A Diet Mimicking Fasting Promotes Regeneration and …zuckerfasten.de/data/documents/Fasting_Bock_2016.pdf · Cell Reports Report A Diet Mimicking Fasting Promotes Regeneration and

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A Diet Mimicking Fasting P
romotes Regenerationand Reduces Autoimmunity and Multiple SclerosisSymptoms
Graphical Abstract

Highlights

d FMD reduces pro-inflammatory cytokines and increases

corticosterone levels

d FMD suppresses autoimmunity by inducing lymphocyte

apoptosis

d FMD promotes regeneration of oligodendrocyte in multiple

MS models

d FMD is a safe, feasible, and potentially effective treatment for

MS patients

Choi et al., 2016, Cell Reports 15, 1–11June 7, 2016 ª 2016 The Author(s)http://dx.doi.org/10.1016/j.celrep.2016.05.009

Authors

In Young Choi, Laura Piccio,

Patra Childress, ..., Friedemann Paul,

Markus Bock, Valter D. Longo

[email protected]

In Brief

Choi et al. show that cycles of a fasting

mimicking diet (FMD) ameliorate disease

severity by suppressing autoimmunity

and stimulating remyelination via

oligodendrocyte regeneration in multiple

sclerosis (MS) mouse models. They also

show that a similar FMD is a safe, feasible,

and possibly a potentially effective

treatment for patients with relapsing-

remitting MS.

mailto:[email protected]

http://dx.doi.org/10.1016/j.celrep.2016.05.009

Please cite this article in press as: Choi et al., A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple SclerosisSymptoms, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.05.009

Cell Reports

Report

A Diet Mimicking Fasting PromotesRegeneration and Reduces Autoimmunityand Multiple Sclerosis SymptomsIn Young Choi,1,10 Laura Piccio,2,10 Patra Childress,3 Bryan Bollman,2 Arko Ghosh,4 Sebastian Brandhorst,1

Jorge Suarez,1 Andreas Michalsen,5 Anne H. Cross,2 Todd E. Morgan,1 Min Wei,1 Friedemann Paul,6,7 Markus Bock,6,7,11

and Valter D. Longo1,4,8,9,11,*1Longevity Institute, School of Gerontology, and Department of Biological Sciences, University of Southern California, Los Angeles,

CA 90089, USA2Department of Neurology and Neurosurgery and Hope Center for Neurological Disorders, Washington University School of Medicine,St. Louis, MO 63110, USA3Global Medicine Program, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA4Department of Neuroscience, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles,CA 90089, USA5Institute of Social Medicine, Epidemiology and Health Economics, Charite University Medicine Berlin, 10117 Berlin, Germany6NeuroCure Clinical Research Center and Clinical and Experimental Multiple Sclerosis Research Center, Department of Neurology, Charite

University Medicine Berlin, 10117 Berlin, Germany7Experimental and Clinical Research Center, a joint cooperation between the Charite Medical Faculty and the Max-Delbrueck Center for

Molecular Medicine, 10117 Berlin, Germany8Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90089, USA9IFOM, FIRC Institute of Molecular Oncology, 20139 Milan, Italy10Co-first author11Co-senior author

*Correspondence: [email protected]://dx.doi.org/10.1016/j.celrep.2016.05.009

SUMMARY

Dietary interventions have not been effective in thetreatment of multiple sclerosis (MS). Here, we showthat periodic 3-day cycles of a fasting mimickingdiet (FMD) are effective in ameliorating demyelin-ation and symptoms in a murine experimental auto-immune encephalomyelitis (EAE) model. The FMDreduced clinical severity in all mice and completelyreversed symptoms in 20% of animals. Theseimprovements were associated with increasedcorticosterone levels and regulatory T (Treg) cellnumbers and reduced levels of pro-inflammatorycytokines, TH1 and TH17 cells, and antigen-present-ing cells (APCs). Moreover, the FMD promotedoligodendrocyte precursor cell regeneration andremyelination in axons in both EAE and cuprizoneMS models, supporting its effects on both sup-pression of autoimmunity and remyelination. Wealso report preliminary data suggesting that anFMD or a chronic ketogenic diet are safe, feasible,and potentially effective in the treatment of re-lapsing-remitting multiple sclerosis (RRMS) patients(NCT01538355).

This is an open access article under the CC BY-N

INTRODUCTION

Multiple sclerosis (MS) is an autoimmune disorder characterized

by T cell-mediated demyelination and neurodegeneration in the

CNS (Friese andFugger, 2005; Pender andGreer, 2007; Sospedra

andMartin, 2005). In experimental autoimmune encephalomyelitis

(EAE), an animal model for MS, activated myelin-specific TH1 and

TH17 cells cross the blood-brain barrier andmigrate into the CNS,

where they are activated by local antigen-presenting cells (APCs)

and promote inflammation (Dhib-Jalbut, 2007; Fletcher et al.,

2010; Goverman, 2009; Hemmer et al., 2002). This inflammatory

process leads to oligodendrocyte death, demyelination, and

axonal damage, which eventually cause neurologic damage (Luc-

chinetti et al., 1999; Raine and Wu, 1993). Although oligodendro-

cyte precursor cells (OPCs) canmigrate to the sites of MS lesions,

they often fail to differentiate into functional oligodendrocytes

(Chang et al., 2002; Wolswijk, 1998). Several MS treatment drugs

have been effective in reducing immune responses, but their

impact on long-term disease progression, accrual of irreversible

neurological disability, and immune system function remains

largely unclear, underlining the need for novel therapeutic strate-

gies (Wingerchuk and Carter, 2014). Therefore, effective treat-

ments for MSmay require not only the mitigation of autoimmunity

butalso thestimulationofoligodendrocyte regenerationand resto-

ration of a functional myelin sheath. Periodic cycles of prolonged

fasting (PF) or of a fasting mimicking diet (FMD) lasting 2 or more

Cell Reports 15, 1–11, June 7, 2016 ª 2016 The Author(s) 1C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

mailto:[email protected]


http://creativecommons.org/licenses/by-nc-nd/4.0/

Figure 1. FMD Cycles Decrease Disease Severity of the MOG35–55-Induced EAE Model

(A) Diagram displaying the time course of the immunization and the diet interventions.

(B) The EAE severity scores of the control diet (EAE CTRL; n = 23), ketogenic diet (EAE KD; n = 13), semi-therapeutic FMD cycles (EAE FMD (S); n = 7), or

therapeutic FMD cycles (FMD(T); n = 23).

(C) Incidence rate of EAE CTRL and EAE FMD (S) (n = 7–23).

(D) EAE severity score in mice for which FMD(T) completely reversed EAE severity, with no observable disease (score = 0; 5 out of 23 mice).

(E) EAE severity score of the best-performing control mice (n = 12) and FMD(T) mice (n = 12).

(F) EAE severity score of the mice treated with FMD after chronic EAE development (EAE CTRL-FMD; n = 6).

(G–M) Spinal cord sections of EAE CTRL and EAE FMD (T) mice with quantification of H&E staining (G), solochrome cyanine staining (H), and MBP (myelin basic

protein)/SMI32 (I) double staining of spinal cord sections isolated at day 14.

Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test, one-way or two-way ANOVA, and Bonferroni post test. Scale bar

represents 200 mm.


days can increase protection of multiple systems against a variety

of chemotherapy drugs in mice and possibly humans (Fontana

et al., 2010; Guevera-Aguirre et al., 2011; Lee et al., 2010; Longo

and Mattson, 2014). Moreover, PF or an FMD reverses the immu-

nosuppression or immunosenescence of either chemotherapy

or aging through hematopoietic stem cell-based regeneration

(Brandhorst et al., 2015; Cheng et al., 2014). Chronic caloric re-

striction, a ketogenic diet (KD), and intermittent fasting have

been shown to help prevent EAE by reducing inflammation and

enhancing neuroprotection when administered prior to disease

induction or signs (Esquifino et al., 2007; Kafami et al., 2010; Kim

do et al., 2012; Piccio et al., 2008), but dietary interventions have

not been reported to be effective as therapies for EAE or MS or

to promote myelin regeneration.

2 Cell Reports 15, 1–11, June 7, 2016

Here, we report on the effects of low-calorie and low-protein

FMD cycles as a treatment in MS mouse models, and we inves-

tigate the mechanisms involved. Furthermore, we report prelim-

inary results on the safety and feasibility of a FMD and a KD in

patients with relapsing-remitting multiple sclerosis (RRMS).

RESULTS

FMD Cycles Reduce Disease Severity in the MOG35–55-Induced EAE ModelWe examined the effects of 3 cycles of a very-low-calorie and

low-protein FMD lasting 3 days every 7 days or a KD continued

for 30 days in EAE-induced by active immunization with myelin

oligodendrocyte glycoprotein 35–55 (MOG35–55) (Figure 1A).


Groups of mice were treated semi-therapeutically (EAE FMD (S),

where FMD treatment started after 10% of the immunized pop-

ulation showed signs of EAE) or therapeutically (EAE FMD (T),

where FMD treatment started after all of the immunized popula-

tion showed signs of EAE). FMD and KD treatment decreased

disease severity compared to that in the control group (Fig-

ure 1B). However, the FMD reduced the mean severity score

to�1, whereas a KD reduced the severity score to�2 at the later

stages (Figure 1B). In the EAE FMD (S) group, FMD treatment not

only delayed the onset of disease but also lowered the incidence

rate (100% versus 45.6%; Figure 1C). In the EAE FMD (T) group,

FMD cycles completely reversed the severity score to 0 in 21.7%

of the cohort (no observable signs; Figure 1D) and reduced the

severity score to <0.5 in >50% of mice (12 out of 23 mice; Fig-

ure 1E). To address whether the FMD cycles also have beneficial

effects in chronic EAE models that have established disease, we

initiated FMD treatment 2 weeks after the initial signs of EAE

were observed (EAE CTRL-FMD). Prior to treatment, the control

diet EAE group (EAE CTRL) and EAE CTRL-FMD cohorts had

similar severity scores (3.19 ± 0.52 versus 3.30 ± 0.27; day 24).

After three FMD cycles, we observed a significant reduction in

severity score in the EAE CTRL-FMD cohort compared to the

EAE CTRL cohort (3.3 ± 0.57 versus 2.1 ± 0.89; day 42; p <

0.05; Figure 1F). As infiltration of immune cells and demyelination

are histopathological hallmarks of EAE and MS, spinal cord sec-

tions from control and FMD(T) mice were stained with H&E to

visualize infiltrating immune cells (Figure 1G) or solochrome

cyanine to visualize myelin (Figure 1H). To assess demyelination

and axonal damage, immunohistochemistry was performed us-

ing antibodies against myelin basic protein (MBP) or dephos-

phorylated neurofilaments (SMI-32; Figure 1I). At day 3, levels

of infiltrating immune cells and demyelination were similar in

the EAE CTRL and EAE FMD groups (Figures 1J and S1H). At

day 14, sections of EAE CTRL mice displayed severe immune

cell infiltration corresponding to demyelinated lesions, reduced

MBP expression, and increased SMI-32 expression (Figures

1J–1M). By contrast, sections of EAE FMD mice at day 14 dis-

played significantly reduced immune cell infiltration and demye-

lination (Figures 1J–1M). Although MBP staining showed no sig-

nificant difference between EAECTRL and EAE FMDmice at day

14 (Figure 1L), neurofilament dephosphorylation in EAE FMD

mice was reduced compared to EAE CTRL mice (Figure 1M).

Overall, these results suggest that FMD cycles reduce EAE dis-

ease severity in part by reducing inflammation and preventing

demyelination and axonal damage.

