Risk factors in Multiple Sclerosis: Detection and Treatment in Daily Life

Risk factors in Mul0ple Sclerosis: Detec0on and Treatment in Daily Life

Caroline Pot Patrice Lalive

Unit of Neuroimmunology and Mul0ple Sclerosis

Geneva University Hospital

16th State of the Art Lucerne, January 11th, 2014

Workshop C

Alimenta0on

Socioeconomic level

Age

Industrialisa0on

Climate

Gene0c Predisposi0on

Race

Geography

Hygiene

Virus

Mul$ple sclerosis

Mul0ple Sclerosis

Mul0factorial origin?

Mul0ple Sclerosis

Mul0ple Sclerosis

•  Environmental risk factors: -> Who is guilty & which level of demonstration ? -> Can we have an impact on it ? -> Counseling for the patient and the family -> Future research direction with high potential

Mul0ple Sclerosis

-> How do you manage “Environmental risk factors” with patients ?

-  Give advices (only if asked) ? -  Blood test ? -  Vit. D ? -  Diet ? -  Smoking ? -  Follow specific protocol?

- > Do you believe this can have an impact on the disease course or the risk of MS?

Mul0ple Sclerosis : Risk factors

1.  Environmental climatic factors and Vitamin D 2.  Smoking 3.  Diet

Obesity Salt intake

4. Gut immunology Parasite infection Gut flora

5. Conclusion

Mul0ple Sclerosis : mul0factorial disease

Gene$c

Twin studies: Monozygo0c twin: 20%

MHC-‐suscep0bility: HLA-‐DRB1 locus non-‐MHC suscep0bility genes:

(IL2RA, IL7R, IRF8, and TNFRSF1A)

Environment

Pathogens (viral exposi0on)

Vitamin (D) Smoking Diet

Commensal flora

Epigene$c

MS

Introduc0on

World Distribu0on of Mul0ple Sclerosis

Introduction

hence limiting ascertainment bias. Treatment costs forchronic diseases such as MS are fully reimbursed and mostcases are captured soon after diagnosis.15

Solar radiation values. For this study, we focused specificallyon UVB, which includes wavelengths of 280 to 320 nm. UVBdata for each MSA region in France were compiled for monthlymeans and annual mean of daily UVB irradiation. The annualmean is an average of monthly values over a 12-month period.Mean monthly values from December to March were used tocalculate the winter mean UVB.

UV data were collected from the Internet-based Solar Radia-tion Database (SoDa) service, a Web site of the MINES Paris-Tech (www.soda-is.com18). This Web site provides a range ofdatabases, algorithms, and time series services for collecting in-formation on solar radiation and associated climate features.19

These databases are produced by the processing of satelliteimages, largely from the Meteosat series of satellites.20,21 UVBis measured by a biometer for solar light which assesses globalirradiance (290 –320 nm) with a measurement error of lessthan 5%.20

The services we employed for the current study are accessedthrough the SoDa Web site, under the “climate normals typical

values” heading. The data used are standard monthly means ofdaily sums for UVB radiation. Model input criteria selected forlatitude and longitude were based on prefecture cities of the 22MSA regions in France. Default model input variables providedby the service were used for ozone levels, altitude, and inclina-tion. The “all sky” UVB model was selected over “clear sky” asthe latter provides the maximum value, whereas the former pro-vides an average value. Output data for global irradiation wereused for analysis as they comprise both diffuse and direct radia-tion. Verification of the models used and a review of the serviceshave been previously published.20,22

A map of France illustrating the variation in intensity of totalirradiation was produced from data available through SoDa ser-vices.18 The quantities illustrated are the annual irradiation(kWh/m2) calculated from 2004. The irradiation data are com-puted from satellite observations which calculate quantity of en-ergy that reaches the ground in Europe (DLR, University ofOldenburg, MINES ParisTech, 2005).

Data analyses. Pearson correlation was used to examine therelationship of overall prevalence rates and annual UVB perMSA region. This was also completed for average winter UVB.Both annual and winter UVB values were then analyzed sepa-rately in males and females. We employed Pearson correlation toassess the association of latitude with MS prevalence, and withmean UVB.

Sex ratio was calculated by dividing the number of femalepatients with MS per 100,000 MSA affiliates by the numberof male cases per 100,000 MSA affiliates for each region.Given that ratios have infinite variance and are problematicvariables to model, they were not used in regression analysis.A linear model with prevalence rate predicted by UVB, sex,and UVB-sex interaction was employed to examine genderdifferences in MS prevalence.

Data were analyzed and graphs produced in SPSS 16.0 (Chi-cago23) and regression modeling was done using R statisticalpackage (Vienna24).

