In the second study we explored the effects of teriflunomide on proliferation and activation of microglia, the primary immune cells in the CNS.
In 2012 teriflunomide has been approved for the treatment of RRMS (Genzyme Corporation, 2012). The main effect of teriflunomide is reported to be anti-proliferative achieved by blocking the enzyme DHODH which is involved in de novo pyrimidine synthesis and hence, limiting the number of activated immune cells infiltrating into the CNS (DI NUZZO et al. 2014).
Several studies have been published on the effects of teriflunomide on T cells, B cells, and macrophages (WILLIAMSON et al. 1995; DAVIS et al. 1996). Pharmacokinetic studies demonstrated that a small percentage of teriflunomide crosses the BBB and reaches the CNS.
Hence, it is possible that it may affect the CNS resident cells. However, very few data suggest direct effects of teriflunomide on neurons and other resident cells in the CNS. For example, it was shown that teriflunomide causes a significant dose-dependent decrease of NO production in LPS/IFN-γ-stimulated primary rat astrocytes (MILJKOVIC et al. 2001). Furthermore, there is proof that teriflunomide modulates microglial activation by causing increased secretion of IL-10 by microglia (KORN et al. 2004). Our main focus was therefore to evaluate effects of teriflunomide on primary rat microglia.
As explained in the introduction (section 1.5) concentrations of approximately 1-3 µM of teriflunomide may reach the CNS and therefore the choice of the doses from 0.25-5 µM was to mimic the situation in vivo where microglia might be exposed to similar doses (LIMSAKUN u. MENGUY-VACHERON 2010). As a proof of principle, we could
Comprehensive discussion
52
demonstrate that these concentrations of teriflunomide inhibited proliferation of activated rat T cells in vitro.
Microglial proliferation is one of the hallmarks of neuroinflammation. In in vitro mixed glial cultures, microglial expansion commences starting from day 4-5 of culture and this process is further enhanced by addition of cytokines such as GM-CSF. Furthermore, several lines of evidence suggest that GM-CSF is a crucial effector cytokine in the CNS that can mediate neuroinflammation (ESEN u. KIELIAN 2007; KOSHIDA et al. 2015). Therefore we investigated proliferation of microglia in GM-CSF-treated mixed glial cultures (day 5) subjected to teriflunomide treatment. Notably, microglia cultured with GM-CSF grew rapidly compared to medium alone. Interestingly, we observed a decreased yield of microglia from GM-CSF-treated cultures co-treated with teriflunomide. We could confirm that this effect was not due to cytotoxicity but rather due to reduced proliferation of microglia.
Since we observed enhanced expression of DHODH-mRNA in microglia in response to LPS/IFN-γ treatment we speculate that the anti-proliferative effect of teriflunomide on activated microglia might be DHODH-dependent. However, at this moment a direct evidence to support this is lacking. It could well be that teriflunomide also inhibits microglial proliferation in a DHODH-independent manner as we observed similar reduction in proliferation of microglia in normal mixed glial cultures, post teriflunomide treatment.
Furthermore, we investigated whether teriflunomide has additional immunomodulatory or suppressive effects on microglia that might explain its beneficial influence on disease activity in MS.
Phagocytosis is one hallmark of activated microglia. In experimental demyelinating diseases it was shown that recruitment of microglia to clear myelin debris by phagocytosis is an essential process for subsequent repair mechanisms (KOTTER et al. 2001; SKRIPULETZ et al. 2013). However, as it was shown that “hyperactivated” microglia tackle viable neurons
Comprehensive discussion
53
during inflammation, others suggested detrimental effects of microglial phagocytosis (BLOCK u. HONG 2005; BROWN u. NEHER 2012). Here, neither resting nor LPS-activated microglia showed changed phagocytic activity after teriflunomide treatment.