FMD Cycles Reduce Infiltration of Immune Cells in theSpinal CordTo investigate the capacity of FMD cycles to reduce potential

autoimmune T cells, we measured circulating white blood cells

(WBCs), lymphocytes, monocytes, and granulocytes in naive,

EAE CTRL, EAE FMD, and EAE FMD:RF (measured 4 days after

returning to a standard ad lib diet) mice after three cycles of the

FMD regimen (Figure 2A). The FMD resulted in a temporary

40%–50% reduction in total WBCs, lymphocytes, monocytes,

and granulocytes. Upon returning to the standard ad lib diet

(EAE FMD:RF), all complete blood counts (CBCs) returned to

either naive or lower levels than those observed in the EAE

CTRL, with the exception of granulocytes, indicating that the

FMDcycles cause bothWBCdeath and regeneration (Figure 2A).

Next, we measured the inflammatory markers associated with

EAE pathophysiology. Day 3 and day 14 spinal cord sections

of the EAE CTRL mice were extensively populated with

CD11b+ cells (Figure 2B). However, at day 14, the EAE FMD

mice displayed a 75% reduction (p < 0.05) in spinal cord-associ-

ated CD11b+ cells compared to mice on the control diet (11.7%

versus 2.8%; Figure 2B). Since myelin-specific effector T cells

migrate into the CNS and initiate demyelination, we investigated

the accumulation of CD4+ or CD8+ T cells in the spinal cord.

A large number of CD4+ T cells were detected in the white matter

of spinal cord sections from the control diet cohort (Figure 2C). In

contrast, the FMD-treated cohort displayed a >4-fold reduction

(p < 0.01) in CD4+ T cells at day 3 (8.6% versus 1.5%; Figure 2C)

compared to the control diet cohort, which remained lower even

at day 14. The FMD group also had reduced CD8+ T cells (day 3:

1.3% versus 0.4%; p < 0.01; Figure 2D) compared to the control

diet group. To investigate whether the FMD affects APCs, we

isolated splenocytes from EAE CTRL and EAE FMD mice at

day 3, stained them for CD11c and F4/80, and characterized

them by flow cytometry. We observed a significant decrease

(p < 0.05) in CD11c+ dendritic cells in the EAE FMD cohort

compared to the EAE CTRL cohort (3.08% ± 0.70% versus

1.46% ± 0.31%), but we did not observe any changes in the

number of F4/80+ macrophage cells in the control or FMD-

treated groups (Figures 2E and S2B). To determine the effects

of the FMD treatment on T cell infiltration in the spinal cord, we

measured T cell activation levels. The number of CD4+ T cells

and CD8+ T cells in EAE CTRL and EAE FMD mice was similar

(Figures S2C and S2D), but the ratio of splenic naive (CD44low)

to activated (CD44high) CD4+ T cells was increased (p < 0.05) in

the FMD group compared to the control group (1.95 versus

3.67; Figure 2F). No difference in CD8+ T cells was observed (Fig-

ure S2E). Moreover, the total number of effector (CD44high and

CD62Llow) T cells was reduced in the FMD compared to the con-

trol group, but the ratio of effector (CD44high and CD62Llow) to

memory T (CD44high and CD62Lhigh) cells did not change (Fig-

ures S2F–S2H). These results indicate that FMD cycles reduce

the number of dendritic cells and increase the relative number

of naive T cells, which may explain the reduced autoimmunity

caused by the FMD.

FMDCycles Induce Autoreactive Lymphocyte Apoptosisand Increase the Number of Naive CellsTo determine whether FMD cycles also reduce the number of

MOG-specific antigen-reactive cells, we used amajor histocom-

patibility complex (MHC) tetramer (MOG35–55/IAb) to identify

antigen-reactive cells after an FMD cycle in vivo. The number

of CD4+ MOG35–55 /IAb+ cells was reduced in the EAE FMD

cohort compared to the EAE CTRL cohort (5.75% ± 0.51%

versus 3.83% ± 0.66% of lymphocytes; * p < 0.05; Figure 2G).

To determine whether the reduced active T cell number is

due to an increase in the number of regulatory T (Treg) cells, we

isolated lymphocytes from draining lymph nodes and spleens

of EAE CTRL or EAE FMD mice and analyzed them for CD4+

CD25+ FoxP3+ Treg cells. The FMD cohort showed a 2-fold in-

crease (p < 0.01) in the number of CD25+ FoxP3+-expressing

Cell Reports 15, 1–11, June 7, 2016 3

(legend on next page)




Treg cells (13.6% ± 4.2% versus 25.1% ± 4.2%; Figure 2H).

Moreover, the FMD cohort showed a 27.8% reduction (p <

0.05) in the number of interferon g (IFN-g)- expressing TH1 cells

(2,974.4 ± 708.0 versus 2,148.1 ± 1,396.1; Figure 2I) and a

46.5% reduction (p < 0.05) in the number of interleukin-17

(IL-17)-expressing TH17 cells (2,535.9 ± 722.0 versus 1,357.1 ±

256.2; Figure 2J), both of which are known to be central media-

tors of EAE. Interestingly, upon re-feeding of the control diet, the

EAE FMD treatment group (EAE FMD:RF) showed a 72.9%

reduction (p < 0.05) in the number of IFN-g-expressing TH1 cells

(2,974.4 ± 708.0 versus 805.8 ± 251.5; Figure 2I) and a 82.9%

reduction (p < 0.05) in the number of IL-17-expressing TH17 cells

(2,535.9 ± 722.0 versus 432.4 ± 117.4; Figure 2J), suggesting

that the FMD can prevent autoimmunity in part by reducing the

levels of pro-inflammatory T cells implicated in EAE.

In order to assess how FMD cycles may reduce the number of

T cells, we measured apoptosis in MOG-specific T cells (CD3+

MOG35–55/IAb) in vivo. We observed a significant increase (p <

0.05) in apoptotic CD3+ MOG35–55/IAb levels in the EAE FMD

cohort compared to the EAE CTRL cohort (28.3% ± 4.94%

versus 39.1% ± 4.79%; Figure 2K), which was consistent with

the major reduction in the number of WBCs and lymphocytes

observed in the FMD group (Figure 2A). To investigate whether

these apoptotic cells are replaced by newly generated cells,

we treated the mice with bromodeoxyuridine (BrdU) during the

re-feeding period (four injections within 48 hr, at 1 mg of BrdU

per injection). Splenocytes were isolated 4 days after the re-

feeding of the regular diet and stained for BrdU (Figure S2I).

We observed no difference in levels of total BrdU+ lymphocytes

(8.11% ± 1.99% versus 12.02% ± 2.72%; Figure S2J), but we

observed a significantly reduced proliferation of TH1 (BrdU+

CD4+IFNg+) (5.74% ± 1.07% versus 3.65% ± 0.63%; *p <

0.05; Figure 2L) and no difference in proliferation of TH17

(BrdU+CD4+IL17+) (4.71% ± 1.53% versus 5.01% ± 1.66%; Fig-

ure S2K). Taken together, these data indicate that FMD cycles

may promote apoptosis of autoreactive T cells, leading to an

increase in the proportion of naive T cells and regulatory

T cells. In addition, FMD cycles may interfere with proliferation

and differentiation of TH1 cells, but not TH17 cells. To investigate

whether the FMD’s effects on CNS infiltrating immune cells are

associated with suppression of TH1- and TH17-dependent cyto-

Figure 2. FMD Cycles Decrease the Number of Infiltrating T Cells in th

(A) Total white blood cell (WBC), lymphocyte, monocyte, and granulocyte counts

mice after three cycles of the FMD and a matched time point for EAE-CTRL mic

(B–D) Spinal cord sections (day 14) and quantification at days 3 and 14 after the fi

mouse).

(E) CD11c+ isolated from EAE CTRL or EAE FMD mice on day 3, and quantificat

(F) CD4+ gated for CD44low or CD44high cells isolated from EAE CTRL or EAE FMD

CD4+ CD44high (active) cells.

(G) CD3+ lymphocytes gated for CD4 and MOG35–55/IAb from EAE CTRL or EAE

(H) CD4+ CD25+ FoxP3+ isolated from EAE CTRL or EAE FMD mice, and quantifi

(I and J) Intracellular staining for either IFNg (I) or IL17(J) after gated for CD4+ of th

(K) Quantification of Annexin V+ apoptotic CD3+ MOG35-55/IAb cells.

(L) Quantification of CD4+IFNg+ of BrdU+ lymphocytes.

(M–O) Serum TNF-a (M), IFN-g (N), and IL-17 levels (O) (pg/ml) in naive, EAE CT

(P) Serum corticosterone levels (ng/ml) before immunization, at the time of sympto

or FMD group.

n = 4–8 per group; mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s

kine production (IL-17, IFN-g, and tumor necrosis factor a

[TNF-a]), we analyzed serum from naive, EAE CTRL, and EAE

FMD mice (Figures 2K–2M). We observed significant reductions

in serum TNF-a (113.3 ± 7.9 versus 79.3 ± 10.5 pg/ml; p < 0.001;

Figure 2M), IFN-g (558.43 ± 124.5 versus 296.0 ± 83.4 pg/ml;

p < 0.001; Figure 2N), and IL-17 (36.8 ± 9.67 versus 20.75 ±

4.2 pg/ml; p < 0.01; Figure 2O). To identify a potential mediator

for the effects of FMD cycles on the suppression of autoimmune

responses, we measured serum corticosterone levels. Cortico-

sterone is a glucocorticoid hormone with broad anti-inflamma-

tory and immunosuppressive effects affecting leukocyte dis-

tribution, trafficking, and death (Ashwell et al., 2000; Herold

et al., 2006; Planey and Litwack, 2000; Vegiopoulos and Herzig,

2007). Serum corticosterone levels were elevated in association

with the first signs of EAE (EAE day 1, before treatment) (data not

shown). FMD treatment caused a further increase in corticoste-

rone levels at day 3 compared to those of controls (245.9 ± 38.8

versus 375.0 ± 94.1 ng/ml; p < 0.01), which returned to EAE basal

levels by day 14 in both groups (Figure 2P). These results indi-

cate that FMD cycles reduce the number of TH1 and TH17

effector cells and the production of pro-inflammatory cytokines.

These effects of the FMDmay be regulated in part by the tempo-

rary elevation of corticosterone levels, dampening of T cell acti-

vation, and reduced APC and T cell infiltration in the spinal cord.

FMD Reverses EAE Symptoms by Reducing the Leveland Reactivity of Established Autoimmune CellsTo determine how the FMD affects the initiation of EAE, spleno-

cytes were isolated from EAE CTRL and EAE FMD mice, re-acti-

vated with MOG35–55 peptide and IL-23 ex vivo, and transferred

into naive recipient mice to induce EAE. The mice were then sub-

jected to either a control diet or FMD cycles (Figure 3A). The

supernatant from ex vivo splenocyte cultures derived from

EAE FMD mice showed no difference in TNF-a levels (110.8 ±

14.9 pg/ml versus 97.1 ± 8.4 pg/ml; Figure 3B) but a major reduc-

tion (p < 0.01) in the levels of IFN-g (342.0± 29.8 pg/ml versus 46.6

± 16.6 pg/ml Figure 3C) and IL-17 (850.5 ± 442.0 pg/ml versus

257.4± 36.4pg/ml; Figure3D). Interestingly, upon in vitro reactiva-

tion, both EAE CTRL and EAE FMD had similar levels of TH1 and

TH17 differentiated cells (Figures 3E and 3F). To determine

whether the immune cells from EAE CTRL and EAE FMD mice

e Spinal Cord

of naive, EAE-CTRL, EAE-FMD, and EAE-FMD:RF (after 3 days of re-feeding)

e.

rst sign of EAE for CD11b+ (B), CD4+ (C), and CD8+ (D) (at least six sections per

ion of cells from the total isolated splenocyte.

mice, and quantification of percent splenocytes in CD4+ CD44low (inactive) or

FMD mice, and quantification of MOG-specific CD4+ cells.

cation of CD25+ FoxP3+ in CD4+ cells.

e naive, EAE CTRL, EAE FMD, EAE FMD:RF and quantification of cell counts.

