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`et al.
`
`Clinical and Experimental Immunology
`
`O R I G I N A L A R T I C L E
`
`doi:10.1111/j.1365-2249.2006.03094.x
`
`Fumaric acid esters are effective in chronic experimental autoimmune
`encephalomyelitis and suppress macrophage infiltration
`
`†
` R. Linker,*
`S. Schilling,* S. Goelz,
`F. Luehder* and R. Gold*
`*Institute for Multiple Sclerosis Research,
`University of Goettingen, Goettingen, Germany
`†
`and
`BiogenIdec Cambridge, MA, USA
`
`Accepted for publication 16 March 2006
`Correspondence: Dr Ralf Gold, Experimental
`and Clinical Neuroimmunology Institute for MS
`Research, Bereich Humanmedizin der Universi-
`taet Goettingen und Gemeinnuetzige Hertie-
`Stiftung, Waldweg 33, D-37073 Goettingen,
`germany.
`E-mail: r.gold@med.uni-goettingen.de
`
`Summary
`
`Fumaric acid esters (FAE) have proven their therapeutic efficacy in psoriasis,
`a Th1 mediated skin disease. More recently, preliminary data have suggested
`an activity in multiple sclerosis (MS) as well. To investigate further possible
`mechanisms of action of these compounds in inflammatory diseases, we stud-
`ied the FAE methyl hydrogen fumarate (MHF) and dimethyl fumarate (DMF)
`in chronic experimental autoimmune encephalomyelitis (EAE) induced by
`immunization of C57BL/6 mice with MOG peptide aa 35–55. Preventive
`treatment with these FAE was delivered twice a day by oral gavage. Both esters
`had a significant therapeutic effect on the disease course and histology showed
`a strongly reduced macrophage inflammation in the spinal cord. Multiparam-
`eter cytokine analysis from blood detected an increase of IL-10 in the treated
`animals. We conclude that the underlying biological activity of FAE in EAE is
`complex and, to elucidate the molecular mechanisms, further investigation is
`needed.
`
`Keywords:
` encephalomyelitis, multiple sclerosis, therapy, inflammation,
`cytokines
`
`Introduction
`
`Multiple sclerosis (MS) is a chronic inflammatory disease of
`the central nervous system (CNS) characterized by the mor-
`phological hallmarks of inflammation, demyelination, loss
`of oligodendrocytes and subsequent axonal/neuronal dam-
`age. Experimental autoimmune encephalomyelitis (EAE) is a
`model disease reflecting some of the typical features of the
`human disease MS. It is characterized by an ascending paral-
`ysis resulting from lymphocytic infiltration of the CNS asso-
`ciated with macrophage and microglia activation [1]. One of
`the key mechanisms thought to contribute to the progression
`of autoimmune demyelination are the effector functions of
`phagocytic cells in the EAE lesion [2,3].
`A well described Th1-mediated skin disease is psoriasis
`[4], which can be effectively treated with Fumaderm®, a mix-
`ture of fumaric acid esters (FAE) including dimethyl fuma-
`rate (DMF) and ethylhydrogen fumarate [5]. After oral
`intake, DMF is rapidly hydrolysed to methyl hydrogen fuma-
`rate (MHF) with MHF possibly being the active metabolite
`[6]. The biological half life of MHF is 36 h, and 30% is
`bound by serum proteins [7]. FAE have been reported to
`→
`induce a Th1
`Th2 shift as part of their treatment effect.
`However, the clinical relevance of this observation is unclear
`
`and the definite molecular mechanisms of DMF and MHF
`actions are poorly understood. MHF, for example, can
`induce interleukin (IL)-10, IL-4 and IL-5 expression in
`γ
`PBMC
`
`in vitro [8] without changing interferon (IFN)-
`, IL-
`12 and IL-2 levels. MHF has also been shown to increases the
`production of IL-4 and IL-5 in T cells
`
`in vitro [9]. Tumour
`α
`necrosis factor (TNF)-
` levels are affected by MHF; initially
`increasing and subsequently decreasing in response to MHF.