Interestingly, at the highest concentration of teriflunomide (5 μM) the mRNA expression of the anti-inflammatory factor IL-10 was increased in LPS-treated microglia, suggesting that teriflunomide may have an anti-inflammatory effect on microglia. Partly, our findings are in line with results from Korn et al., who showed that teriflunomide triggers cultured rat microglia to enhance IL-10 release (KORN et al. 2004). In the same study, it has been reported that teriflunomide increases the expression of the co-stimulatory molecule CD86 (KORN et al. 2004) which has been associated with the induction of T cell proliferation and activation (BECHMANN et al. 2001). However, we observed an opposite effect of teriflunomide on CD86 expression with a slight but significant decrease in CD86 expression in LPS-activated microglia following 72 h teriflunomide treatment. A clear explanation for this contrasting finding is lacking and could be attributed to difference in teriflunomide doses or differences in the mixed glial cell culture and microglia isolation protocols. Although the effects we observed here were only small, it hints at the potential of teriflunomide in conferring an anti-inflammatory phenotype to microglia. Nevertheless, no significant effects of teriflunomide were observed on the mRNA expression of pro-inflammatory mediators such as iNOS and TNF-α in LPS/IFN-γ-treated microglia. This is contrasting to the reports showing that low concentrations of teriflunomide might exert pro-inflammatory effects by increasing production of pro-inflammatory factors (e.g. TNF-α, IL-1β) in peripheral monocytes and cultured macrophages from synovial fluid of rheumatoid arthritis (RA) patients (CUTOLO et al. 2003; POPA L et al. 2010).
Comprehensive discussion
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NF-κB, a key mediator in the pro-inflammatory response, is induced in macrophages and microglia in response to various external stimuli (e.g. LPS) (BALDWIN 1996). Several drugs are known to target NF-κB activation (EPINAT u. GILMORE 1999; MC GUIRE et al.
2013). Some of the existing MS therapies have been linked to altered NF-κB activation both by acting locally in the CNS and by interfering with peripheral immune activation. For example, responsiveness of MS patients to IFN-β was linked to NF-κB activation in monocytes and granulocytes (ZULA et al. 2011). Also, laquinimod, an oral immunomodulatory drug that in Phase III trials was demonstrated to be effective in patients with relapsing-remitting MS (BRUCK u. ZAMVIL 2012; COMI et al. 2012) has been shown in the model of cuprizone-induced demyelination to act through inhibition of NF-κB activation in astrocytes (BRUCK et al. 2012). In our experiments NF-B activation in response to LPS-treatment was unaffected in microglia following teriflunomide treatment.
As mentioned before the inhibition of the DHODH is mediated at nanomolar concentrations of teriflunomide (FOX et al. 1999), DHODH-independent effects such as inhibition of protein tyrosine kinases and NF-κB activation are achieved only at higher (µM) concentrations in vitro (XU et al. 1995; XU et al. 1996). Interestingly, in previous studies it was shown that teriflunomide inhibited the TNF-induced NF-κB activation in a human T cell line with maximum inhibition at 5-10 µM (MANNA u. AGGARWAL 1999; MANNA et al.
2000). In contrast, Zeyda and colleagues found that much higher concentrations of teriflunomide (200 µM) failed to inhibit critical early T cell signaling events in human peripheral blood T cells, including phosphorylation of ERK1/2 and activation of NF-κB via degradation of IκBα (ZEYDA et al. 2005). Therefore, it is likely that the effects of teriflunomide could underlie a different dose-dependent responsiveness and furthermore could be different in cell lines in comparison to primary cells as used in our investigations.
Comprehensive discussion
55
Taken together, we provide evidence for potential anti-proliferative mode of action of teriflunomide on microglia, in addition to its proposed action to inhibit proliferation of activated T and B cells in the periphery. Thus teriflunomide may target also microglia in neuroinflammatory diseases, including MS.
5.3. Conclusion
The aim of this thesis was to investigate the effects of disease-modifying therapies on primary immune cells to gain knowledge on the mechanism of action.
Our findings demonstrate that in vitro application of recombinant IFN-β1b reduces CXCL12/CXCR4-mediated migration of primary human T cells from healthy individuals and RRMS patients. It was clearly indicated that IFN-β1b can impair the functional T cell response to CXCR4 by downregulating its expression in human primary T cells. However, it is still not clear whether our findings are relevant in MS patients and whether CXCR4 has a unique function in the treatment with IFN-β1b. Second, our data suggest that teriflunomide might also act by inhibiting the proliferation of microglia in the CNS.
We believe the current findings are highly relevant for the understanding of the mode of action of drugs used in the treatment of MS.
56
Summary
57 6. Summary
Tanja Wostradowski
Deciphering the mode of action of drugs used in the treatment of multiple sclerosis Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). A key event of its pathology is the infiltration of activated leukocytes into the CNS.