RESULTS Characteristics of dataset. The populationsize, prevalence rates, sex ratio, and latitude for eachof the 22 MSA regions in France are given in table 1.For each region, typical values of solar UVB areshown for annual mean (monthly values averagedover a 12-month period) and winter mean (averageof December to March monthly values, table 1). MSprevalence rates increased with increasing latitudeand decreasing UVB values. Latitude was stronglycorrelated with prevalence rates (r ! 0.78, p "0.001) and with annual global UVB (r ! #0.96,p " 0.001) in the 22 regions of France we studied.The correlation of MS prevalence with longitude wasnot significant (data not shown).

Prevalence, gender, and UVB. Descriptively, it can beseen that overall prevalence rates for MS are relatedto the intensity of solar UV radiation in the corre-sponding regional districts of France (figure 1). MSprevalence was strongly associated with annual globalUVB (r ! #0.80, p " 10#5) and winter mean (r !#0.87, p " 10#6; table 2).

Figure 1 Annual mean ultraviolet B (Wh/m2) radiation in France

Multiple sclerosis prevalence rates (per 100,000) for each Mutualite Sociale Agricole re-gion are shown.

Table 2 Association of regional age-standardized MS prevalence rates withannual and winter means of daily UVB irradiation

MSprevalence

UVB annual mean UVB winterb mean

Correlationcoefficient p Valuea

Correlationcoefficient p Valuea

Total #0.80 "0.001 #0.87 "0.001

Female #0.76 "0.001 #0.80 "0.001

Male #0.46 0.032 #0.56 0.006

Abbreviations: MS ! multiple sclerosis; UVB ! ultraviolet B.a Threshold for significance was set at p " 0.05.b Winter: December to March average.

Neurology 76 February 1, 2011 427

Neurology. 2011 Feb 1;76(5):425-31. 1. Environmental risk factor

MS prevalence rates (per 100 000 for each region of France)

Environmental clima0c risk factors for MS

mechanism is favoured by some authors, who suggest that vitamin

D positively influences the activity of regulatory T lymphocyte

cells, restoring a better ratio between the lymphocytes Th2

(protective) and Th1 (aggressive); the overall effect being a

decrease in inflammation (Cantorna, 2006, 2008; Smolders

et al., 2008a). It should be noted that this mechanism is analogous

to the mechanism of interferon-b, used as an immunomodulator in

multiple sclerosis therapy, and that a potentiation exists between

the beneficial effects of interferon-b and 1,25(OH)2D analogue

used together in EAE (Van Etten et al., 2007). However, it may

be that the effects of 1,25(OH)2D in EAE result from other

mechanisms.

Possible specific effect of UVB,independent of vitamin DIt has recently been reported that UVB itself may also have a

beneficial effect in EAE that could be independent of the

25(OH)D serum level and vitamin D mechanism, the authors

suggesting that this immunological UVB effect could account for

the assumed immunological effect of vitamin D previously

reported in EAE as well as in multiple sclerosis (Becklund et al.,

2010). However, this as yet unique study will need additional

confirmation since a transitory significant increase in the

25(OH)D serum level was nevertheless observed in the mice trea-

ted with UVB. Furthermore, UVB could have produced

1,25(OH)2D directly in the mouse skin (Lehman et al., 2001;

Reichrath, 2007), this finally resulting, via the draining lymph

nodes and the general immune system, in a general positive im-

munosuppressive effect (Gorman et al., 2007; Loser and Beissert,

2009) whatever the 25(OH)D serum level. Lastly, a possible spe-

cific action of UVB does not exclude a parallel immunological

effect of 1,25(OH)2D in EAE, an effect that has previously been

shown in many different studies in which UVB did not play any

role (see above and Niino et al., 2008).

Epidemiological findings

Effect of latitude on the risk of multiplesclerosisThe effect of latitude on the risk of multiple sclerosis has long

been known and is universally acknowledged, the prevalence of

the disease being minimal at the equator and increasing with

either North or South latitude (Handel et al., 2010) (Fig. 3).

This effect is observed on a world scale (Gale and Martyn,

1995; Alonso and Hernan, 2008; Sloka et al., 2009), at a contin-

ental level (Kurtzke, 1995; Puggliatti et al., 2006), in large coun-

tries, such as the USA (Kurtzke et al., 1985, Kurtzke, 2008), the

former Soviet Union (Boiko et al., 1995) and Australia (Van der

Mei et al., 2001; Taylor et al., 2010) and even in comparatively

Figure 3 Environmental climatic risk factors for multiple sclerosis and links between them. The r- and P-values illustrate the example ofFrance and correspond to the Pearson correlation tests reviewed in this article or performed by the authors, based on data for Frenchregions concerning (A) mean latitude, (B) mean global annual sunshine (Suri et al., 2007), (C) mean serum level of vitamin D in normaladults (Chapuy et al., 1996) and (D) multiple sclerosis prevalence in French farmers (Vukusic et al., 2007); r and P in black = data from22 regions; r and P in red = data from nine regions. Modified from Pierrot-Deseilligny (2009).

