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In dieser Arbeit wurde LOX-1 der Ratte kloniert und rekombinant dargestellt. Es wurden polyklonale Antikörper gegen zwei Bereiche von LOX-1 generiert. Der zytosolische Bereich von LOX-1 wird in vitro phosphoryliert. Ischämie führt im Rattenherzen zu vermehrter Expression von LOX-1. Vorbehandlung der Tiere mit Sildenafil kann diesen Effekt vermindern. LPS-Induzierte Sepsis führt im Rattendarm zu vermehrter Expression von LOX-1 auf mRNA-Ebene.
Expression of the T cell regulatory molecule ICOS (CD278) and LICOS (CD275) on human blood cells
(2006)
Expression of the T cell regulatory molecule ICOS (CD278) and LICOS (CD275) on human blood cells Summary General bacterial infections, which can lead to the clinical picture of sepsis, are a major concern in intensive care units (ICU) and mortality remains high. Recent data have shown that, besides an overreaction of the immune system, also immunosuppression also plays a role in the pathogenesis of sepsis. Immunosuppression has been documented in patients with polytrauma, stroke and burn wounds, which all confer a high risk of severe bacterial infection. Moreover, it has been shown that T cells have an important role in sepsis. A shift of a Th1 dominated T cell response towards a Th2 response has been described as a potential mechanism of immune suppression in patients with sepsis. One of the molecules on the surface of T cells that is involved in the Th2-mediated immune response is the Inducible Costimulator of T cells (ICOS). Its ligand, LICOS, is expressed on the surface of B cells and monocytes. ICOS ligation induces the production of anti-inflammatory cytokines, especially of IL-10. However, nothing is known about the expression of ICOS on T cells and that of LICOS on APCs in patients with severe trauma and stroke. Therefore, in the present study, in a first step, a recombinant human LICOS-Ig fusion protein was generated, which was then used as an antigen for the generation of anti-LICOS monoclonal antibodies. In three fusion experiments, 5,000 primay clones were screened and a single hybridoma was obtained, which produced monoclonal antibodies that specifically reacted with recombinant LICOS, both in form of the LICOS-Ig fusion protein and on the surface of a cell line transfected with a full-length LICOS transgene. Since, it turned out that the antibodies did not bind with high affinity to wild type LICOS, as it is expressed on primary human blood cells, phenotypic analyses were carried out with another anti-LICOS monoclonal antibody, which had become commercially available. Next, the expression of HLA-DR, CD86, LICOS, and ICOS, on the surface of monocytes (CD14+), B cells (CD19+) and T cells (CD3+, CD4+) in whole blood was measured by flow cytometry. Six patients with severe trauma and nine stroke patients were compared with 32 healthy donors. On CD14+ monocytes from healthy donors, the expression levels of HLA-DR and CD86 were over 90%, while the expression of LICOS was much lower (7,5%). In critically ill patients, HLA-DR, CD86 and LICOS expression were strongly reduced. CD86 and HLA-DR were co-regulated, while HLA-DR and LICOS were not. In healthy donors, virtually all B cells expressed HLA-DR and the majority of them co-expressed LICOS (72%), while only a small fraction were CD86+ (14%). After trauma and stroke, HLA-DR, as well as LICOS expression on these cells remained normal; CD86 had a tendency towards being downregulated in most of the trauma patients, while most of the stroke patients exhibited normal CD86+ levels. The levels of HLA-DR and LICOS on T cells in trauma and stroke patients were low and very similar to those of healthy donors. The fraction of CD3+ T lymphocytes or their CD4+ subpopulation, which expressed measurable levels of ICOS (64% and 48%, respectively), did not change after stoke or trauma. However, within the ICOS+ T cell population two subpopulations could be distinguished: ICOSbright and ICOSdim T cells. Interestingly, the ICOSbright subpopulation, but not the ICOSdim and ICOSnegative subpopulations, was markedly increased in all trauma patients and in most of the stroke patients. Given that CD86 was co-regulated with HLA-DR on monocytes it appears that, similar to HLA-DR, CD86 expression could discriminate between patients with a low and high risk of sepsis. In contrast, because of its low basal expression on monocytes and its low signal-noise ratio, LICOS expression levels are not informative. Since ICOS expression on T cells is tightly connected to IL-10 secretion, the high proportion of ICOS bright cells in critically ill patients might contribute to the high IL-10 serum concentrations, which have been reported to be linked to immunosuppression in these patients.
Hintergrund: Die Peritonitistherapie beinhaltet häufig die Abdominallavage mit dem lokalen Antiinfektivum Polyhexanid, als 0,05% Lösung, die häufig zu einer Hypotension führt. Methode: Wir untersuchten in gesunden Ratten die Auswirkungen einer Peritoneallavage mit Polyhexanid oder NaCl (je 10ml über 10min; Polyhexanid n=5, NaCl n=5) auf den Blutdruck und registrierten bei 7 weiteren Tieren die Durchmesser submukosaler Blutgefäße einer Ileumschlinge vor und nach lokaler Superfusion mit Polyhexanid mittels intravitaler Fluoreszenzmikroskopie. In isometrischen Präparationen der thorakalen Rattenaorta mit und ohne Endothel, die mit Phenylephrin(PE) oder KCl 20 oder 40 mmol kontrahiert worden waren, wurden die Effekte von Polyhexanid auf den Gefäßtonus bestimmt. Resultate: Es wurde festgestellt, dass Peritoneallavage mit Polyhexanide zu einem Abfall des mittleren arteriellen Drucks führte, während Superfusion mit Polyhexanid eine lokaleVasodilatation der Gefäße der Darmwand bewirkte. In vitro relaxierte Polyhexanide Aortenringe, die mit Phenylephrin kontrahiert worden waren, Präparationen +End reagierten empfindlicher Inkubation mit dem NO-Synthetasehemmer N(G)-nitro-L-arginine methyl ester oder dem Guanylylcyclasehemmer 1H-(1,2,4)Oxodiazolo-(4,3-a)quinoxalin-1-one führte jeweils zu einer partiellen Hemmung der relaxierenden Wirkung. Die relaxierenden Effekte von Polyhexanid konnten in Präparationen ohne Endothel durch keinen der Kaliumkanalblocker Charibdotoxin, Tetraäthylammoniumchlorid, Glibenclamid oder 4-Aminopyrridine verhindert werden. Polyhexanid zeigte keine Wirkung auf Aortenpräparate, die mit 40mmol KCl kontrahiert worden waren. Schlussfolgerung: Es ist zu folgern, dass Polyhexanid NO freisetzen, Kaliumkanäle aktivieren und darüber zu einer Vasodilatation führen kann, die dann eine Hypotension bewirkt.