Interferon (IFN)-β1b is licensed for the treatment of relapsing forms of MS to reduce the frequency of clinical exacerbations. The effect of IFN-β is thought to be mediated by the modulation of immune cells including an effect on leukocyte infiltration of the CNS. The chemokine CXCL12 and its receptor CXCR4 are involved in this process of transmigration into the CNS. Although IFN-β has been shown to decrease CXCL12-induced cell migration in PBMCs, it is not clear whether IFN-β plays a role in CXCL12-induced migration of T cells.
Inhibition of CXCR4-dependent leukocyte migration could be one of the mechanisms of immunomodulation by IFN-β1b. T cells have been shown to express the type I IFN receptor complex (IFNAR1/2) that is necessary for IFN signaling. Our objective was to investigate a crosstalk of key elements in IFN-β and CXCR4 signaling cascade and functional CXCL12-dependent response in T cells after in vitro IFN-β treatment, and to determine differences in CXCR4 expression and function in MS patients compared to healthy controls.
We could demonstrate that IFN-β1b (1000 U/ml) reduced CXCL12-dependent migration of primary human T cells from healthy individuals and RRMS patients in vitro.
IFN-β1b pretreatment did not lead to a reduction of either ERK1/2 or AKT phosphorylation.
However, CXCR4 surface expression was reduced after 24 h of IFN-β1b pretreatment as measured by flow cytometry. Quantitative real time-PCR revealed a similar reduction in CXCR4-mRNA. The IFN-β1b induced reduction in migration and CXCR4 expression might not mediated by early intracellular signaling events downstream of CXCR4 activation
Summary
58
induced by CXCL12. The requirement of several hours’ exposure to IFN-β1b supports a transcriptional regulation. Interestingly, T cells of MS patients showed a lower CXCR4 expression than healthy individuals in vivo, which was not further reduced in patients under IFN-β1b therapy.
A main characteristic of neuroinflammatory diseases such as MS is microglial proliferation and activation. The role of microglia in MS pathogenesis is highly debatable since both beneficial as well as detrimental functions have been proposed. To gain insights on effects of teriflunomide on microglial activation and proliferation, in vitro experiments with rodent microglial cultures were performed.
We analyzed whether teriflunomide influences cellular reactions and immune function of microglia by treatment with physiologically relevant concentrations (0-5 µM). Besides investigations on proliferation of microglia we examined the polarization of microglia into a pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotype under the treatment of teriflunomide. For the proliferation assay, mixed glial cell cultures were stimulated by different concentrations of teriflunomide together with GM-CSF. Here, we concluded that teriflunomide exerted significant inhibition of microglial proliferation in mixed glial cultures activated with GM-CSF. Only treatment with 5 µM teriflunomide slightly increased expression of the anti-inflammatory cytokine IL-10 from activated microglia. In addition, we found that teriflunomide leads to significantly decreased expression of co-stimulatory molecule CD86 in LPS-activated microglia; however, teriflunomide did not modulate pro-inflammatory phenotype of primary rat microglia. Furthermore, we observed no influence of teriflunomide on the phagocytic activity of unstimulated or LPS-activated microglia.
Taken together, a better characterization of molecular pathways of either IFN-β or teriflunomide might help to identify new drug targets for an add-on therapy to improve the efficacy of a MS therapy or differentiate between treatment responder from non-responder.
Zusammenfassung
59 7. Zusammenfassung
Tanja Wostradowski
Aufklärung der Wirkungsweise verschiedener Therapeutika zur Behandlung der Multiplen Sklerose
Die Multiple Sklerose (MS) ist eine chronisch-entzündliche Autoimmunerkrankung des zentralen Nervensystems (ZNS), die überwiegend durch eine T-Zell vermittelte Autoimmunreaktion gegen Myelinantigene hervorgerufen wird. Entsprechend den Erkenntnissen aus experimentellen Modellen, geht man davon aus, dass es bei der MS zu einer Aktivierung autoreaktiver T-Zellen und einem Funktionsverlust der Blut-Hirnschranke kommt. Dies führt zur Infiltration, lokalen Reaktivierung und Proliferation verschiedener Immunzellen, die eine lokale Entzündungskaskade initiieren können. Kennzeichen der MS sind folglich Inflammation, Demyelinisierung und axonale Pathologien, die sich in vielfältigen neurologischen Defiziten widerspiegeln. Obwohl periphere T-Lymphozyten lange als Ziel verschiedener MS-Therapeutika im Mittelpunkt standen, gewinnen zunehmend Therapien an Bedeutung, die zusätzlich gegen Zellen im ZNS wie Mikroglia und Astrozyten gerichtet sind.
Interferon (IFN)-β ist seit mehr als 20 Jahren eine wirksame, sichere Behandlungsmethode der schubförmigen und teils der sekundär-progredienten Multiplen Sklerose. IFN-β beeinflusst das Entzündungsgeschehen, indem es unter anderem die Einwanderung von Lymphozyten in das ZNS reduziert. Es wurde gezeigt, dass IFN-β1b, vermittelt über die Expression von RGS1 (negative regulator of G-protein signaling), die Chemokin CXCL12-induzierte Migration von mononuklearen Zellen des peripheren Blutes (PBMCs) hemmt. Dennoch ist der Wirkmechanismus von IFN-β nur teilweise verstanden.
Demgegenüber stehen neue Therapieformen zur peroralen Applikation für die Behandlung
Zusammenfassung
60
von MS, wie z.B. Teriflunomid, dessen Effekte auf das Immunsystem und auf Zellen des ZNS bis dato nur unzureichend untersucht sind.
Teriflunomid ist, wie IFN-β, ein Immunmodulator. Der genaue Wirkmechanismus von Teriflunomid ist bisher nicht vollständig geklärt. Es wirkt durch die nichtkompetitive, reversible Blockierung des mitochondrialen Enzyms Dihydroorotat-Dehydrogenase (DHODH), einem Schlüsselenzym der Pyrimidin-Biosynthese. So wird insbesondere die Proliferation aktivierter T- und B-Zellen gehemmt. Verschiedene Studien zeigten zudem, dass durch Teriflunomid die Produktion einiger pro-inflammatorischer Zytokine durch Makrophagen reduziert wird.
Die vorliegende Arbeit befasst sich mit möglichen Wirkmechanismen verschiedener Therapeutika zur Behandlung der MS. Einerseits kann CXCL12-abhängige T-Zell Migration in vitro/in vivo in MS-Patienten durch IFN-β beeinflusst werden, anderseits kann die Aktivierung und Proliferation von Mikroglia durch physiologisch relevante Konzentrationen von Teriflunomid (0-5 µM) moduliert werden.
Unsere Ergebnisse aus dem ersten Teil der Arbeit zeigen deutlich eine Hemmung der CXCL12-abhängige Migration von humanen T-Zellen von gesunden Probanden und von RRMS Patienten (in vitro) durch IFN-β nach 20 h Behandlung. In Untersuchungen der CXCR4/CXCL12-abhängigen und IFN-β1b-induzierten Signalkaskaden konnten wir zeigen, dass β1b ausgewählte Signalwege nicht beeinflusst. Im Gegensatz dazu reduziert IFN-β1b die Expression des Rezeptors CXCR4 in T-Zellen von gesunden Probanden und von
RRMS Patienten in vitro. Eine Hemmung der Migration bei MS-Patienten in vivo war nicht ersichtlich. Daraus kann man schließen, dass der aufgedeckte Mechanismus auf der Herunterregulierung der CXCR4 Expression auf Transkriptionsebene basieren könnte.
Im zweiten Teil der Arbeit haben wir untersucht, ob Teriflunomid neben den beschriebenen Effekten auf Leukozyten auch die Proliferation und Funktion von Mikroglia
Zusammenfassung
61
beeinflussen kann. Wir konnten zeigen, dass Teriflunomid die Proliferation von aktivierten Mikroglia aus gemischten Gliazellkulturen hemmt. Darüber hinaus konnten wir einen minimalen Einfluss von Teriflunomid auf immunmodulatorische Faktoren in aktivierten Mikroglia feststellen. Zu einem zeigte Teriflunomid eine leichte, aber signifikante Hochregulierung des anti-inflammatorischen Mediators IL-10, zum anderem eine Reduktion des co-stimulierenden Moleküls CD86 nach Aktivierung mit LPS. Andererseits inhibierte Teriflunomid nicht den NF-κB Signalweg, was mit einer Reduktion von inflammatorischen Zytokinen und Adhäsionsmolekülen einhergegangen wäre. Unsere Ergebnisse legen nahe, das Teriflunomid keine bedeutende Rolle in der Regulation inflammatorischer Funktionen einnimmt.