Hypovitaminosis D in multiple sclerosis Brain 2010: 133; 1869–1888 | 1875

at Institut universitaire de hautes etudesinternationales - Bibliotheque on D

ecember 18, 2013

http://brain.oxfordjournals.org/D

ownloaded from

Brain 2010: 133; 1869-1888

Environmental clima0c risk factors for MS

1. Environmental risk factor and Vitamin D

Mul0ple Sclerosis

Vitamin D ?

Vitamin D and Multiple Sclerosis: risk of developing MS

Neurology 2004 Jan 13, vol. 62 (1) 60-65

Prospective study Nurses’ Health Study

Women who took vitamin D ( ≥400 interna0onal units/day)

had a 40% lower risk of MS than women who did not use vitamin D supplements.

(rela0ve risk 0.59, 95% CI 0.38-‐0.91)

JAMA. 2006;296(23):2832-2838.

Prospective, nested case-control study among more than 7 million US military personnel

1. Vitamin D

Genome Wide Association Studies (GWAS)

Nature. 2011 Aug 10;476(7359):214-9

cyp27B1

cyp24A1

1. Vitamin D

Genome Wide Association Studies (GWAS)

1. Vitamin D

Ann Neurol 2010(68):193-203. Neurology. 2012 Jul 17;79(3):261-6.

August and nadirs in mid February. The fitted sinu-soidal curve resulted in a geometric mean 25-OH-Dconcentration of 69 nmol/L. There was a consider-able variation in mean levels between patients (coef-ficient of variation 41%).

Association between serum 25-OH-D concentrationsand exacerbation risk. Exacerbation rates were foundto decrease with increasing levels of serum 25-OH-Dconcentrations (figure 2A). For the low (!50 nmol/L), medium (50 –100 nmol/L), and high ("100nmol/L) category the monthly exacerbation rateswere 0.15 (95% confidence interval [CI] 0.12–0.20), 0.10 (95% CI 0.08–0.14), and 0.07 (95% CI0.05–0.12), respectively. The risk of an exacerbation

was significantly increased in the group with low se-rum 25-OH-D concentrations (!50 nmol/L) com-pared to the group with high serum concentrations("100 nmol/L). Rate ratios for the low and mediumgroup were 2.0 and 1.4, respectively (p for trend #0.007).

In univariate analysis it was also found that infec-tions were associated with the risk of an exacerbation.The exacerbation rate within an at-risk period was2.1-fold increased (95% CI 1.6–2.8, p ! 0.001, fig-ure 2B). Simultaneous evaluation of categories of lev-els of serum 25-OH-vitamin and infections showedthat both factors were related to the exacerbation rate(table 2). Also the effect of one factor did not dependon the other (interaction: p # 0.18).

Other characteristics (gender, age, EDSS, use ofinterferon-!, and number of exacerbations in the2-year period before entry into the study) were notsignificantly associated with the exacerbation rates.This applied in univariate (all p " 0.18) as well asmultivariable analysis (all p " 0.17). In particular,the effect of vitamin D on exacerbations was notmodified by interferon use (p # 0.78 for the interac-tion effect).

No significant differences among the 4 seasonswere found regarding exacerbation rates, in univari-ate, or in multivariate analysis.

Analyzing logarithmically transformed serum 25-OH-D concentrations on a continuous scale showedthat a doubling of serum 25-OH-D concentrationscaused a decrease of the exacerbation rate by 27%(95% CI 8%–42%, p # 0.008) (adjusted for theeffect of infections). Adding quadratic and cubicterms of the linear predictor, i.e., log(serum concen-tration), did not significantly improve the fit of themodel, indicating the linearity of the association andthe absence of a threshold.

Analyzing the 9 dropouts it was found that thedropout rate did not significantly correlate withthe cumulative number of exacerbations duringthe study (Cox regression: p # 0.29), nor with anybaseline characteristic.

DISCUSSION In the present study we show thatlower 25-OH-D levels are significantly associatedwith a higher exacerbation risk in patients withrelapsing-remitting MS. In the category of low 25-OH-D levels, the risk for an exacerbation was 2 timeshigher than in the category of high levels. This asso-ciation was log linear without a threshold effect; adoubling of serum 25-OH-D concentrations low-ered the exacerbation risk by 27%. Adjustment forpotential confounders, including infection, gender,disability (EDSS), and use of immunomodulatorytherapy, did not alter this association. In particular,

Figure 2 Monthly exacerbation rates for the different groups

(A) Monthly exacerbation rates for the different groups of serum 25-hydroxy-vitamin D(25-OH-D) concentrations, and (B) for at-risk period for infections. Error bars denote95% confidence intervals. Serum 25-OH-D concentrations: p (trend) # 0.007; infec-tions p ! 0.001.

Table 2 Association between exacerbation rate and serum 25-OH-Dconcentrations and infection according to multivariable analysis

Relative exacerbationrate

95% