub(50) value for CD4 super(+) T-cell adhesion was calculated to be 0.03 mu M and the ID sub(50) value for CD4 super(+) T-cell penetration 1.21 mu M. 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.
    Keywords: Medicine ; Biology
    ISSN: 0966-3274
    E-ISSN: 1878-5492
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  • 6
    Language: English
    In: World journal of gastroenterology, 14 April 2005, Vol.11(14), pp.2080-7
    Description: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.
    Keywords: Cell Culture Techniques -- Methods ; Hepatocytes -- Cytology
    ISSN: 1007-9327
    E-ISSN: 22192840
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  • 7
    Language: English
    In: Transplantation, 27 December 2003, Vol.76(12), pp.1735-41
    Description: The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 microM, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.
    Keywords: Cell Adhesion -- Drug Effects ; Endothelium, Vascular -- Physiology ; Mycophenolic Acid -- Analogs & Derivatives
    ISSN: 0041-1337
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  • 8
    In: Transplantation, 2000, Vol.70(1), pp.236-240
    Description: Interaction of endothelial P-selectin with sialyl Lewis-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 E 2 (PGE 2) 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 PGE 2-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 PGE 2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.
    Keywords: Cd4-Positive T-Lymphocytes -- Physiology ; Dinoprostone -- Pharmacology ; Endothelium, Vascular -- Cytology ; P-Selectin -- Biosynthesis;
    ISSN: 0041-1337
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  • 9
    Language: English
    In: International Journal of Molecular Medicine, May 2008, Vol.21(5), pp.611-620
    Description: Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks (‘autocrine’ setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.
    ISSN: 1107-3756
    Source: Spandidios Publications (Spandidos Publications Ltd.)
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  • 10
    In: International Journal of Molecular Medicine, 05/01/2008
    ISSN: 1107-3756
    E-ISSN: 1791-244X
    Source: CrossRef
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