`Other
`
`in vitro studies have shown that DMF can inhibit the
`transcription of many pro-inflammatory cytokines and this
`inhibition appears to correlate with a blockade of the TNF-
`κ
`induced nuclear translocation of a NF-
`B p65. MHF has
`κ
`been reported to inhibit LPS-induced NF-
`B activation in
`dendritic cells (DC) and endothelial cells
`
`in vitro [10,11].
`Moreover, DC differentiation is inhibited by both DMF and
`MHF in a dose-dependent manner and the capacity of DC to
`stimulate lymphocytes in culture is reduced after DMF treat-
`ment [12]. However, since therapeutic concentrations of
`FAE
`in vivo
` are unknown and may differ considerably from
`those used in
`
`in vitro experiments, the clinical relevance of all
`these
`
`in vitro results remains to be determined.
`Recently FAE have been discussed as therapeutic tools for
`autoimmune diseases beyond psoriasis. An initial study
`describes the rather dramatic effect of FAE on magnetic res-
`
`© 2006 British Society for Immunology,
`
`
`
`Clinical and Experimental Immunology,
`
`145:
`
` 101–107
`
`101
`
`Page 1 of 7
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`Page 1 of 7
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`Coalition v. Biogen
`IPR2015-01993
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`S. Schilling et al.
`
`onance imaging (MRI) inflammation in a small number of
`MS patients [13]. However, knowledge on the mechanisms
`in vivo
` is extremely limited. Our goal was to investigate the
`action of DMF and MHF given preventively in chronic
`MOG-induced EAE of the C57BL/6 mouse, a model that
`resembles many features of progressive neurological destruc-
`tion in MS. In addition to observing the effects on clinical
`disease course, multi parameter cytokine profiling of longi-
`tudinal blood samples was applied to screen for molecular
`changes during treatment and histological analysis was used
`to extend our understanding of
`in vivo
` mechanisms.
`
`Materials and methods
`
`Animals
`
`Female C57BL/6 mice were purchased from Harlan Labora-
`tories (Harlan Winkelmann, Borchen, Germany) for all fol-
`lowing experiments. Animals were 8–12 weeks old and body
`weight was in the range 20–30 g. Animals were housed in an
`IVC facility with controlled light cycle and were given com-
`mercial food pellets and water ad libitum. All experiments
`were approved by the Lower Saxony state authorities for ani-
`mal experimentation.
`
`Induction and clinical evaluation of EAE
`
`For induction of EAE, mice received s.c. injections in
`µ
`the flanks and tail base of 50
`g MOG 35–55 peptide
`(synthesized at Charité Berlin, Department for peptide- and
`protein-chemistry) in PBS emulsified in an equal volume of
`complete Freund’s adjuvant (CFA) containing
`Mycobacte-
`rium tuberculosis
` H37RA (Difco, Detroit MI, USA) at a final
`concentration of 0·5 mg/ml. Two injections of pertussis toxin
`(List Biological Laboratories Inc., California, USA; 200 ng
`per mouse i.p) were given on days 0 and 2. Animals were
`weighed and scored for clinical signs of disease on a daily
`basis. Disease severity was assessed using a scale ranging
`=
`from 0 to 10; scores were as follows [14]: 0
` normal;
`=
`=
`1
` reduced tone of tail; 2
` limp tail, impaired righting;
`=
`=
`=
`3
` absent righting; 4
` gait ataxia; 5
` mild paraparesis of
`=
`=
`hindlimbs; 6
` moderate paraparesis; 7
` severe paraparesis
`=
`=
`=
`or paraplegia; 8
` tetraparesis; 9
` moribund; 10
` death. In
`accordance to Lower Saxony animal protection laws, mice
`were sacrificed in case of paraplegia (score 7 or higher). Ani-
`mals that had to be terminated because of paraplegia were
`consecutively rated as ‘7’ despite their absence in the further
`experiment.
`
`Treatment
`
`µ
`O
`l 0·08% Methocel/H
`The medication was diluted in 200
`2
`as vehicle and administered by oral gavage starting from day
`3 post immunization (p.i) until termination. Each treatment
`group consisted of 8 animals: vehicle alone as a negative
`
`control, 5 mg/kg body weight DMF twice a day, 15 mg/kg
`body weight DMF twice a day, 5 mg/kg body weight MHF
`twice a day. The compounds were obtained via Fumapharm
`AG. MHF, which is highly acidic, was given as calcium salt to
`avoid acidosis. The lower DMF dose and the MHF dose cor-
`related to the dose used in human psoriasis in clinical trials.
`The threefold higher dosage of DMF was used to compensate
`for body surface disparity of mice. Oral gavage was used to
`ensure exact dosing and to avoid compound degradation.
`
`‘Multi-analyte profiling’ (MAP)
`µ
`l) were obtained under general anaes-
`Plasma samples (50
`thesia from retro-orbital sinus of all mice before immuniza-
`tion, at the peak of the disease (day 11) and in partial
`remission (day 21). The plasma protein concentration of 60
`cytokines and other markers was measured by MAP testing
`<
`(Rules Based Medicine, Austin, TX, USA;
`http://www.
`>
`rulesbasedmedicine.com
`).
`
`Histology
`
`In the early chronic phase, day 27 p.i., animals were deeply
`anaesthesized with ketamine/xylazine hydrochloride and
`transcardially perfused with saline followed by 4% of
`paraformaldehyde. The complete spinal cord was carefully
`removed and 8–10 axial sections were further processed for
`routine paraffin embedding [14]. Early terminated animals
`were excluded from histology due to low comparability of
`different disease stages.
`Paraffin sections were subjected to haematoxylin/eosin
`(H&E) staining to assess parameters of inflammatory infil-
`µ
`trates. Immunohistochemistry was performed with 5
`m
`paraffin sections as described previously [14]. If necessary,
`antigen unmasking was achieved by heat pretreatment of
`sections for 30 min in 10 mM citric acid buffer in a micro-
`wave oven (850 W). After inhibition of unspecific binding
`°
`with 10% BSA, sections were incubated overnight at 4
`C
`with the appropriate primary antibody in 1% BSA. Second-
`ary antibodies were used as indicated below. After blockade
`of endogenous peroxidase with H
`O
`, the peroxidase-based
`2
`2
`ABC detection system (DAKO, Hamburg, Germany) was
`employed with diaminobenzidine tetrahydrochloride (DAB)
`as the chromogenic substrate. Specificity of staining was
`confirmed by omitting the primary antibody as a negative
`control. T cells were labelled with rat anti-CD3 (Serotec;
`Wiesbaden, Germany; 1: 200) and macrophages with rat
`anti-mouse Mac-3 (Pharmingen; 1 : 200), each with a rabbit
`anti-rat secondary antibody (Vector via Linavis, Wertheim,
`Germany; 1 : 200).
`Quantitative evaluation of histopathological changes was
`essentially performed as described earlier [14]. Coded sec-
`tions from cervical, thoracic and lumbar spinal cord were
`evaluated by a blinded observer by means of overlaying a ste-
`reological grid and counting mean CD3 and Mac-3 positive
`
`102
`
`© 2006 British Society for Immunology,
`
`
`
`Clinical and Experimental Immunology,
`
`145:
`
` 101–107
`
`Page 2 of 7
`
`Page 2 of 7
`
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`2
`) with the most
`cells within 3 visual fields (each 0·096 mm
`intense pathology under a 400 fold magnification.
`
`Table 1.
`severity.
`
`Disease incidence and termination rate because of disease
`
`Fumarate therapy in experimental autoimmune encephalomyelitis
`
`Statistical analysis
`
`Analysis of the clinical course was performed using Two-way
`anova
` (GraphPad Prism program, California, USA). The
`Mann–Whitney U-test was performed for histological anal-
`yses (SPSS program, SPSS, Chicago, IL, USA). All data are
`±
`given as mean
` SEM. P-values were considered significant at
`<
`<
`<
`
`P
` 0·05, highly significant at
`
`P
` 0·01 or
`
`P
` 0·001.
`
`Results
`
`EAE clinical course of FAE treated C57BL/6 mice
`
`EAE was induced in C57BL/6 mice using MOG 35–55. Three
`days after disease induction, before the first symptoms
`occurred, mice were treated preventively with either carrier
`(control), 5 mg/kg DMF, 15 mg/kg DMF or 5 mg/kg MHF
`twice a day and the progression of clinical disability was
`ascertained. Since the pilot experiments did not consistently
`show a robust treatment effect of 5 mg/kg DMF, a higher
`dose group (15 mg/kg DMF) was included in the subsequent
`experiments.
`Symptoms of EAE started after 12·3 days (mean) p.i. in
`the control group, after 13·9 days in the 5 mg/kg DMF
`group, after 14·8 days in the 15 mg/kg DMF group and after
`13·1 days in the MHF group (Fig. 1a), the delay of the FAE
`treated groups was not significant. The results of two inde-
`pendent experiments were pooled regarding clinical scores
`=
`and histology in order to attain larger group sizes (
`
`n
` 16
`=
`mice per group for control and MHF,
`
`n
` 15 for DMF
`5 mg/kg and DMF 15 mg/kg) (Fig. 1a). There was a dose
`dependent, highly significant benefit from preventive treat-
`ment with DMF, with the 15 mg/kg dose being more effec-
`<
`tive (
`P
` 0·0001 compared to control, two-way
`anova
`) than
`<
`the 5 mg/kg dose (
`P
` 0·001 compared to the control
`group). The difference between DMF 5 mg/kg and 15 mg/
`<
`kg was also significant (
`P
` 0·0001). The most favourable
`<
`course was seen within the MHF treated group (
`P
` 0·0001
`compared to control). The disease incidence and the num-
`ber of mice reaching the most severe clinical score, where
`they had to be terminated due to animal protection laws are
`given in Table 1. Usually one day before the first EAE symp-
`toms occur, animals loose 1–2 g of body weight, which is
`partly due to cytokine stress. Figure 1b shows the extent of
`weight loss in the different groups, which resembles,
`although less sensitively, the clinical course in these ani-
`mals. There was no significant difference between 5 mg/kg
`DMF and control mice. The weight difference between con-
`<
`trol and MHF treated animals was significant (
`P
` 0·001,
`two-way
`anova
`).
`The mean termination time point was 13·3 days for con-
`trol animals, 15·5 days for 5 mg/kg DMF, 15·5 days for
`
`Control
`
`DMF
`5 mg/kg
`
`DMF
`15 mg/kg
`
`Incidence of EAE
`1st exp.
`2nd exp.
`All
`
`100%
`88%
`94%
`
`85%
`88%
`87%
`
`Termination (reaching paraplegia or more)
`1st exp.
`13%
`43%
`2nd exp.
`50%
`13%
`All
`31%
`27%
`
`100%
`100%
`100%
`
`14%
`38%
`27%
`
`MHF
`
`75%
`75%
`75%
`
`38%
`13%
`25%
`
`Values represent percentage of affected animals per group.
`
`15 mg/kg DMF and 14·3 days for 5 mg/kg MHF, the
`>
`observed delay is not significant (
`
`P
` 0·05, Mann–Whitney
`
`U-test).
`
`Histology
`
`The beneficial clinical effect of FAE was consistent with the
`results from histological studies performed on day 27 p.i.
`(Fig. 2): Infiltration of CD3 positive cells (T-cells) into the
`spinal cord was slightly reduced at both dosages of DMF
`=
`=
`(
`P
` 0·14 for the 5 mg/kg group,
`
`P
` 0·12 for the 15 mg/kg
`group, Mann–Whitney
`
`U-test), and significantly by MHF
`<
`(
`P
` 0·01). In addition, significantly fewer Mac-3 positive
`cells (macrophages/microglia) were found in the spinal cord
`<
`of DMF 15 mg/kg and MHF treated animals (
`P
` 0·01 and
`<
`
`P
` 0·001, respectively). DMF 5 mg/kg also appeared to
`reduce the number of Mac-3 positive macrophages in the
`spinal cords of these mice, however, this effect was not sta-
`tistically significant (Fig. 3a,b).
`
`Multi-analyte profiling (MAP) results
`
`As an attempt to understand the biology underlying the clin-
`ical and histological observations, plasma levels of 60 pro-
`teins (including cytokines and other inflammation markers)
`were determined using MAP. A control experiment was per-
`formed for validation of the method, in which plasma sam-
`ples were taken from 5 healthy mice at 3 time points, the
`plasma from the last time point was divided and the two
`fractions were analysed separately. Standard errors were low
`for over 30 markers, it was more than 10% for eotaxin,
`α
`β
`growth hormone, IL-1
`, IL-2, IL-4, IL-11, IP-10, MIP-1
`,
`γ
`myoglobin, SCF, GM-CSF, IFN-
`, IL-12p70, IL-17, IL-3, IL-
`α
`6, IL-7, MIP-2, OSM, RANTES, TIMP-1 and TNF-
` (not
`shown). Intra-animal variability is shown for IL-5, IL-10,
`α
`γ
`TNF-
` and IFN-
` (Fig. 4). SEM was low for IL-10 and IL-5
`γ
`α
`and rather high for IFN-
`. TNF-
` was below the detection
`level. Therefore while a high reproducibility in most param-
`eters can be assumed, there may be variability in those cytok-
`ines listed above.
`
`© 2006 British Society for Immunology,
`
`
`
`Clinical and Experimental Immunology,
`
`145:
`
` 101–107
`
`103
`
`Page 3 of 7
`
`Page 3 of 7
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`

`

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`
`S. Schilling et al.
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`
`Discussion
`
`In a chronic mouse model for MS we showed that the
`fumaric acid esters DMF and MHF given preventively, can
`effectively reduce disease activity. This finding was further
`underscored by histological data showing a dramatic
`decrease in inflammatory cell infliltrates. Our observations
`in EAE are consistent with the successful use of FAE (a mix-
`ture with the main compound DMF) in psoriasis, a classic
`CD4+ Th1 T cell mediated disease [5]. Similar to psoriasis,
`the MOG 35–55 induced EAE of the mouse has a predomi-
`nance of Th1 mechanisms [15] underlying its pathology.
`This is probably due to CpG motifs in the DNA of Mycobac-
`terium tuberculosis from complete Freund’s adjuvant used
`for immunization. The infiltrate seen in this model is dom-
`inated by an influx of macrophage/microglial cells and secre-
`tion of proinflammatory cytokines [16,17].