RL, and EAE FMD mice on day 3 after the first sign of EAE.

m occurrence, or 3 or 14 days after the initial symptom appeared in the control

t test, one-way ANOVA, and Bonferroni post test. Scale bar represents 200 mm.


Figure 3. Antigen-Activated Splenocytes

from EAE-CTRL and the EAE-FMD Mice

Had Similar Encephalitogenic Effects

(A) Diagram for the adoptive transfer EAE model.

(B–D) Quantification of TNF-a (B), IFN-g (C), and

IL-17 (D) (pg/ml) in the supernatant from ex vivo

cultures of splenocytes from naive, EAE CTRL,

andEAEFMDmice eitherwith orwithoutMOG35–55

and IL-23 re-activation.

(E and F) Quantification of TH1 or TH17 (repre-

sented by percentage of CD4+) from lymphocyte

culture of EAE CTRL and EAE FMD mice with or

without MOG35–55 and IL-23 re-activation.

(G) Incidence rate of adoptive transfer EAE

groups.

(H) EAE severity score of adoptive transfer EAE

groups.

n = 5–6 per group; mean ± SEM. *p < 0.05, **p <

0.01, and ***p < 0.001, Student’s t test, one-way

ANOVA, and Bonferroni post test.


have similar encephalitogenic effects, we transferred splenocytes

from either donor group (EAE CTRL or EAE FMD) into naive recip-

ients (A,EAECTRLdonor tocontrol diet recipient; andC,EAEFMD

donor to control diet recipient). This resulted in a similar disease

incidence rate (Figure 3G) and an equally severe EAE disease

severity by day 20 (2.38± 0.48 versus 2.70± 0.75; Figure 3H), indi-

cating that the FMDdid not affect the development and function of

reactive immunecells in vivoorexvivo.However,whenFMDtreat-

ment was initiated after transfer of control donor splenocytes (B,

EAE CTRL donor to naive mice with FMD treatment), recipient

mice displayed a delayed disease onset (day 12 versus day 16


post-transfer; Figure 3G) and a major

reduction in EAE severity scores

compared to control mice (2.38 ± 0.48

versus 0.75 ± 0.87; Figure 3H). Taken

together, these results suggest that T cell

priming in response to myelin antigen

occurred normally in the EAE CTRL and

EAEFMDgroups, but the FMDcan reduce

the level of the existing autoimmunity.

FMD Cycles StimulateRemyelination by PromotingOligodendrocyte RegenerationTo investigatewhether the reduceddemy-

elination in FMD mice may also be related

to enhanced oligodendrocyte regenera-

tion, we first carried out a quantitative im-

age analysis of NG2+ (an oligodendrocyte

progenitor cell [OPC] marker) and GST-

p+ (a mature oligodendrocyte marker) in

spinal cord sections from control or FMD

mice (Figure 4A). We observed no differ-

ence in the number of NG2+ OPCs in sec-

tions taken from EAE CTRL and EAE FMD

mice (Figure S3A). However, at day 14, the

number of GST-p+ oligodendrocytes was

reduced in the EAE CTRL group, but not in the EAE FMD group

(886.7 ± 41.6 versus 1,273 ± 200.3; cells per spinal cord section

area; p < 0.01; Figure 4B). To assess whether the normal levels

of mature oligodendrocytes in the EAE FMD group were due to

enhanced regeneration and/or differentiation, EAE CTRL or EAE

FMD mice were injected with BrdU at the time of re-feeding

(day 10). We observed a major increase (p < 0.01) in the percent-

age of cells that are double positive for BrdU+ and GST-p+ in the

EAE FMD group compared to the EAE CTRL group (42.9% ±

11.2% versus 83.0% ± 13.2%; p < 0.01), suggesting that the

FMD promotes oligodendrocyte differentiation from precursor

Figure 4. FMD, which Protects the Mouse Spinal Cord from Loss of Oligodendrocytes and Enhances Remyelination, Is Safe and Potentially

Effective in the Treatment of MS Patients

(A–C) Spinal cord sections isolated at day 14 and quantification for GST-p (mature oligodendrocyte) and BrdU (A), TUNEL and NG2 (oligodendrocyte precursor

cells) (B), and TUNEL and GST-p (C) in naive, EAE-CTRL, or EAE-FMD mice.

(F–H) Sections from the corpus callosum region and quantification of cuprizone treated brains, stained with Luxol Fast Blue of the naive control, end of 5 weeks of

cuprizone diet (week 0), cuprizone (5 weeks) plus regular chow (2 weeks), and cuprizone (5 weeks) plus FMD cycle (2 weeks).

(I and J) Section from the corpus callosum region and its quantification of the cuprizone treated brains stained with GST-p+ of cuprizone (5 weeks) plus regular

chow (2 weeks), and cuprizone (5 weeks) plus FMD (2 weeks). Quantification is normalized to percent naive GST-p+ level.

(K–N) Change in quality of life at 3months in terms of overall quality of life (K), change in health (L), physical health composite (M), andmental health composite (N).

The dotted line represents a threshold that is thought to be clinically important (R5 points). Data represent mean ± standard error of the difference (SED);

*p < 0.05, Mann-Whitney U test. An increase of R5 points is considered clinically important.

At least 12 sections per mouse were used for quantification; n = 4; mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA and Bonferroni post test.




cells (Figure 4C). To assess the effects of the FMD on either OPCs

or mature oligodendrocytes, sections were stained with TUNEL,

an apoptotic marker, and GST-p+ or NG2+ (Figure 4D). We

observed a significant increase in the number of TUNEL+ NG2+

(11.2 ± 12.2 versus 1.9 ± 1.4 cells/section) and TUNEL+ GST-p+

(18.8±15.2versus2.9±5.3cells/section) cells in thecontrol group

compared to the FMD group (p < 0.05; Figures 4E and 4F). Taken

together, these results indicate that the FMD not only stimulates

regeneration and differentiation of oligodendrocytes but also pro-

tects OPCs and mature oligodendrocytes from apoptosis.

To investigate whether the FMD-dependent stimulation of

oligodendrocyte differentiation and remyelination can occur in-

dependent of the observed effects on T cell number and activity,

we used the cuprizone-induced demyelinating mouse model

(Ransohoff, 2012; Torkildsen et al., 2008). Addition of 0.2%

(w/w) cuprizone to the regular mouse diet for 5–6 weeks results

in demyelination in the corpus callosum followed by sponta-

neous remyelination upon re-feeding with regular chow. After

5 weeks of cuprizone treatment, mice were switched to either

the control diet or FMD cycles for 5 weeks, and some were

euthanized weekly to assess the degree of myelination by Luxol

fast staining and GST-p+ (Figures 4G and 4I). As expected, after

5 weeks of the cuprizone diet, a significant reduction in myelin

staining was observed in the corpus callosum compared to the

naive controls (Figures 4H and 4J). After two cycles, the FMD-

treated group displayed increased myelin staining and an

increased number of GST-p+ oligodendrocytes compared to

the control diet group (Figures 4H and 4J). However, at later

time points, we did not observe differences in spontaneous

re-myelination between the control diet and FMD cohorts, as it

is well established that cuprizone-dependent myelin damage

can be fully reversed after removal of the toxin (Figures S3C

and S3D). These results indicate that the FMD promotes OPC-

dependent regeneration and accelerates OPC differentiation

into oligodendrocytes while enhancing remyelination indepen-

dently of its modulation of the inflammatory response.

A Randomized Pilot Trial to Test the Effects of a FMD orKD in Relapsing-Remitting MS Patients: Evidence forSafety and FeasibilityA randomized,parallel-group, three-armpilot trial (NCT01538355)

was conducted to assess the safety and feasibility of FMD or KD

treatment on health-related quality of life (HRQOL) in RRMS pa-

tients. 60 patients were randomly assigned to a control diet (CD;

n = 20), KD for 6 months (n = 20), or a single cycle of a modified

human FMD for 7 days (n = 20) followed by a Mediterranean diet

for 6 months (Figure S4). Baseline characteristics were balanced

among the three groups (Tables S1 andS2). The FMD and KD co-

horts displayed clinicallymeaningful improvements in theHRQOL

summary scales at 3months, which included the overall quality of

life (Figure 4K) change in health (Figure 4L), a physical health com-

posite (Figure 4M), and a mental health composite (Figure 4N).

Also, similar changes were observed in the total HRQOL scales

at different time points (Figure S5). Adverse events (AEs) and

serious adverse events (SAEs) were reported for 92% (8%) of

CD cohort individuals, 78% (16%) of FMD cohort individuals,

and 78% (11%) of KD cohort individuals (Table S5). The most

common AE was airway infection, and the most frequent SAE


was lower urinary tract infection. No indication of an increase in

liver enzymes exceeding the normal range was observed in any

of the three treatment groups. Also, the interventions were well

tolerated, as evidenced by high compliance rates (CD, 60%;

KD, 90%; and FMD, 100%). During the 6-month study period,

we observed a total of eight relapses: four in the CD group, one

in the KD group, and three in the FMD group. In addition to

increased b-hydroxybutyrate levels in plasma, we observed a

slight reduction in lymphocytes and WBC counts and detected

amild reduction in expandeddisability status scale (EDSS) scores

in the FMD and KD groups (measured at baseline, month 3, and

month 6; Tables S1 and S6). Thus, there was an inverse associa-

tion between EDSS andHRQOL scores (Table S7). InMSpatients

the FMD treatment lead to an over 20% drop in the total lympho-

cyte count (baseline versus day8; TableS4) in 72%of the patients

(13 of 18 FMD-treated patients). WBC counts returned to the

baseline levels after these patients were switched to the Mediter-

ranean diet (month 3). Based on the mouse studies, these results

raise the possibility that the FMD alleviated symptoms in MS

patients by reducing the number of autoimmune lymphocytes.

Overall, our study indicates that the administration of FMD and

KD is safe, feasible, and potentially effective, but further studies,

including analyses such as magnetic resonance imaging (MRI),

blinded clinical assessments, and immune assays, are required

to determine efficacy.

DISCUSSION

An FMD administered every week was effective in ameliorating

EAE symptoms in all mice and completely reversed disease pro-

gression in a portion of animals after the onset of EAE signs. By

contrast, the KD had more modest effects and did not reverse

EAE progression in mice. FMD cycles appear to be effective in

the treatment of EAE in mice by (1) promoting oligodendrocyte

precursor-dependent regeneration and (2) reducing the levels

of microglia/monocytes and T cells contributing to autoimmunity

and encephalomyelitis. Our results support an FMD-mediated

anti-inflammatory effect possibly involving the upregulation of

AMPK or the downregulation of mTORC1, which sense nutrient

availability and dictate cell fate (Laplante and Sabatini, 2012). It

was shown thatmTORC1couples immune signals andmetabolic

programming to establish Treg cell function (Zeng et al., 2013). In

fact, treatment with the mTORC1 inhibitor rapamycin or the

AMPK activator metformin attenuates EAE symptoms by modu-

lating effector T cells andTreg cells and restricting the infiltrationof

mononuclear cells into the CNS (Esposito et al., 2010; Nath et al.,

2009). Therefore, FMD treatment could interfere with T cell prolif-

eration and differentiation and with recruitment of other immune

cells, resulting in a decreased recruitment at lesion sites (Fig-

ure 5). Some of these effects of the FMD may be triggered by

endogenous glucocorticoid production. Glucocorticoids are

used to treat MS relapses, but they are generally administered

in short bursts, since they can cause AEs such as osteoporosis

and metabolic syndrome (Brusaferri and Candelise, 2000; Ce

et al., 2006; Roth et al., 2010; Uttner et al., 2005). The FMD may

avoid these adverse effects by promoting additional and coordi-

natedendogenous responses. Importantly, FMDcyclesalso acti-

vated OPCs, resulting in myelin regeneration, as demonstrated

Figure 5. A Simplified Model of FMD-Mediated Effects on Immune Suppression, Oligodendrocyte Regeneration, and Differentiation in MS

FMD treatment promotes endogenous glucocorticoid production, increases Treg cell numbers, blocks T cell activation, and promotes T cell death. In the lesion

area, FMD treatment reduces autoimmune T cell and microglia infiltration and promotes oligodendrocyte-precursor-dependent regeneration and differentiation

of myelinating oligodendrocytes, which engage with demyelinated axons to promote the formation of myelin sheaths.


by accelerated remyelination rate in the cuprizone model (Fig-

ure 5). Notably, because it is the alternation of FMD cycles and

re-feeding and not the FMDalone that promotes the regeneration

and replacement of autoimmune cells with naive cells, the use of

chronic restriction or even a chronic KD may not be effective, or

as effective, in the treatment of EAE and MS.

