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.

Influence of polyclonal ATGs on expression of adhesion molecules: an experimental study.

BACKGROUND: The aim of our study was to assess the influence of polyclonal antithymocyte globulins (ATGs) on the expression of adhesion molecules on lymphocytes, neutrophils, and thrombocytes by means of flow cytometry. ATGs are employed in various regimens for solid organ transplantation. Immunosuppression with ATGs may influence the expression of adhesion molecules on thrombocytes, lymphocytes, and neutrophils due to nonspecific antibodies directed against myeloid and nonmyeloid cells. MATERIAL AND METHODS: Depletion, activation, and expression of adhesion molecules on thrombocytes (CD41, CD42, CD62p and CD107a), neutrophils, and lymphocytes (CD11, CD18, CD62L) were studied in vitro in whole blood of healthy volunteers by means of flow cytometry after incubation with different dosages of three ATGs. RESULTS: Our data showed no ATG-mediated cytotoxic activity against platelets. ATGs were able, however, to induce activation of platelets through increased expression of P-selectin and hLAMP-1. ATGs also influenced the expression of adhesion molecules on lymphocytes and neutrophils by reducing the expression of CD62L. Furthermore, the effects of ATG on CD11/CD18 were dependent on the dosage and type of ATG. CONCLUSION: Polyclonal ATGs induced expression of adhesion molecules and activation of unstimulated thrombocytes as well as reduced the expression of adhesion molecules on lymphocytes and neutrophils. Increased adhesion of thrombocytes may be responsible for the undesirable side effects observed in clinical practice such as thrombocytopenia. However, reduction in the expression of adhesion molecules on lymphocytes and neutrophils may decrease the effects of ischemia/reperfusion injury.

Can we continue research in splenectomized dogs? Mycoplasma haemocanis: old problem--new insight.

We report the appearance of a Mycoplasma haemocanis infection in laboratory dogs, which has been reported previously, yet, never before in Europe. Outbreak of the disease was triggered by a splenectomy intended to prepare the dogs for a hemorrhagic shock study. The clinical course of the dogs was dramatic including anorexia and hemolytic anemia. Treatment included allogeneic transfusion, prednisone, and oxytetracycline. Systematic follow-up (n = 12, blood smears, antibody testing and specific polymerase chain reaction) gives clear evidence that persistent eradication of M. haemocanis is unlikely. We, therefore, had to abandon the intended shock study. In the absence of effective surveillance and screening for M. haemocanis, the question arises whether it is prudent to continue shock research in splenectomized dogs.

Evaluation of a system for the perfusion of isolated, rodent organs.

Perfusion of isolated organs is a common experimental approach. However, the surfaces of the perfusion system might alter the components of the blood and thereby negatively affect organ function. The aim of this study was to minimize the influence of the perfusion system on the blood components and to evaluate the system. Pressure and flow in the perfusion system consisting of a roller-pump, reservoir, oxygenator, hemo-filter and bubble-trap with a total tubing length of 4.5 m are controlled by a computer software (DASYLAB, Datalog, Moenchengladbach, Germany) via a transducer connected to the system. The organ to be perfused is positioned under a microscope (Orthoplan, Leica, Bensheim, Germany), allowing the investigation of microcirculatory parameters. The images raised are recorded on video tapes. To evaluate the system it was perfused with human blood (Hct 28 to 30%) for 90 min. Heparin (n = 6) or citrate (n = 6) served as anti-coagulants. The disappearance of cells from the blood was determined at time points 0, 1, 5, 10, 15, 20, 30, 45, 60, 75 and 90 min by means of a cell counter (AC T8, Coulter Beckmann, Krefeld, Germany). Cell activation was assessed by analysis of the expression of L- and P-selectin and CD11b. The activation of the complement system was examined by measuring the serum levels of the complement factors C3c and C4. There was no significant loss or activation of the blood cells at any of the above given time points. The serum levels of the complement factors remained within the physiological range and showed no changes throughout the whole experiments. Thus, the perfusion system does not have a negative influence on the blood and its individual components, and is therefore a reliable tool for perfusion experiments.

Mechanisms of cytokine cascade activation in patients with sepsis: normal cytokine transcription despite reduced CD14 receptor expression.

BACKGROUND: Lipopolysaccharide causes activation of monocytes/macrophages with excessive secretion of cytokines resulting in hypotension and shock in patients with sepsis. Lipopolysaccharide may induce these responses by interacting with lipopolysaccharide-binding protein and then binding to the cell surface protein CD14 or by acting directly with CD11-CD18 on monocytes/macrophages. The role of CD14 and CD11-CD18 in the activation of macrophages with enhanced cytokine transcription in patients with septic shock remains to be determined. METHODS: To study this, heparinized blood was obtained from 16 patients with septic shock on days 0, 1, 3, 5, 7, and 10 and compared with 20 control patients. The expression of CD14 and CD11b on monocytes in whole blood was measured by direct immunofluorescence and flow cytometry. Moreover, whole blood was stimulated with lipopolysaccharide (1 microgram/ml) for 0, 1, 2, 4, 8, and 24 hours, and messenger RNA expression for tumor necrosis factor-alpha, interleukin-beta (IL-1 beta), and IL-6 was determined on isolated peripheral blood mononuclear cells with Northern blot analysis. RESULTS: Both CD14 expression and receptor density on monocytes from whole blood were markedly suppressed (-63% on day 3; p < 0.05) in the septic group compared with controls. Although CD11b expression was also decreased (-24% on day 1; p < 0.05), receptor density on monocytes was slightly increased in the septic group in comparison with the control group. Kinetics and intensity of messenger RNA expression for tumor necrosis factor-alpha, IL-1 beta, and IL-6 were similar in both groups. CONCLUSIONS: These data indicate that in patients with septic shock, lipopolysaccharide-mediated signaling and cytokine transcription are unchanged despite a significant reduction of CD14 expression and density on monocytes. Thus, lipopolysaccharide-induced activation of monocytes from patients with sepsis may occur through direct binding of lipopolysaccharide to the CD11-CD18 complex or other lipopolysaccharide receptors, whereas binding of the lipopolysaccharide-lipopolysaccharide-binding protein complex to the CD14 receptor may not play a pivotal role in sepsis.