Zusammenfassend konnten wir feststellen, dass die Wirkmechanismen verschiedener Substanzen zur Behandlung der MS, in vitro sowohl einen Einfluss auf Immunzellen aus der Peripherie als auch auf Zellen des ZNS nehmen können, sofern sie in der Lage sind in vivo die Bluthirnschranke zu überwinden. Die Ergebnisse unterstreichen Effekte von IFN-β und Teriflunomid auf wichtige Zelltypen, die abhängig von den gegebenen Bedingungen Einfluss auf den Krankheitsverlauf der MS haben könnten.
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References
63 8. References
ALBERT, M., J. ANTEL, W. BRUCK u. C. STADELMANN (2007):
Extensive cortical remyelination in patients with chronic multiple sclerosis.
Brain Pathol 17, 129-138 ASCHERIO, A. (2013):
Environmental factors in multiple sclerosis.
Expert Rev Neurother 13, 3-9
BALAK, D. M., G. J. HENGSTMAN, A. CAKMAK u. H. B. THIO (2012):
Cutaneous adverse events associated with disease-modifying treatment in multiple sclerosis: a systematic review.
Mult Scler 18, 1705-1717 BALDWIN, A. S., JR. (1996):
The NF-kappa B and I kappa B proteins: new discoveries and insights.
Annu Rev Immunol 14, 649-683 BAR-OR, A. (2014):
Teriflunomide (Aubagio®) for the treatment of multiple sclerosis.
Exp Neurol 262 Pt A, 57-65
BECHMANN, I., S. PETER, M. BEYER, U. GIMSA u. R. NITSCH (2001):
Presence of B7--2 (CD86) and lack of B7--1 (CD(80) on myelin phagocytosing MHC-II-positive rat microglia is associated with nondestructive immunity in vivo.
FASEB J 15, 1086-1088
BEN-NUN, A., N. KAUSHANSKY, N. KAWAKAMI, G. KRISHNAMOORTHY, K. BERER, R. LIBLAU, R.
HOHLFELD u. H. WEKERLE (2014):
From classic to spontaneous and humanized models of multiple sclerosis: impact on understanding pathogenesis and drug development.
J Autoimmun 54, 33-50
BERAHOVICH, R. D., B. A. ZABEL, M. E. PENFOLD, S. LEWEN, Y. WANG, Z. MIAO, L. GAN, J.
PEREDA, J. DIAS, SLUKVIN, II, K. E. MCGRATH, J. C. JAEN u. T. J. SCHALL (2010):
CXCR7 protein is not expressed on human or mouse leukocytes.
J Immunol 185, 5130-5139
BERGHMANS, N., H. HEREMANS, S. LI, E. MARTENS, P. MATTHYS, L. SOROKIN, J. VAN DAMME u.
G. OPDENAKKER (2012):
Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes.
J Neuroinflammation 9, 243
BLEUL, C. C., R. C. FUHLBRIGGE, J. M. CASASNOVAS, A. AIUTI u. T. A. SPRINGER (1996):
A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1).
J Exp Med 184, 1101-1109
BLOCK, M. L. u. J. S. HONG (2005):
Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism.
Prog Neurobiol 76, 77-98
BO, L., C. A. VEDELER, H. NYLAND, B. D. TRAPP u. S. J. MORK (2003):
Intracortical multiple sclerosis lesions are not associated with increased lymphocyte infiltration.
Mult Scler 9, 323-331
References
64
BRINKMANN, V., A. BILLICH, T. BAUMRUKER, P. HEINING, R. SCHMOUDER, G. FRANCIS, S.
ARADHYE u. P. BURTIN (2010):
Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.
Nat Rev Drug Discov 9, 883-897 BROWN, G. C. u. J. J. NEHER (2012):
Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'.
Trends Biochem Sci 37, 325-332
BRUCK, W., R. PFORTNER, T. PHAM, J. ZHANG, L. HAYARDENY, V. PIRYATINSKY, U. K. HANISCH, T. REGEN, D. VAN ROSSUM, L. BRAKELMANN, K. HAGEMEIER, T. KUHLMANN, C. STADELMANN, G. R. JOHN, N. KRAMANN u. C. WEGNER (2012):
Reduced astrocytic NF-kappaB activation by laquinimod protects from cuprizone-induced demyelination.