Finally, we report that the administration of the FMD and

KD in MS patients was safe and well tolerated and resulted

in high compliance. We observed positive effects of FMD cy-

cles or KD treatment in RRMS based on changes in self-re-

ported HRQOL and a mild improvement in EDSS (Table S6).

However, the lack of a proper Mediterranean diet control

makes it difficult to establish whether FMD cycles alone

are sufficient to produce these effects. In addition, MRI ana-

lyses and adequately blinded clinical assessments (EDSS

and multiple sclerosis functional composite [MSFC]), as well

as immune function analyses would greatly enhance the

strength of the clinical findings. Because, unlike for the mouse

experiments, the FMD was only administered to patients only

once, it will be important to test the effects of multiple FMD

cycles on MS patients in larger, randomized, and controlled

trials.

EXPERIMENTAL PROCEDURES

EAE Model

C57Bl/6 (10-week-old female) mice were purchased from The Jackson Lab-

oratory and immunized subcutaneously with 200 mg MOG35–55 (GenScript)

mixed 1:1 with supplemented complete Freund’s adjuvant followed by

200 ng pertussis toxin (PTX; List Biological Laboratories) intraperitoneally

(i.p.) at days 0 and 2. For adoptive transfer, spleens from active immunized

mice were isolated and red blood cells (RBCs) were lysed. Spleen cells were

cultured in the presence of MOG35–55 (20 mg/ml) with rmIL-23 (20 ng/ml) for

48 hr. Cells were collected and re-suspended in PBS, and 15 million cells

were injected intravenously. See Supplemental Experimental Procedures

for a detailed description of disease severity scoring. All experiments

were performed in accordance with approved Institutional Animal Care

and Use Committee (IACUC) protocols of the University of Southern

California.

Mouse Fasting Mimicking Diet

Mice were fed ad lib with irradiated TD.7912 rodent chow (Harlan Teklad),

containing 15.69 kJ/g digestible energy (animal-based protein 3.92 kJ/g,

carbohydrate 9.1 kJ/g, and fat 2.67 kJ/g). The experimental FMD is based

on a nutritional screen that identified ingredients that allow high nourish-

ment during periods of low calorie consumption. The FMD diet consists

of two different components, day 1 diet and day 2–3 diet, that were fed

in this order, respectively. See Supplemental Experimental Procedures

for a detailed explanation of the FMD. Mice consumed all the supplied

food on each day of the FMD regimen and showed no signs of food aver-

sion. After the end of FMD, we supplied TD.7912 chow ad lib for 4 days

before starting another FMD cycle. Prior to supplying the FMD, animals

were transferred into fresh cages to avoid feeding on residual chow and

coprophagy.

Clinical Trial Design

This study was a three-armed, parallel-group, single-center, controlled,

and randomized clinical pilot trial to assess the effects of dietary interven-

tions on HRQOL in RRMS patients. The permuted-block randomization

was generated online at http://randomization.com. An investigator blind


http://randomization.com


to the randomization plan determined the patients’ randomization number

before they underwent the randomization step. This study is registered at

http://www/clicaltrials.gov as NCT01538355. The study was approved by

the local ethics committee. All participants gave informed written consent

according to the 1964 Declaration of Helsinki. See Supplemental Experi-

mental Procedures for detailed descriptions of the clinical trial and diet

compositions.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

five figures, and seven tables and can be found with this article online at

http://dx.doi.org/10.1016/j.celrep.2016.05.009.

AUTHOR CONTRIBUTIONS

I.Y.C., L.P., M.W., and V.D.L. designed mouse experiments. I.Y.C., S.B., and

P.C. performed the mouse experiment. I.Y.C., L.P., P.C., B.B., and A.G.

performed and processed immunohistochemistry. I.Y.C., L.P., and B.B. per-

formed qualitative and quantitative analysis. I.Y.C. and J.S. performed fluores-

cence-activated cell sorting (FACS) analysis. I.Y.C. processed the cytokine

assay. A.M., F.P., and M.B. designed the human study; M.B. acquired human

clinical data; A.M., F.P., and M.B., analyzed and interpreted data; and M.B.

performed, interpreted, and presented the statistical analysis. A.M., F.P.,

M.B., A.H.C., T.E.M., M.W., and V.D.L. were involved in discussing the results

and editorial support. I.Y.C., M.B., and V.D.L. wrote the paper. All authors

discussed the results and commented on the manuscript.

CONFLICTS OF INTEREST

The University of Southern California has licensed intellectual property to

L-Nutra that is under study in this research. As part of this license agreement,

the University has the potential to receive royalty payments from L-Nutra.

V.D.L. has equity interest in L-Nutra, a company that develops medical food.

ACKNOWLEDGMENTS

We thank Dr. Stephen Hauser for insightful comments, Dr. Pinchas Cohen for

assistance with fluorescence microscopy, and Nadine Krueger and Gabi Rahn

for technical assistance. L.P. is a HarryWeaver Neuroscience Scholar of the Na-

tional Multiple Sclerosis Society (NMSS, JF 2144A2/1) and is funded by Fonda-

zione Italiana Sclerosi Multipla (FISM; 2014/R/15) and the Office of the Assistant

Secretary of Defense for Health Affairs, through the Multiple Sclerosis Research

Program, under award number W81XWH-14-1-0156. The opinions, interpreta-

tions, conclusions, and recommendations express in this article are those of

the author and are not necessarily endorsed by the Department of Defense.

Themouse studywas fundedbyNational InstitutesofHealth (NIH)/National Insti-

tute on Aging (NIA) grant AG034906 (to V.D.L.). The human study was funded by

Meylin Projekt e.V. and Familie ErnstWendt StiftungStadt Koeln,whichwere not

involved in any decision-making processes relating the study or its participants.

The work of F.P. is supported by Deutsche Forschungsgemeinschaft (DFG Exc

257). The content is solely the responsibility of the authors and does not neces-

sarily represent theofficial viewsof theNIAorNIH.TheUniversityofSouthernCal-

ifornia has licensed intellectual property to L-Nutra that is under study in this

research. As part of this license agreement, the university has the potential to

receive royalty payments from L-Nutra. V.D.L. has equity interest in L-Nutra, a

company that develops medical food.

Received: May 22, 2015

Revised: February 20, 2016

Accepted: April 26, 2016

Published: May 26, 2016

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Cell Reports, Volume 15

Supplemental Information

A Diet Mimicking Fasting Promotes

Regeneration and Reduces Autoimmunity

and Multiple Sclerosis Symptoms

In Young Choi, Laura Piccio, Patra Childress, Bryan Bollman, Arko Ghosh, SebastianBrandhorst, Jorge Suarez, Andreas Michalsen, Anne H. Cross, Todd E. Morgan, MinWei, Friedemann Paul, Markus Bock, and Valter D. Longo

Supplemental Figure 1

0 570

80

90

100

110

120

10 15 20 25 30

EAE-CTRL

EAE-FMD

EAE-KD

Days after Initial Symptoms

BW

(%

)

0

1

2

3

**

D30D3

EAE-CTRL

EAE-FMD

**

Deg

ree o

f D

em

yelin

ati

on

(a.u

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D30D3

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EAE-CTRL

EAE-FMD**

SM

I32

(# o

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ive P

ixels

/mm

2)

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D30D3

EAE-CTRL

EAE-FMD

MB

P

(# o

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ixels

/mm

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Inflam

mato

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filt

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(a.u

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EAE CTRL

0

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*** EAE-CTRL

EAE-FMD

*** ***

Mean

clin

ical E

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EAE FMD

a b c d

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(# o

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ixels

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rum

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(n

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300Naive

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***

O

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rate

(m

M)

l m n

EAE CTRL EAE FMD

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0 5 10 15 200

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EAE Caloric Restricted

Mean

Clin

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AE

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Figure S1. The beneficial effect of MOG-induced EAE disease progression persist a long

term. Related to Figure 1

a. Body weights of control mice (Black), mice fed with ketogenic diet (Red), and mice fed with

FMD-Thr (Blue) (% BW ± S.E.M.).

b. Serum IGF-1 (ng/mL) level of naive, EAE Day1 before treatment, CTRL and FMD at Day3

and D14 (mean± S.E.M.; p < 0.05; t-test).

c. Serum glucose (mg/dL) level of naive, EAE Day1 before treatment, CTRL and FMD at

Day3, Day7 and D14 (mean± S.E.M.; p < 0.05; t-test).

d. Serum ketone body-βOH Butyrate (mM) level of naive, CTRL and FMD at Day3 and D14.

e. Average clinical score at different time points 3 days, 14 days, or 30 days post initial sign

(n=23; *** p < 0.001; t-test).

f. EAE severity score of control diet (EAE CTRL), and the same dietary composition of the

control diet but matching the calories of the FMD (EAE CR; n=6)] (mean ± S.E.M.).

g. Quantification of MBP of EAE CTRL and EAE FMD on Day14 (n = 8, mean ± S.E.M.).

h-n. The representative staining (h-j) and quantification (k-n) of (h) H&E, (i) solochrome

cyanide, (j) MBP and SMI32 at D3 and D30 of control and FMD (n = 8, mean ± S.E.M., * p <

0.05, ** p<0.01; t-test; Scale bar represents 200 µm).


Nai

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200

400

600

800

1000*

NAIVE

EAE-CTRL

EAE-FMD

Seru

m IF

N (

pg

/mL

)

0

10

20

30

40

50NAIVE

EAE-CTRL

EAE-FMD

*

Se

rum

IL

-17

(p

g/m

L)

n

Naive EAE FMD FMD-RF0

50

100 Lymph%

Mon%

Gran%

% o

f W

BC

b

EAE C

TRL

EAE F

MD

0

5

10

15

20EAE CTRL

EAE FMD

F4

/80

+ C

ells

gEAE CTRL EAE FMD

CD44

CD

62

L

e

EAE C

TRL

EAE F

MD

0

2

4

6

CD

4+IL

17

+

(% o

f B

rdU

)

i j k

EAE C

TRL

EAE F

MD

0

5

10

15

20 p=0.07

Brd

U+ (

% o

f li

ve c

ell

s)

Figure S2. FMD modulate immune response by reducing CD4 T cell activation. Related to

Figure 2

a. % Lymphocyte, monocyte, and granulocyte of total white blood cell of Naïve, EAE CTRL,

EAE FMD, and EAE FMD-RF (n=4-6; Mean ± S.E.M.).

b. Quantification of F4/80+ cells (% of total splenocyte) of EAE CTRL and EAE FMD (n=4;

mean ± s.e.m.).

c. % of CD3+ CD4+ splenocyte of control or FMD. FMD treatment had no significant

difference in % of CD3+ cells (n=4; mean ± s.e.m.).

d. % of CD3+ CD8+ splenocyte of control or FMD. FMD treatment had no significant

difference in % of CD3+ cells (n=4; mean ± s.e.m.).

e. CD8+ splenocyte activation level (CD8+CD44High) of naïve, CTRL and FMD (n=4; mean ±

s.e.m.).

f. Representative flow cytometry plot of CD44 and CD62L and quantification of EAE CTRL

and EAE FMD (n=4; mean ± s.e.m.).

g-h. % ratio of effector (CD44High and CD62Llow) to memory T-cells (CD44High and CD62LHigh)

population from (g) CD4+CD44high T cells, and (h) CD8+CD44high T cells (n=4, mean ± s.e.m.).

