Influence of polyclonal antithymocyte globulins on the expression of adhesion molecules of isolated human umbilical vein endothelial cells.

OBJECTIVES: Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents applied for the treatment and prevention of organ rejection after transplantation. ATGs induce complement-mediated cell death in T lymphocytes and decrease leukocyte adhesion. However, little is known about the effects of ATGs on endothelial cells (EC). Our aim was to study the influence of ATGs upon the expression of adhesion molecules on human umbilical vein endothelial cells (HUVECs) after stimulation with tumor necrosis factor (TNF)-alpha. MATERIAL AND METHODS: HUVECs obtained from umbilical cords were incubated with ATGs before and after 6-hour stimulation with TNF-alpha. The group incubated without ATG served as the controls. Another group was not stimulated with TNF-alpha. By flow cytometry, we analyzed the expression of several adhesion molecules: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM), platelet EC adhesion molecule (PECAM), and CD62E. Statistical analysis used analysis of variance. RESULTS: After TNF-alpha stimulation, the EC surface expression of ICAM-1 and CD62E was reduced, although not significantly, in treated as compared with untreated cells. The expression of ICAM-1 and CD62E was similar in the unstimulated groups. The expression of VCAM, PECAM, CD55, and CD58 was not modified by ATG treatment. CONCLUSION: Our results demonstrated that ATGs insignificantly reduced the expression of adhesion molecules in HUVECs. The effect of ATGs on stimulated HUVECs remains unclear, probably due to the lack of effector cells.

Antithymocyte globulins bind to human umbilical vein endothelial cells.

OBJECTIVE: Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents used for the treatment of organ rejection after transplantation. ATGs induce complement-mediated cell death of T lymphocytes and may decrease leukocyte adhesion. However, little is known about their effects on endothelial cells (ECs). Our aim was to study whether they bind to human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: HUVECs obtained from umbilical cords were cultured and incubated with ATGs. A group incubated without ATG served as controls. All groups were coincubated with an anti-rabbit-IgG. Binding of ATGs to HUVECs was investigated by means of flow cytometry. Statistical analysis was performed using one-way analysis of variance (ANOVA). RESULTS: ATGs were bound to HUVECs with or without prior stimulation by tumor necrosis factor-alpha (TNF-alpha). Binding of ATGs to HUVECs was >99% in all treated groups. The mean intensity of fluorescence was constant in the ATG-treated groups. CONCLUSIONS: Our results demonstrated that ATGs bind to HUVECs before and after stimulation with TNF-alpha. Binding of ATGs to HUVECs suggests an independent endothelial mechanism of ATG action.

Diaspirin crosslinked hemoglobin: evaluation of effects on the microcirculation of striated muscle.

Hemoglobin-based oxygen carriers such as diaspirin-crosslinked hemoglobin (DCLHb) have been proposed for blood substitution due to their plasma expansion and oxygen transport capacity. This study investigates the effects of DCLHb on the microcirculation of striated muscle after moderate topload infusion and isovolemic exchange transfusion in awake hamsters. The skinfold chamber model in hamsters and intravital fluorescence microscopy were used for analysis of vessel diameter, red blood cell velocity (RBCV), leukocyte sticking to the microvascular endothelium, and macromolecular leakage in striated skin muscle. In each animal, arteriolar and postcapillary vessel segments were chosen and sequentially recorded on videotape (baseline). Animals were subjected to either topload infusion (10% of blood volume) or isovolemic exchange transfusion (hct 30%) of DCLHb followed by measurements at 10, 30, and 60 min thereafter. In vivo visualization of plasma and leukocytes was performed using FITC-dextran 150,000 and rhodamine 6G, respectively. No significant changes of vessel diameter and RBCV were observed after topload infusion or isovolemic exchange transfusion with DCLHb, either in postcapillary venules or in arterioles when compared with baseline values. Leukocyte sticking and macromolecular leakage were not found enhanced after administration of DCLHb. We conclude that the introduction of DCLHb-bound oxygen into the tissue does neither stimulate leukocyte adhesion nor impair endothelial integrity.

Relation of early Photofrin uptake to photodynamically induced phototoxicity and changes of cell volume in different cell lines.

For efficacy of photodynamic therapy, selective uptake and retention of photoactive substances has been postulated. Therefore, measurements were performed to find out whether the photosensitiser Photofrin is taken up differently in malignant and non-malignant cells in vitro. In addition, the sensitivity of malignant cells and non-malignant cells to photodynamic exposure was investigated, by quantifying viability and volume alterations of the cells. Bovine aortic endothelial cells, mouse fibroblasts and amelanotic hamster melanoma cells were suspended in a specially designed incubation chamber under controlled conditions (e.g. pH, pO2, pCO2 and temperature). After establishing constant baseline conditions, the cellular fluorescence intensity per cell volume, indicative of the uptake of Photofrin, and cell volume were assessed by flow cytometry, and cell viability was quantified by the trypan blue exclusion test. Photodynamic exposure of cells was performed using an argon-pumped dye laser system via a 600 microns optical fibre at energy density of 4 Joules at the cell surface (40 mW/cm2, 100 s). In comparison to endothelial and fibroblast cells, the melanoma cells exhibited no increased uptake of Photofrin, and no enhanced sensitivity to photodynamic therapy (PDT). However, the fluorescence intensity/volume of endothelial cells was two to three times higher at each concentration of the photosensitiser. Following PDT, reduction in cell viability was dependent on the concentration of Photofrin, and directly correlated with fluorescence intensity per cell volume. In addition, the cells of all three lines, treated by PDT, revealed dose-dependent changes in cell volume. Melanoma cells exhibited the most excessive increase. It is suggested that selective uptake of photosensitiser in vitro is not characteristic for tumour cells. The high uptake of Photofrin by endothelial cells may indicate that the vascular endothelium is a major target for PDT, leading to cessation of tumour blood flow and subsequent destruction of tumour tissue. In addition, PDT-induced swelling of tumour cells might represent and effect synergistically impairing tumour perfusion, and thereby promoting tumour death.