`Symptoms of EAE occurred 10–12 days (mean) after
`administration of the first FAE dose. The clinical course was
`ameliorated in treated animals from the beginning and con-
`tinued until the end of the experiment. An extended obser-
`vation was not performed in this study, spinal cord
`inflammation in all remaining animals was analysed in the
`early chronic phase (day 27). One of the most interesting
`observations of this study was a clear reduction in macroph-
`age infiltration of the spinal cord in the treated groups, a
`finding which corroborated the more favourable clinical
`course of treated animals. As was seen in the clinical course
`of the disease, this was dose dependent for DMF and most
`pronounced for MHF. The inhibition of T cell infiltration
`was less obvious, but still significant in the MHF treated
`group. In addition to their antigen presenting function,
`macrophages/microglia cells are active players in myelin
`destruction in EAE as well as in MS, and phagocytosis of
`myelin is a marker for ongoing demyelination [18]. Toward
`this end, activated microglia secrete pro-inflammatory
`cytokines such as IL-1, IL-6, LT, MIP-1α and others [19] and
`proteinases which seem to be involved in the breakdown of
`the blood–brain barrier [20]. It is of note that, so far, the
`observed clinical effect of FAE in MS is the strong reduction
`of new Gadolinium enhancing lesions [13]. Histopatholog-
`ical studies made on brain biopsies by Brück et al. [21] have
`clearly shown that Gadolinium enhancement correlates with
`macrophage infiltration.
`The underlying mechanisms by which FAE exert its bio-
`logical effects are not yet understood, although there is some
`data published from in vitro studies and from the use in pso-
`riasis. The key target seems to be immune cells, and it is pre-
`sumed that a significant aspect of its effect is mediated by a
`Th2 polarization of CD4+ T cells. Consistent with this
`assumption is a study that shows that the release of IL-4 and
`IL-10 by lymphocytes stimulated with herpes simplex virus
`antigen is increased by treatment with DMF in vitro [22].
`Alteration of cell survival has also been postulated as a pos-
`sible mode of action for FAE. It has been shown that DMF
`
`Control, 5/16 terminated
`DMF 5 mg/kg, 4/15 terminated
`DMF 15 mg/kg, 4/15 terminated
`Ca-MHF, 4/16 terminated
`
`2
`
`4
`
`6
`
`8 10 12 14 16 18 20 22 24 26 28 30
`Days
`
`Weight
`
`Control
`DMF 5 mg/kg
`DMF 15 mg/kg
`Ca-MHF
`
`(a)
`10
`
`9 8 7 6 5 4 3 2 1 0
`
`0
`
`Clinical score + SEM
`
`(b)
`22
`
`21
`
`20
`
`19
`
`18
`
`Weight [g]
`
`17
`0
`
`2
`
`4
`
`6
`
`8 10 12 14 16 18 20 22 24 26 28 30
`Days
`
`±
`Fig. 1.
` standard error of the mean
`(a) Mean EAE disease scores
`(SEM) of C57BL/6 mice treated with DMF 5 mg/kg (upright triangle),
`DMF 15 mg/kg (inverted triangle), MHF (diamond) and carrier alone
`(square). Preventive treatment was started on day 3. The treatment
`<
`effect was significant for MHF (
`P
` 0·0001 compared to the control
`
`group, two-way anova
`) and also for DMF, with the 15 mg/kg dose being
`<
`<
`more effective (
`P
` 0·0001) than the 5 mg/kg dose (
`P
` 0·001). (b) Mean
`weight and SEM of C57BL/6 mice which were treated with DMF 5 mg/
`kg (upright triangle), DMF 15 mg/kg (inverted triangle), MHF (dia-
`mond) and carrier alone (square) in the experiment shown in (a). DMF
`5 mg/kg does not differ much from the control group, whereas the DMF
`15 mg/kg group already exhibits lower weight in the beginning. MHF
`treated animals loose significantly less weight than the control animals
`<
`(
`P
` 0·001, two-way
`anova
`).
`
`In one of the earlier experiments where mice were treated
`with carrier, 5 mg/kg DMF and 5 mg/kg MHF, samples were
`taken from each group (8 animals/group) before immuniza-
`tion, at the onset of disease (day 11) and in partial remission
`(day 21) for MAP analysis. In this experiment, IL-5 (Fig. 5)
`was significantly increased by DMF in the late phase
`<
`γ
`α
`(
`P
` 0·05, Mann–Whitney
`
`U-test) while IFN-
`, TNF-
` and
`IL-4 were below detection level in most cases (not shown).