i. BrdU injection timeline. BrdU was given during the re-feeding period (4 injections within 48

hours, 1 mg of BrdU / injection).

j. Quantification of BrdU+ lymphocytes of EAE CTRL and EAE FMD (n=4, mean ± s.e.m.).

k. Quantification of CD4+IL17+ BrdU+ lymphocytes of EAE CTRL and EAE FMD (n=4, mean ±

s.e.m.).

l-n. Serum levels of (l) TNF-α, (m) IFN-γ, and (n) IL-17 at Day25


a b

EAE-C

TRL

EAE-F

MD

0

50

100

150

200

*

TU

NE

L+ C

ells

/ s

ec

tio

n

d

0

200

400

600 **Naive

EAE-CTRL

EAE-FMD

D3 D14

# N

G2

+ C

ells

/ s

ec

tio

n

e

0

25

50

75

100

***Naive

Cup (5 weeks)

Cup+CTRL

Cup+FMD

Weeks after Cuprizone Withdrawal

Week1 Week3 Week5

Lu

xo

l F

as

t S

tain

ing

% C

trl P

ixe

l

0

500

1000

1500 ***

**

D3 D14

# G

ST

/mm

2

0

25

50

75

100

Naive

Cup (5 weeks)

Cup + CTRL

Cup + FMD

Weeks after Cuprizone Withdrawal

Week1 Week3 Week5

***

**

*

GS

T

+/

DA

PI

in C

C

(%)

of

Naiv

e

c

Figure S3. FMD increases oligodendrocyte differentiation and protection of OPC and

oligodendrocytes. Related to Figure 4

a. Quantification of number of NG2+ (oligodendrocyte precursor cells) at D3 and D14.

b. Quantification of number of GSTπ+ (matured oligodendrocyte) at D3 and D14.

c. Quantification of number of TUNEL+ cells sowing a significantly increased number of

apoptotic cells in control but not in FMD on D3 (n=6; *p<0.05; t-test).

d. Luxol fast staining quantification (% Naïve control pixel) of naïve mice, cuprizone fed (5

weeks) mice, control diet fed mice and FMD cycle fed mice at week 1, week3 and week

5 upon withdrawal of cuprizone diet.

e. GSTπ quantification (% Naïve of # of GSTπ+ / DAPI+ in corpus callosum) of naïve mice,

cuprizone fed (5 weeks) mice, control diet fed mice and FMD cycle fed mice at week 1,

week3 and week 5 upon withdrawal of cuprizone diet.


Figure S4. Schematic diagram displaying the time course of clinical trial and the diet

interventions. Related to Figure 4

A randomized parallel-group 3 arm pilot trial (NCT01538355) was with relapsing-

remitting MS patients. 60 patients were randomly assigned to: control diet (CD), KD for 6

months or a single cycle of modified human FMD for 7 days followed by a Mediterranean diet

for 6 months

Supplemental Figure 5.

1 3 60

5

10

15

*

Month

Me

an

Ch

an

ge

fro

m B

as

elin

e

Ph

ys

ica

l H

ea

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Co

mp

os

ite

(M

S-5

4)

1 3 6-5

0

5

10

15

* *

Month

Me

an

Ch

an

ge

fro

m B

as

elin

e

Me

nta

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ea

lth

Co

mp

os

ite

(M

S-5

4)

1 3 6-15

-10

-5

0

5

10

15

**

*

*

Month

Me

an

Ch

an

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m B

as

elin

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Ph

ys

ica

l F

un

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(M

S-5

4)

1 3 60

5

10

15

20

Month

Me

an

Ch

an

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fro

m B

as

elin

e

En

erg

y / F

ati

gu

e (

MS

-54

)

1 3 6-40

-30

-20

-10

0

10

20

30

40 *

*

Month

Me

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Ro

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ati

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s P

hy

sic

al (M

S-5

4)

1 3 6-10

0

10

20

30

*

Month

Me

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Bo

dily

Pa

in (

MS

-54

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1 3 6-10

-5

0

5

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*

*

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Me

an

Ch

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Ov

era

ll Q

ua

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of

Lif

e (

MS

-54

)

1 3 6-20

-10

0

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30

* **

*

*

Month

Me

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(M

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Me

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He

alt

h P

erc

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tio

n (

MS

-54

)

1 3 6-5

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Month

Me

an

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as

elin

e

Se

xu

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(M

S-5

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1 3 6-10

-5

0

5

10

15

Month

Me

an

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m B

as

elin

e

So

cia

l F

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(M

S-5

4)

1 3 6-10

-5

0

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Month

Me

an

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m B

as

elin

e

He

alt

h D

istr

es

s (

MS

-54

)

1 3 60

5

10

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Month

Me

an

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m B

as

elin

e

Em

oti

on

al W

ell-B

ein

g (

MS

-54

)

1 3 6-20

-10

0

10

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Month

Me

an

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an

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m B

as

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e

Ro

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imita

tio

ns

Em

otio

na

l (M

S-5

4)

1 3 6-10

-5

0

5

10

15

Month

Me

an

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an

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m B

as

elin

e

Co

gn

itiv

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(M

S-5

4)

1 3 6-10

0

10

20

30

Month

Me

an

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an

ge

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m B

as

elin

e

Sa

tis

fac

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ex

ua

l F

un

ctio

n (M

S-5

4)

CDFMDKD

a b c

d e f

g h i

j k l

m n o

p

Figure S5. The MS-54 scores at Month 1, Month3 & Month6. Related to Figure 4

This pilot clinical feasibility trial revealed potentially positive effects on HRQOL based on self-

reports for both FMD and KD. Mean change from baseline of CD, FMD, and KD at month 1, 3

and 6 of physical health composite (a), mental health composite (b), physical function (c),

energy/fatigue (d), role of limitation physical (e), bodily pain (f), overall quality of life (g),

change in health (h), health perception (i), sexual function (j), social function (k), health distress

(l), emotional well-being (m), role limitations emotional (n), cognitive function (o), and

satisfaction sexual function (p) are shown. Dotted line represents threshold which is thought to

be clinically important (≥ 5 points) in MS-54 outcome. FMD was performed only once which

resulted in a maximum effect size at month 3; thus study time between Month 3 and 6 is

suggested to be a washout period of FMD treatment (mean ± SED; * p<0.05; Mann-Whitney-U

test. Increase of ≥ 5 points are considered as clinically important). At month 3 and 6, the CD had

negligible effect sizes (0.04 to 0.08) and no clinically meaningful impact (mean change from

baseline (MCB) > 5) (Norman et al., 2003, Rudick et al., 2007, Kappos et al., 2014) on physical

health composite (PHCS; 0.22+11.4; MCB + SD) and on mental health composite (MHCS;

1.88+19.9) and on most sub-scales of the MS-54 (Supplemental Fig. 5; Supplemental Table

3). In contrast, patients in the FMD group showed clinically meaningful improvement on MS-54

scores with medium to large effect sizes (0.4 to 0.5) after 3 months, including increases on PHCS

(7.27+4.3), MHCS (8.85+14.2) and on 10 out of 14 of sub-scales (Supplemental Fig. 5;

Supplemental Table 3). After 6 months, the KD group showed medium to large effect sizes (0.3

to 0.5) and clinically meaningful improvement in PHCS (8.37+11.0), MHCS (6.27+14.8) and on

11 out of 14 of sub-domains (Fig. 4k-n; Supplemental Fig. 4; Supplemental Table 3).

Supplementary Table 1. Summary of demographics and primary outcome at baseline.

Related to Figure 4 and Supplementary Figure 4.

Supplementary Table 2. Summary of secondary outcome parameters at baseline. Related

to Figure 4, Supplementary Figure 4 and 5.

Supplementary Table 3. Mean change from baseline (MCB) in Multiple Sclerosis Quality

of Life (MS-54) scores. Related to Figure 4 and Supplementary Figure 5.

Supplementary Table 4. Mean Change from Baseline (MCB) in white blood cells and

lymphocytes. Related to Figure 4 and Supplementary Figure 5.

Supplementary Table 5. Adverse events and safety parameters. Data are number of events

(number of individuals). Related to Supplementary Figure 5.

Supplementary Table 6. Change from baseline in expanded disability severity scales

(EDSS). Related to Supplementary Figure 5

Supplementary Table 7. Correlation analysis between baseline MS-54 scores and EDSS

scores. Related to Supplementary Figure 5.

Supplementary Table 1: Summary of demographics and primary outcome at baseline. Data are mean (SD), number (%) or median (inter quartile range).

Baseline data of secondary outcomes are given in supplementary table 1. *Kruskal Wallis test for comparison between the three groups was performed. Baseline data were available for 48 patients deviations are given in brackets (n=control diet, fasting mimicking diet, ketogenic diet).

Baseline characteristics

Total (n=48)

SD IQR CTRL (n=12)

SD IQR FMD

(n=18) SD IQR

KD Diet (n=18)

SD IQR *p-

value

Age in years 44.8 10.4 50.5 10.4 44.4 11.1 41.3 8.2 0.0505

Gender F/M 38/10

(79/21) 9/3

(75/25) 15/3

(83/17) 14/4

(78/22) 0.848

Expanded disability status score 3 2.0-4 2.5 1.5-4 4 2.4-4 3 2.4-3.5 0.2305

Disease Duration in years 8.9 7.3 9.9 9.2 11 7.7 6.3 4.3 0.0882

Relapse rate 12 months prior study outset 0.4 0.5 0.33 0.65 0.39 0.5 0.44 0.5 0.6849

No immune modulating drugs 11 (23) 3 (25) 2 (11) 6 (33) 0.3

Glatirameracetate 15 (31)

7 (58)

6 (33)

2 (11)

0.025

Interferon beta 1a 9 (19) 1 (8) 6 (33) 2 (11) 0.1

Interferon beta 1b 3 (6)

0

1 (6)

2 (11)

0.5

Fingolimod 4 (8) 0 1 (6) 3 (17) 0.2

Natalizumab 4 (8)

1 (8)

1 (6)

2 (11)

0.8

Intravenous immuneglobulin 2 (4) 0 1 (6) 1 (6) 0.7

BMI 26.7 5.5 27.3 6.9 26 4.8 26.9 5.3 0.9047

Weight Kg 78 17.9 80.2 22.7 74.5 13.9 80 18.4 0.5792

Percent Body Fat 36.6 10.4 38.03 10.57 35.73 9.912 36.49 11.27 0.8568

Primary outcome parameters

Physical Health Composite (n=12,13,13) 67.4 15.2 73.09 8.786 59.61 15.55 69.93 17.19 0.0701

Mental Health Composite (n=12,17,15) 71.1 16.8 75.42 13.56 64.21 19.08 75.47 14.71 0.0925

Physical Function (n=12,18,16) 75.9 25.5 87.9 18.4 67.4 28.81 76.5 23.76 0.0636

Health Perception (n=12,17,17) 54.8 19.9 57.5 16.86 49.9 16.68 57.7 17 0.2738

Energy/Fatigue (n=12,17,17) 46.2 17.3 52.33 19.18 40.47 15.48 47.53 16.79 0.2425

Role Limitations Physical (n=12,18,15) 58.6 37.5 85.42 24.91 42.36 28.16 56.67 44.79 0.0072

Pain 72.7 25 83.2 21.9 59.4 25.3 79 21.7 0.0187

Sexual Function (n=12,14,16) 74.6 27.2 59.68 32.13 79.78 21.36 81.26 25 0.1479

Social Function 79.2 18.8 80.56 16.41 79.39 18.76 78.22 21.05 0.9897

Health Distress (n=12,18,16) 73.3 18.1 75.03 11.91 64.78 22.07 81.68 12.6 0.0619

Overall Quality of Life 67.9 15.6 75.51 13.13 60.11 16.46 70.65 13.28 0.0262

Emotional Well Being (n=12,17,17) 69.5 17.2 71.67 18.25 64.47 19.89 72.94 12.77 0.4105

Role Limitations Emotional (n=12,18,17) 75.2 37.1 80.56 26.44 64.81 44.97 82.36 33.57 0.455

Cognitve Function (n=12,18,16) 70.2 18.2 74.75 13.62 68.4 19.35 68.75 20.16 0.5023

Change in Health 47.9 18.5 50 10.66 41.67 19.17 52.78 20.81 0.2247

Satisfaction with Sexual Function (n=12,16,16) 46.2 31.3 40.28 31.36 44.78 33.74 52.09 29.75 0.5493

Supplementary Table 2

Summary of secondary outcome parameters at baseline. Data are mean (SD). *Kruskal Wallis test for comparison

between the three groups was performed. Baseline data were available for 48 patients deviations are given in brackets

(n=control diet. fasting mimicking diet. ketogenic diet).

