Quantifizierung der IFN-γ-mRNA in Lymphknotenzellen von Lewis-Ratten mit Experimenteller Autoimmuner Enzphalomyelitis (EAE) und ihre Beeinflussung durch Kaliumkanalmodulatoren

Kirsten Wissel

ISBN 978-3-89722-429-2
230 pages, year of publication: 2000
price: 40.50 €
Abstract: Alkoxypsoralens, known as DNA photomodifying agents, have been shown to block voltage-dependent K+ channels (Kv) as well as to alleviate functional deficits in certain multiple sclerosis (MS) patients in a manner similar to 4-aminopyridine. Since Kv channel blockers are known to inhibit T cell-mediated immune responses both in vitro and in vivo, we investigated the effects of 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), Margatoxin (MgTX), known as specific blocker of the Kv channel subtype Kv1.3, and Quinidine, blocking agent of Ca2+- and voltage-dependent K+ channels, by (1) proliferation of lymph node cells (LNC) from Lewis rats challenged for experimental autoimmune encephalomyelitis (EAE) by immunisation with spinal cord homogenate as measured by 3H-thymidine incorporation and (2) IFN-y gene expression of LNC as measured by quantitative RT-PCR. The effects of the K+ channel blockers were determined in unstimulated (control), guinea pig (GP) myelin- and Concanavalin A (ConA)-stimulated LNC at different phases of the disease. For accurate quantitation of small differences in IFN-g mRNA levels we established a non-competitive RT-PCR that based on the internal standardization by using a synthetic homologous internal RNA standard which was added to a certain number of LNC prior to the RNA extraction. The co-preparation of both internal RNA standard and wildtype total RNA was followed by co-amplification in the same PCR and co-quantification by differential liquid phase-hybridization/ELISA in microtiter plates. A linear measuring range was achieved comprising up to 40fold changes of wildtype mRNA copy numbers in cell cultures, and the accuracy of quantitation method was confirmed by a variance of about 23 %. The maximum IFN-g wildtype mRNA copies per lymph node cell in the control and GP-myelin cultures were found at the height of disease. Contrastly the highest number of IFN-g mRNA copies in ConA-cultures was attained in the early phase. We found suppressive effects of 5-MOP and 8-MOP on cell proliferation as well as on gene expression of IFN-g in control, GP-myelin and ConA-stimulated cultures. Thereby a significant stronger inhibition of 5-MOP compared to 8-MOP was monitored. Quinidine exhibited high blocking effects on cell proliferation and IFN-g gene expression in all cell cultures. Contrastly MgTX did not suppress significantly the cell proliferation nor IFN-g gene expression in control as well as in in vitro stimulated cell cultures. Due to our findings we conclude that 5-MOP, 8-MOP and Quinidine interfere with voltage-controlled signal transduction in T-lymphocytes and might therefore suppress immune responses in autoimmune diseases of the central nervous system. In particular the alkoxypsoralens and their derivates without phototoxic effects are new candidates for further studies on K+ channel blocking immunosuppressive drugs. The agents may exert a dual beneficial effect on demyelinating diseases like MS due to their ability to attenuate the inflammatoryprocess and to improve axonal conductivity.

  • Interferongamma-Genesepression
  • EAE/MS
  • Therapie f. MS-Patienten
  • Validierung der Quantifizierung
  • noncompetitive RT-PCR

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