`IL-10 (Fig. 6) levels tend to be higher (P > 0·05) in DMF and
`MHF treated animals.
`
`104
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`© 2006 British Society for Immunology,
`
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`
`Clinical and Experimental Immunology,
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`145:
`
` 101–107
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`Fumarate therapy in experimental autoimmune encephalomyelitis
`
`(a)
`
`(c)
`
`(b)
`
`(d)
`
`control/Mac-3
`
`control/CD3
`
`MHF/Mac-3
`
`MHF/CD3
`
`Fig. 2. Representative areas from spinal cord cross sections show reduced infiltration of macrophages (a,c) and T cells (b,d) in control animals (a,b)
`compared to MHF treated animals (c,d) after immunohistochemistry. Nuclei were counterstained with haematoxylin, 5 µm sections.
`
`can inhibit the nuclear entry of NF-kappaB [11] and induce
`apoptosis in lymphocytes and dendritic cells [23].
`With Multi Parameter Cytokine Analyses, we could not
`confirm a relevant change in cytokine expression in plasma
`samples which would point to one of the known patterns.
`
`IL-4 and IFN-γ were generally low or even below the detec-
`tion limit, IL-5 was only significantly increased by DMF in
`the chronic phase. In cell culture studies, MHF has been
`reported reduce the production of IFN-γ by DC in a dose
`dependent manner [10] however, the concentrations used in
`
`Macrophages (Mac-3)
`
`(b)
`300
`
`200
`
`100
`
`0
`
`control
`
`D M F 15 m g/kg
`D M F 5 m g/kg
`
`M H F
`
`Mean cell count/mm2+ SEM
`
`T cells (CD3)
`
`control
`
`D M F 15 m g/kg
`D M F 5 m g/kg
`
`M H F
`
`(a)
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Mean cell count/mm2+ SEM
`
`Fig. 3. Blinded quantification of inflammation in spinal cord during chronic EAE (27 days p.i). Control n = 11, 5 mg/kg DMF n = 11, 15 mg/kg DMF
`n = 11, MHF n = 12. Bars show mean cell count per mm2. (a) The infiltration of CD3 + cells (T cells) was nonsignificantly reduced with DMF and
`significantly reduced with MHF (P < 0·01, Mann–Whitney U-test). (b) The infiltration of Mac-3+ cells (macrophages) was reduced nonsignificantly
`with 5 mg/kg DMF, significantly with 15 mg/kg DMF (P < 0·01, Mann–Whitney U-test) and highly significantly reduced with MHF (P < 0·001).
`
`© 2006 British Society for Immunology, Clinical and Experimental Immunology, 145: 101–107
`
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`

`IL-10
`
`300
`
`200
`
`100
`
`Concentration [ng/ml] + SEM
`
`0
`
`pretreat
`
`ctrl peak
`
`ctrl late
`
`M H F peak
`
`M H F late
`
`D M F peak
`
`D M F late
`
`Fig. 6. IL-10 levels during EAE with FAE therapy by MAP-analysis. IL-
`10 was non-significantly elevated in the FAE treated groups. Shown are
`mean ± SEM.
`
`Another source for IL-10 are Th2-polarized T cells. Although
`these data were gained from blood samples and may not
`exactly reflect changes in lymphoid tissue or the spinal cord
`in situ, as they were obtained from animals after in vivo
`administration of FAE, they may give us insight into
`more physiological activities of these potentially valuable
`therapeutics.
`Further studies are needed to learn more about the mech-
`anisms involved in the observed clinical effect of DMF and
`MHF in the EAE model. Moreover, FAE are promising for
`clinical trials in larger MS cohorts.
`
`Acknowledgements
`
`The technical support by Silvia Seubert and Annette Horn is
`greatly appreciated. Supported by funds from the Medical
`Faculty, University of Goettingen, and by research grants
`from Fumapharm AG and BiogenIdec AG.
`
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