Baseline characteristics

Total

(n=48)

SD

IQR

Control

Diet

(n=12)

SD IQR

Fasting

Mimicking

Diet (n=18)

SD

IQR

Ketogenic

Diet (n=18)

SD

IQR *p-value

RR systolic 120.8 15.5 121.3 16.5 125.5 16 115.7 13.3 0.1334

RR diastolic 72.4 9.1 70 13 73.9 6 72.3 9 0.6298

Energy intake in kcal per day

(n=9,12,12) 1686 456 1912 340 1484 413 1718 514 0.1048

Carbohydrate intake per day in

g (n=9,12,12) 179.1 59.8 208.4 70.8 163.5 50.5 172.7 56.4 0.2465

Fat intake per day in g

(n=9,12,12) 68.5 20.9 76.3 16.3 59 17.4 72.1 24.8 0.1305

Protein intake per day in g

(n=9,12,12) 72.6 27.8 79.2 37.4 62.8 22.36 77.31 23.69 0.3136

Beck Depression Inventory

(n=10,15,17) 9.0 6.8 7.6 6.2 11.3 7.7 7.8 6.1 0.2463

Fatigue Severity Scale 4.3 1.7 3.6 1.9 5.2 1.1 3.9 1.8 0.0235

Modified Fatigue Impact Scale

(n=9,16,18) 33.1 17.6 24.1 19.2 38.3 15.7 32.9 17.5 0.2474

Visual Analogue Scale Fatigue

(n=12,16,18) 4.0 2.3 3.6 2.2 4.1 2.5 4.1 2.3 0.7991

Multiple Sclerosis Functional

Composite 3 z-score 0.16 0.69 -0.081 0.7 -0.027 0.89 0.431

Multiple Sclerosis Functional

Composite 2 z-score 0.29 0.74 -0.099 0.64 -0.093 0.88 0.0906

Timed 25 foot Walk Test in sec. 6.0 6.2 5.7 4.7 5.5 2.1 6.6 9.4 0.3019

9-hole peg test in sec. 21.8 7.2 20.8 3.0 21.9 4.700 22.5 8.1 0.8745

Paced Auditory Serial Addition

Test 3 45.1 10.7 48.6 10.1 42.4 11.7 45.5 10 0.2689

Paced Auditory Serial Addition

Test 2 33.0 9.9 39.9 11.5 29.9 8.8 31.4 8 0.0371

ALT - U/l 23.6 14.6 20.7 8 26.9 18.3 22.2 13.9 0.6689

AST - U/l 24.2 8.4 24.5 7.3 25.1 8.6 23.2 9.2 0.6862

y-GT U/l 27.2 38.7 20.9 13.8 36.1 60.2 22.6 16.4 0.804

White Blood Cells count/nl 6.451 2.266 6.097 1.17 6.523 2.742 6.614 2.381 0.8949

Lymphocytes count/nl 1.943 1.028 1.877 0.6592 1.912 1.142 2.018 1.152 0.9287


Mean change from baseline (MCB) in Multiple Sclerosis Quality of Life (MS-54) scores. Comparison for rates of intra- and inter-group change in MS-54

measures. We considered for statistical analysis results of patients with fully completed data sets.*Friedmann test for intra-group gradient. **Mann-Whitney-

U test for inter-group differences. Clinically relevant changes are indicated in bold.

MS-54

Domains

Control Diet

(CD)

Fasting Mimicking

Diet (FMD)

Ketogenic Diet

(KD)

FMD - CD KD - CD

n MCB SD *p n MCB SD *p n MCB SD *p Differences (95%CI) **p Differences (95%CI) **p

Physical

Health

Composite

(PHCS)

Baseline 73.09 8.79 59.61 15.55 <0.05 69.93 17.19 Mean Lower Upper Mean Lower Upper

Month 1 12 1.33 6.05 13 3.44 8.86 13 2.43 8.61 2.11 -4.22 to 8.44 1.09 -5.11 to 7.30

Month 3 12 0.42 8.37 13 7.27 7.52 13 4.93 13.92 6.85 0.28 to 13.42 <0.05 4.51 -5.10 to 14.12

Month 6 12 0.22 11.41 13 4.33 8.81 13 8.37 11.02 4.11 -4.29 to 12.50 8.15 -1.13 to 17.43

Mental

Health

Composite

(MHCS)

Baseline 75.42 13.56 64.21 19.08 75.47 14.71 <0.05

Month 1 12 -0.05 7.03 17 9.65 13.66 15 0.59 9.35 9.69 1.69 to 17.69 <0.05 0.64 -6.07 to 7.35

Month 3 12 1.15 14.38 17 8.85 14.16 15 3.46 14.18 7.70 -3.33 to 18.72 <0.05 2.31 -9.07 to 13.69

Month 6 12 1.88 19.90 17 2.69 15.80 15 6.27 14.84 0.81 -12.8 to 14.42 4.39 -9.37 to 18.15

Physical

Function

Baseline 87.90 18.40 67.40 28.81 76.50 23.76 <0.005

Month 1 12 -1.25 6.44 18 -8.48 15.70 16 -0.87 13.28 -7.23 -15.8 to 1.32 0.38 -8.18 to 8.95

Month 3 12 -7.50 11.77 18 3.72 14.21 16 5.69 12.03 11.22 1.06 to 21.38 <0.05 13.19 3.84 to 22.55 <0.005

Month 6 12 -6.67 14.03 18 4.27 9.79 16 7.26 13.07 10.94 2.05 to 19.83 <0.05 13.92 3.34 to 24.51 <0.005

Health

Perception

Baseline 57.50 16.86 49.90 16.68 57.70 17.00

Month 1 12 3.75 15.24 17 2.42 11.63 17 -2.65 12.55 -1.33 -11.6 to 8.90 -6.40 -17.0 to 4.20

Month 3 12 0.42 13.39 17 0.40 13.36 17 3.97 18.86 -0.02 -10.4 to 10.33 3.55 -9.48 to 16.59

Month 6 12 6.57 13.51 17 0.36 12.53 17 6.62 15.54 -6.20 -16.2 to 3.81 0.05 -11.4 to 11.46

Energy /

Fatigue

Baseline 52.33 19.18 40.47 15.48 <0.05 47.53 16.79 <0.01

Month 1 12 0.00 7.24 17 7.71 15.07 17 3.76 9.54 7.71 -0.97 to 16.39 3.76 -2.63 to 10.16

Month 3 12 0.67 17.55 17 9.65 12.89 17 10.35 18.82 8.98 -2.59 to 20.56 9.69 -4.48 to 23.85

Month 6 12 4.23 23.87 17 1.41 13.78 17 10.82 19.79 -2.81 -17.2 to 11.55 6.60 -10.1 to 23.27

Baseline 85.42 24.91 42.36 28.16 <0.005 56.67 44.79

Role

Limitations

Physical

Month 1 12 -

4.167 23.44 18 0.69 36.50 15 6.67 39.49 4.86 -19.6 to 29.30 10.83 -15.8 to 37.46

Month 3 12 -2.08 29.11 18 28.47 37.33 15 8.33 48.80 30.56 4.34 to 56.77 <0.05 10.42 -22.5 to 43.37

Month 6 12 -

14.58 27.09 18 13.19 32.22 15 15.00 44.12 27.78 4.64 to 50.91 <0.05 29.58 -0.40 to 59.57

Pain

Baseline 83.20 21.90 59.40 25.30 <0.05 79.00 21.70

Month 1 12 3.18 9.68 18 12.68 27.23 18 5.37 19.56 9.51 -4.98 to 24.00 2.20 -10.3 to 14.71

Month 3 12 2.36 18.96 18 16.90 24.27 18 4.27 20.04 14.55 -2.51 to 31.60 <0.05 1.91 -13.1 to 16.89

Month 6 12 -0.84 20.26 18 11.19 20.94 18 6.40 17.81 12.03 -3.75 to 27.82 7.23 -7.1 to 21.60

Sexual

Function

Baseline 59.68 32.13 79.78 21.36 81.26 25.00

Month 1 12 5.63 17.38 14 1.79 10.41 16 -2.43 8.69 -3.83 -15.2 to 7.57 -8.06 -19.7 to 3.59

Month 3 12 8.61 18.39 14 -1.20 12.16 16 8.84 13.76 -9.81 -22.3 to 2.64 0.23 -12.2 to 12.70

Month 6 12 11.85 17.21 14 5.96 19.46 16 0.52 14.73 -5.89 -20.9 to 9.10 -11.3 -23.8 to 1.09

Social

Function

Baseline 80.56 16.41 79.39 18.76 78.22 21.05

Month 1 12 5.56 10.25 18 -2.77 21.15 18 5.11 15.42 -8.33 -20.2 to 3.59 -0.45 -10.8 to 9.95

Month 3 12 4.17 8.35 18 2.55 20.59 18 6.49 20.10 -1.61 -14.5 to 11.27 2.33 -10.3 to 14.93

Month 6 12 1.38 12.74 18 4.41 17.29 18 7.43 12.74 3.03 -8.93 to 14.98 6.05 -3.68 to 15.77

Health

Distress

Baseline 75.03 11.91 64.78 22.07 81.68 12.60

Month 1 12 -0.54 16.76 18 8.84 14.88 16 1.941 12.44 9.38 -2.56 to 21.33 2.27 -8.35 to 12.88

Month 3 12 3.09 15.88 18 5.48 14.45 16 4.662 16.05 2.39 -9.08 to 13.86 1.05 -10.7 to 12.84

Month 6 12 4.15 16.50 18 5.72 22.14 16 6.234 14.94 1.57 -13.8 to 16.93 2.08 -10.2 to 14.34

Overall

Quality of

Life

Baseline 75.51 13.13 60.11 16.46 <0.05 70.65 13.28

Month 1 12 -4.36 13.78 18 7.88 9.98 18 0.80 16.71 12.24 3.37 to 21.11 <0.05 5.16 -6.76 to 17.08

Month 3 12 -1.33 8.37 18 10.87 11.48 18 3.53 14.61 12.20 4.28 to 20.11 <0.005 4.86 -4.71 to 14.43

Month 6 12 -0.86 14.58 18 5.51 12.85 18 5.03 17.47 6.37 -3.98 to 16.71 5.89 -6.62 to 18.41

Emotional

Well-

Being

Baseline 71.67 18.25 64.47 19.89 72.94 12.77

Month 1 12 2.67 7.69 17 11.53 17.94 17 2.59 12.40 8.86 -1.24 to 18.96 -0.08 -8.38 to 8.23

Month 3 12 1.67 15.77 17 7.53 17.54 17 3.18 18.93 5.86 -7.17 to 18.89 1.51 -12.2 to 15.21

Month 6 12 3.25 21.34 17 4.94 13.23 17 5.53 16.49 1.69 -11.5 to 14.85 2.28 -12.1 to 16.68

Role

Limitations

Emotional

Baseline 80.56 26.44 64.81 44.97 82.36 33.57

Month 1 12 0.00 28.43 18 12.97 38.17 17 -7.85 32.34 12.97 -13.5 to 39.43 -7.85 -31.7 to 15.98