Acta Neuropathol 124, 411-424 BRUCK, W. u. S. S. ZAMVIL (2012):
Laquinimod, a once-daily oral drug in development for the treatment of relapsing-remitting multiple sclerosis.
Expert Rev Clin Pharmacol 5, 245-256
BRUNEAU, J. M., C. M. YEA, S. SPINELLA-JAEGLE, C. FUDALI, K. WOODWARD, P. A. ROBSON, C.
SAUTES, R. WESTWOOD, E. A. KUO, R. A. WILLIAMSON u. E. RUUTH (1998):
Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.
Biochem J 336 (Pt 2), 299-303
CHANG, E. Y., B. GUO, S. E. DOYLE u. G. CHENG (2007):
Cutting edge: involvement of the type I IFN production and signaling pathway in lipopolysaccharide-induced IL-10 production.
J Immunol 178, 6705-6709
CHEN, M., G. CHEN, S. DENG, X. LIU, G. J. HUTTON u. J. HONG (2012):
IFN-beta induces the proliferation of CD4+CD25+Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells in the treatment of multiple sclerosis.
J Neuroimmunol 242, 39-46 CHENG, W. u. G. CHEN (2014):
Chemokines and chemokine receptors in multiple sclerosis.
Mediators Inflamm 2014, 659206 CLAUSSEN, M. C. u. T. KORN (2012):
Immune mechanisms of new therapeutic strategies in MS: teriflunomide.
Clin Immunol 142, 49-56
CLAYCOMB, K. I., K. M. JOHNSON, P. N. WINOKUR, A. V. SACINO u. S. J. CROCKER (2013):
Astrocyte regulation of CNS inflammation and remyelination.
Brain Sci 3, 1109-1127
COMI, G., D. JEFFERY, L. KAPPOS, X. MONTALBAN, A. BOYKO, M. A. ROCCA u. M. FILIPPI (2012):
Placebo-controlled trial of oral laquinimod for multiple sclerosis.
N Engl J Med 366, 1000-1009
COMPSTON, A. u. A. COLES (2008):
Multiple sclerosis.
Lancet 372, 1502-1517
CONFAVREUX, C., P. O'CONNOR, G. COMI, M. S. FREEDMAN, A. E. MILLER, T. P. OLSSON, J. S.
WOLINSKY, T. BAGULHO, J. L. DELHAY, D. DUKOVIC, P. TRUFFINET u. L. KAPPOS (2014):
Oral teriflunomide for patients with relapsing multiple sclerosis (TOWER): a randomised, double-blind, placebo-controlled, phase 3 trial.
Lancet Neurol 13, 247-256
References
65
CONFAVREUX, C., S. VUKUSIC, T. MOREAU u. P. ADELEINE (2000):
Relapses and progression of disability in multiple sclerosis.
N Engl J Med 343, 1430-1438
CUTOLO, M., A. SULLI, P. GHIORZO, C. PIZZORNI, C. CRAVIOTTO u. B. VILLAGGIO (2003):
Anti-inflammatory effects of leflunomide on cultured synovial macrophages from patients with rheumatoid arthritis.
Ann Rheum Dis 62, 297-302
DAVIS, J. P., G. A. CAIN, W. J. PITTS, R. L. MAGOLDA u. R. A. COPELAND (1996):
The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase.
Biochemistry 35, 1270-1273
DE WEERD, N. A. u. T. NGUYEN (2012):
The interferons and their receptors--distribution and regulation.
Immunol Cell Biol 90, 483-491
DENDROU, C. A., L. FUGGER u. M. A. FRIESE (2015):
Immunopathology of multiple sclerosis.
Nat Rev Immunol 15, 545-558
DENIC, A., B. WOOTLA u. M. RODRIGUEZ (2013):
CD8(+) T cells in multiple sclerosis.
Expert Opin Ther Targets 17, 1053-1066 DHIB-JALBUT, S. u. S. MARKS (2010):
Interferon-beta mechanisms of action in multiple sclerosis.
Neurology 74 Suppl 1, S17-24
DHIB-JALBUT, S., S. SUMANDEEP, R. VALENZUELA, K. ITO, P. PATEL u. M. RAMETTA (2013):
DHIB-JALBUT, S., S. SUMANDEEP, R. VALENZUELA, K. ITO, P. PATEL u. M. RAMETTA (2013):