Month 3 12 2.78 33.23 18 12.97 34.57 17 3.92 28.58 10.19 -15.8 to 36.19 1.14 -22.5 to 24.79

Month 6 12 2.78 41.33 18 -1.85 44.99 17 -3.93 48.42 -4.63 -37.9 to 28.65 -6.70 -42.0 to 28.62

Cognitive

Function

Baseline 74.75 13.62 68.40 19.35 68.75 20.16

Month 1 12 0.25 7.34 18 2.08 15.46 16 2.73 15.97 1.83 -8.01 to 11.68 2.48 -6.88 to 11.85

Month 3 12 -1.31 12.88 18 5.21 8.64 16 3.25 16.28 6.52 -1.51 to 14.55 4.57 -7.16 to 16.29

Month 6 12 -1.05 15.95 18 2.43 15.77 16 7.03 16.59 3.49 -8.61 to 15.58 8.08 -4.73 to 20.90

Change in

Health

Baseline 50.00 10.66 41.67 19.17 0.01 52.78 20.81 <0.05

Month 1 12 -2.08 16.71 18 12.50 23.09 18 11.11 26.04 14.58 -1.31 to 30.48 <0.05 13.19 -4.24 to 30.63

Month 3 12 -4.17 20.87 18 15.28 22.91 18 16.67 30.92 19.44 2.55 to 36.34 0.01 20.83 -0.09 to 41.76 <0.05

Month 6 12 -2.08 19.82 18 13.89 19.60 18 14.58 29.78 15.97 0.94 to 31.00 <0.05 16.67 -3.43 to 36.76

Satisfaction

with Sexual

Function

Baseline 40.28 31.36 44.78 33.74 52.09 29.75

Month 1 12 -3.47 10.92 16 7.29 21.92 16 0.9375 15.62 10.77 -2.31 to 23.85 4.41 -6.45 to 15.27

Month 3 12 6.93 21.87 16 15.63 31.91 16 0.01 21.09 8.70 -13.4 to 30.76 -6.93 -23.7 to 9.89

Month 6 12 4.15 28.56 16 14.59 30.36 16 3.14 27.37 10.44 -12.8 to 33.68 -1.01 -22.9 to 20.87


Mean Change from Baseline (MCB) in white blood cells and lymphocytes. Comparison for rates of intra- and inter-group change in outcome measures. *

Wilcoxon matched-pairs signed rank test for intra-group comparison. **Mann-Whitney-U test for inter-group differences.

Laboratory

parameters

Control Diet (CD) Fasting Mimicking

Diet (FMD)

Ketogenic Diet (KD) FMD - CD KD - CD

n MCB SD *p n MCB SD *p n MCB SD *p Differences (95%CI) **p Differences (95%CI) **p

White Blood

Cells

count/nl

Baseline 6.10 1.17 6.52 2.74 6.61 2.38 Mean Lower Upper Mean Lower Upper

Month 1 12 0.19 1.22 18 -0.94 2.13 0.08 18 -0.44 1.42 -1.13 -2.53 to 0.27 0.09 -0.63 -1.66 to 0.40

Month 3 12 0.49 1.21 18 -0.18 1.73 18 -0.27 1.95 -0.67 -1.85 to 0.52 -0.76 -2.06 to 0.53

Month 6 12 0.81 1.77 18 -0.48 2.06 18 -0.72 1.75 -1.29 -2.78 to 0.20 0.07 -1.52 -2.86 to -0.18 <0.05

Lymphocytes

count/nl

Baseline 1.88 0.66 1.91 1.14 2.02 1.15

Month 1 12 0.12 0.35 18 -0.30 0.42 <0.05 18 -0.13 0.70 -0.42 -0.72 to -0.11 <0.05 -0.24 -0.69 to 0.21

Month 3 12 0.34 0.47 <0.05 18 0.05 0.50 18 -0.09 0.64 -0.29 -0.66 to 0.08 -0.43 -0.87 to 0.01 <0.05

Month 6 12 0.31 0.51 <0.05 18 -0.19 0.83 18 -0.20 0.60 0.08 -0.50 -1.05 to 0.06 0.07 -0.51 -0.94 to -0.08 <0.01

Supplementary Table 5 Adverse events and safety parameters. Data are number of events (number of individuals). Control Diet (CD), Fasting Mimicking Diet (FMD), Ketogenic Diet (KD).

CD FMD KD

Total adverse events 21 (11) 20 (14) 24 (14)

Respiratory tract infection 13 (9) 8 (7) 13 (12)

Transient reduced gait performance 0 6 (6) 0

Periapical periodontitis 1 (1) 1 (1) 0

Depression 0 1 (1) 0

Diarrhea 3 (3) 0 3 (3)

Dizziness 0 1 (1) 0

Feel cold 0 0 1 (6)

Headache 0 2 (2) 2 (2)

Nausea 0 0 2 (2)

Pain 4 (3) 1 (1) 2 (2)

Liver enzymes exceeding reference range 3fold 0 0 0

Total serious adverse events 1 (1) 3 (3) 2 (2)

Carpal tunnel syndrome 0 1 (1) 0

Ureteric colic 0 0 1 (6)

Lower urinary tract infection 1 (8) 2 (2) 1 (1)

Supplemental Table 6

Change from Baseline in Expanded Disability Severity Scale (EDSS).

Data are median (IQR). Mann-Whitney-U test for comparison between *CD

and FMD or **CD and KD. CD=Control Diet. FMD=Fasting Mimicking Diet.

KD=Ketogenic Diet.

CD FMD KD p* p**

Difference

at month 3 0 (0 to 0) 0 (-1 to 0) 0 (-0.5 to 0) <0.05 <0.05

Difference

at month 6

0 (0 to

0.5) 0 (-0.5 to 0.1)

-0.5 (-1.5 to

0) <0.05 <0.01

Supplemental Table 7

Correlation analysis between baseline MS-54 scores and EDSS score of the 48 RRMS patients.

Deviations are given in brackets.

EDSS

rs p-value

Physical Health Composite (n=39) -0.624 <0,0001

Mental Health Composite (n=45) -0.255 0.09

Physical Function (n=47) -0.821 <0,0001

Health Perception (n=48) -0.242 0.1

Energy/Fatigue (n=47) -0.301 <0,05

Role Limitations Physical (n=46) -0.468 <0.001

Pain (n=48) -0.308 <0,05

Sexual Function (n=43) -0.105 0.5

Social Function (n=48) -0.32 <0,05

Health Distress (n=47) -0.104 0.5

Overall Quality of Life (n=48) -0.279 0.05

Emotional Well-Being (n=47) -0.111 0.5

Role Limitations Emotional (n=47) -0.143 0.3

Cognitive Function (n=47) -0.306 <0,05

Change in Health (n=48) -0.091 0.5

Satifaction with Sexual Function (n=46) -0.209 0.2

Supplementary Experimental Procedures

Fasting mimicking diet (Mouse).

On day 1, mice consume about 50% of their normal caloric intake (7.87 kJ/g). On days 2-3 mice

consume about 10% of their normal caloric intake (1.51 kJ/g). On average, mice consumed 11.07

kJ (plant-based protein 0.75 kJ, carbohydrate 5.32 kJ, fat 5 kJ) on each day of the FMD regimen.

Ketogenic Diet (Mouse).

The ketogenic diet was purchased from BioServ (F3666).

Cuprizone model.

C57BL/6 mice (5-weeks-old-female) were purchased from Charles River. Mice were fed 0.2%

w/w cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma) mixed into a ground standard

rodent chow (Harlan). Curprizone diet was maintained for 5 weeks; thereafter mice were put on

normal chow or FMD cycles (3 days of FMD followed by 4 days of regular chow) for another 5

weeks. 0, 1, 2, 3, 4 and 5 weeks after cuprizone withdrawal animals were euthanized. Brains

were perfused with 4% Paraformaldehyde (PFA), extracted, fixed in 4% PFA, paraffin-

embedded, sectioned and stained as described in the immunohistochemistry section.

EAE clinical disease severity score.

Clinical EAE was graded on a scale of 1-5 by established standard criteria as follows: score 0, no

observable disease; score 1, complete loss of tail tone; score 2, loss of righting; score 3, one hind

limb paralysis; score 4, both hind limbs paralysis; score 5, moribund/dead.

BrdU Injection.

BrdU (Sigma) was prepared in PBS (10 mg/mL stock solution) and intra peritoneal injected

according to experiment schedules. Two different BrdU injection schedules were conducted: For

oligodendrocyte precursor cells in the spinal cord, BrdU was injected 4 times daily (50 mg/kg).

For immune cell proliferation, 4 injections (1 mg/injection) was i.p. injected to mice 48 hours

prior to euthanasia.

In vitro T-cell assay.

Active EAE induced mice were sacrificed on Day 13 and spleens were removed. Blood was

collected for sera. Spleens were teased apart to single cell suspensions, RBC were lysed and

splenocytes were isolated by centrifugation. Cells were suspended in RPMI 1620 (Gibco)

supplemented with 10% fetal bovine serum and counted. Cells were plated at 2 x 105 cells per

well on a 96-well plate and treated with either 20 ug/ml MOG peptide or 10 ng/ml phorbol

myristate acetate (PMA) and 300 ng/ml ionomycin for 48 h to stimulate cytokine production.

Cytokine secretion was blocked during the last 5 h by treatment with monensin. FACS Analysis.

FACS analyses for different immune cell population were performed following standard

protocol. Freshly harvested splenocytes were stained with different immune cell markers (see

supplemental material and methods), followed by standard protocol for AnnexinV, intracellular

or BrdU staining. Analysis was performed with BD FACS diva on LSR II.

Statistical Analysis [Mouse study].

All data are expressed as the mean ± SEM. For mice, all statistical analyses were two-sided and

P values <0.05 were considered significant (* p<0.05, ** p<0.01, *** p<0.001). Differences

among groups were tested either by Student t-test comparison or one-way ANOVA followed by

Bonferroni post-test using GraphPad Prism v.5. Competing risk analysis was performed to assess

statistical differences in the rate of deaths.

Immunohistochemistry, antibodies and quantification.

Spinal cords were isolated from mice following standard protocols. Briefly, spinal cords were

fixed in 4% PFA overnight and stored in 0.5% sodium azide at 4 °C. Sectioning (5 µm) were

performed at the USC Histology Core. For paraffin embedded sections, the spinal cords were

fixed in 4% PFA, dehydrated in sequential concentrations of ethanol, cleared in xylene,

infiltrated, and then embedded with paraffin. Tissue was transversely cut in 9 µm sections.

Sections were de-paraffinized with xylene, and then hydrated with water. Antigen retrieval was

performed by placing sections in 0.1M citric acid pH 6, and boiled for 4 minutes in the

microwave. Slides were allowed to cool, coated with blocking solution (5% horse serum and

0.1% triton-X in PBS) for 1 hour at RT, stained with primary antibodies (See supplementary

material and methods) overnight at 4° C. For fresh frozen sections, the spinal cord sections were

air-dried and fixed in 4% PFA for 15 min, and washed with PBS containing 0.5% Tween-20.

Sections were permeabilized in 1% NP40 in PBS for 15 min, washed and blocked in 5% donkey

serum in 0.4% Triton for 1 hour, and incubated with various primary antibodies (See

supplementary material and methods) and incubated overnight in 4° C. Following day, sections

were washed, stained with secondary antibodies. Sections were washed with PBS, and covered

with Fluorshield Mounting Medium with DAPI (Vector Labs). Apoptotic cells were detected

using In Situ Cell Death Kit (Roche) following the manufacturer’s protocol. Images were

acquired and analyzed using a Nikon Eclipse 90i fluorescent and bright field microscope, along

with Metamorph 7.7 software. Images from the same spinal cord sections were “stitched” using

ImageJ Fiji. A minimum of 8 stitched spinal cord sections was quantified using ImageJ

(National Institute of Health). Raw images were digitally contrasted in the same way for each

stain within each time point. The digitally contrasted images were duplicated, and the region of

interest (ROI) was manually drawn on the image. Each image was thresholded equally for each

stain, and the region statistics tool was used to calculate the number of positive fluorescent pixels

located within the ROI. Antibodies used for Spinal cord immunostainings: anti-SMI32 (abcam,

ab28029, 1:1000), rabbit anti-MBP (zymed, 18-0038, 1:200), goat anti-GSTπ (Abcam, ab53943,

1:200), rabbit anti-NG2 (Millipore, AB5320, 1:100), rat anti-BrdU (Serotec, MCA2060, 1:200),

rat anti-CD4 (eBioscience, 14-0041-86, 1:100), rat anti-CD8 (abcam, ab22378, 1:100) and rat

anti-CD11b (Serotec, MCA711G, 1:200). Secondary antibodies include donkey anti-mouse

Alexafluor 647 (abcam, ab150103, 1:500) and donkey anti-rabbit Alexafluor 488 (abcam,

ab150069, 1:500).

Antibodies used for FACS analysis: CD3 Alexa700 (ebioscinece), CD4 PE-cy5 (ebioscinece),

CD44 APC (ebioscience), CD62L PE-cy7 (ebioscience), CD8 Alexa488 (ebioscience), CD11C

PE-cy7 (ebioscience), F4/80 PE-cy5 (ebioscience), CD25 PE-cy7 (ebioscience), FoxP3

Alexa488 (ebioscience), IL17 PE-cy7 (ebioscience) and IFNγ APC (ebioscience).

Demyelination Scoring.

Myelin was stained, using 9 μm sections, with solochrome cyanine as previously described

(Kiernan, 1984). Briefly, sections were stained for 90 minutes with Eriochrome Cyanine R

(Sigma; St. Louis, MO). Sections were washed in tap water, and then differentiated for 30 s in

10% iron (III) chloride (Sigma). Next, sections were counterstained with Van Gieson's stain for 2

min, washed, dehydrated in sequential concentrations of ethanol, cleared in xylene, and cover

slipped. Stained tissue was manually scored for the amount of demyelination on a scale from 0-5

(0=no demyelination, 1=one region of subpial white matter affected, 2=multiple regions of

subpial white matter affected, 3=multiple regions of white matter affected beyond subpial region,

4=parenchymal region affected, less than half of total white matter, 5=parenchymal region

affected, more than half of total white matter).

H&E Scoring.

Stained tissue was manually scored for the amount of inflammatory infiltration on a scale from

0-5 (0=no infiltration, 1=a few infiltrates in the leptomeninges, 2=organization of infiltrates

around blood vessels, 3=extensive perivascular cuffing with extension into the underlying

parenchyma, 4=infiltration in the parenchymal region, less than half of total white matter,

5=infiltration in the parenchymal region, more than half of total white matter).

Serum physiological biomarkers and cytokines.

Prior to blood collection, mice were withheld food for up to 4 hours. Serum was stored at -80°C.

β-hydroxybutyrate was measured with a colorimetric assay kit following the manufacturer’s

protocol (#700190, Cayman Chemical). IGF-1 (R&D), TNF-α (R&D), IFN-γ (R&D), and IL-17

(R&D) was measured following the manufacturer’s protocol.

Detailed Procedures for Clinical Trial.

Patients / Inclusion and Exclusion Criteria.

Patients were randomly allocated to I) KD for 6 months or II) 7 days FMD followed by

Mediterranean diet for 6 months or III) CD for 6 months. All patients met the following criteria:

Age > 18 < 67 years, stable disease modifying therapy (DMT) for at least six months prior to

inclusion, no DMT for at least six months or naive to therapy, EDSS < 6.5 and BMI > 18 < 45.

Exclusion criteria were primary or secondary progressive forms of MS, clinically relevant heart,

lung, liver, and kidney diseases, pregnancy or breast-feeding, other neurologic disorders, cancer,

weight loss therapy in the month prior to screening, relapse or steroid pulse therapy < 30 days prior

to screening, diabetes or other metabolic defects, bulimia, anorexia and drug abuse

Study Settings.

The study was approved by the local ethics committee and all participants gave informed written

consent according to the 1964 Declaration of Helsinki. RRMS patients fulfilling the current panel

criteria were prospectively recruited from our neurology outpatient clinic and via public

information all over Germany (Polman et al., 2005)

Interventions.

HRQOL in RRMS patients who met the inclusion criteria (n=60) were randomly assigned to three

study dietary interventions (n=20 per group): control diet (normal caloric standard diet), fasting

mimicking diet (FMD), and KD. To evaluate the food intake, a 115 item dietary self-record with

additional gaps for unlisted and individual foods or liquids and quantities prospectively over a

period of 7 days before baseline and between all other visits were recorded (Optidiet software

Version 5.1 GOE mbH, Büro Linden, Linden, Germany).

Modified human fasting mimicking diet.

A single cycle of the modified fasting mimicking diet consist of Day 1 – pre-fasting followed by

Day 2-8 – very low calorie diet. Day 1-prefasting consists of an 800 kcal (about 40% of normal

caloric intake similar to mouse Day1 FMD) monodiet (fruit, rice, or potatoes) by preference of

individuals. On the following day patients were recommended to use an oral laxative, Natrium

Sulfuricum (20-40 g). FMD consisted of 100 ml vegetable broth or vegetable juice with 1

tablespoon of linseed oil 3 times daily, plus additional calorie-free liquids. The daily calorie intake

was predefined with 200 – 350 kcal (10-18% of normal caloric intake similar to mouse Day 2-3

FMD). Patients were advised to drink 2-3 L of unsweetened fluids each day (water, and herbal

teas) and to use an enema if tolerated. After the 7-day fasting period solid foods were stepwise

reintroduced for three days, starting with a steamed apple at day 8. After the fasting and refeeding

period a normocaloric, plant-based Mediterranean diet was maintained until study end.

Low Glycemic Load Ketogenic Diet (KD).

Patients received KD for 6 months. Patients were recommended an average daily intake of < 50g

carbohydrates, > 160g fat and < 100g protein intake daily. Patients received detailed information

about nutritional facts, glycemic load and learned how to handle carbohydrates by an experienced

nutritional coach during group based workshops on 3 weekends. Details of the study specific KD

will be analyzed and published separately.

Control Diet.

Patients on CD met the criteria of a regular diet in German population as described in the “National

Nutrition Survey II” (http://www.was-esse-ich.de/index.php?id=44). We advised patients to stay

on their regular diet.

Assessments and Outcome Measures.

All participants were assessed for HRQOL at baseline, next visit occurred between days 3 to 17

after study outset (All FMD group members were assessed before breaking the FMD at day 8),

another visit was set after 3 months and the last regular visit proceeded after 6 months.

Primary Outcome Measures.

To measure HRQOL we used the generic and disease specific Multiple Sclerosis Quality of Life-

54 (MS-54) questionnaire consisting of 54 items. 2 composite scores concealing physical (PHCS)

and mental health (MHCS) are calculated from 12 directly obtained scales: Physical Health, Health

Perception, Energy/Fatigue, Role Limitations Physical, Pain, Sexual Function, Social Function,

Health Distress, Overall Quality of Life, Emotional Well Being, Role Limitations Emotional,

Cognitive Function. Additionally the MS-54 includes 2 single item scales: Change in Health and

Satisfaction with sexual functions. This instrument is validated for the German population, has

shown good reliability and validity within MS patients. We observed in our trial that patients with

a baseline EDSS score of > 2 < 3 compared with those in patients with baseline scores < 2 showed

> 5 points lower mean PHCS and MHCS scores per 0.5 EDSS incline. Thus we defined a change

of > 5 points to be clinically meaningful, which is in accordance with other studies (Rudick et al.,

2007, Michalsen and Li, 2013, Kappos et al., 2014)

Anthropometric Measurements.

Weight, fat and lean mass was specified by Air-Displacement Plethysmography (Bod Pod, Life

Measurements, Inc. Concord, CA), BMI was calculated as weight (kg)/height²(m). Seated blood

pressure was taken twice on each arm. All data were collected by trained study personnel only.

Depression.

The Beck depression inventory (BDI) was administered at each visit.

Fatigue.

Fatigue was measured with modified fatigue impact scale (MFIS), fatigue severity scale (FSS) and

visual analogue scale fatigue (VASF) at every visit.

Neurological Disability.

At baseline, month 3 and month 6, the EDSS(Kurtzke, 1983) and the Multiple Sclerosis Functional

Composite MSFC(Cutter et al., 1999) were performed to assess neurological disability. The

examiner was not blinded to treatment allocation.

Safety and other laboratory parameters.

Prior to blood collection all patients were on an overnight fast and samples were always taken at

the same time + 1h. Aspartate transaminase (AST), alanine transaminase (ALT), gamma-

glutamyltransferase (GT), WBCs, and lymphocytes were analyzed referring to international

standards at all visits. Other laboratory parameters as described in clinicaltrials.gov will be

analyzed and published separately.

Statistical Analysis [Human study].

Due to the small sample size of the pilot study, sufficiently powerful statistical analysis was not

possible. Baseline characteristics of the three intervention groups were compared using Kruskal-

Wallis tesw to determine uniformity of subjects. The primary end point, MS-54, and relevant

secondary endpoints were analyzed using non parametric Friedmann’s test for comparing intra

group differences at 3 or 4 time levels or Wilcoxon matched-pairs signed rank test at 2 time levels.

Next we performed baseline correction to increase the comparability between the groups. Thus we

calculated within each group the mean change from baseline (MCB) differences for every visit

and in a further step we compared MCB scores between all three groups using non parametric

Kruskal-Wallis test followed by Mann-Whitney-U test for pair wise comparisons. Associations

between variables were assessed using Spearman’s rank correlation coefficient (rs). All graphs are

based on mean and standard error of the mean (SEM) data. Because of the exploratory character

of the study analysis, alpha-adjusting was not performed. The test level for statistical significance

of differences between and within the treatment groups was defined as p = 0.05 (two-sided) for all

tests. For statistical analyses the following software was used: SPSS, version 20 (IBM, Armonk,

New York, US) and Graph Pad Prism, Version 5.0 (GraphPad Software, CA, US)

Supplemental References

Cutter GR, Baier ML, Rudick RA, Cookfair DL, Fischer JS, Petkau J, Syndulko K, Weinshenker BG, Antel JP, Confavreux C, Ellison GW, Lublin F, Miller AE, Rao SM, Reingold S, Thompson A, Willoughby E (1999) Development of a multiple sclerosis functional composite as a clinical trial outcome measure. Brain : a journal of neurology 122 ( Pt 5):871-882.

Kappos L, Gold R, Arnold DL, Bar-Or A, Giovannoni G, Selmaj K, Sarda SP, Agarwal S, Zhang A, Sheikh SI, Seidman E, Dawson KT (2014) Quality of life outcomes with BG-12 (dimethyl fumarate) in patients with relapsing-remitting multiple sclerosis: the DEFINE study. Multiple sclerosis 20:243-252.

Kurtzke JF (1983) Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33:1444-1452.

Michalsen A, Li C (2013) Fasting therapy for treating and preventing disease - current state of evidence. Forschende Komplementarmedizin 20:444-453.

Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, Lublin FD, Metz LM, McFarland HF, O'Connor PW, Sandberg-Wollheim M, Thompson AJ, Weinshenker BG, Wolinsky JS (2005) Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria". Annals of neurology 58:840-846.

Rudick RA, Miller D, Hass S, Hutchinson M, Calabresi PA, Confavreux C, Galetta SL, Giovannoni G, Havrdova E, Kappos L, Lublin FD, Miller DH, O'Connor PW, Phillips JT, Polman CH, Radue EW, Stuart WH, Wajgt A, Weinstock-Guttman B, Wynn DR, Lynn F, Panzara MA, Affirm, Investigators S (2007) Health-related quality of life in multiple sclerosis: effects of natalizumab. Annals of neurology 62:335-346.

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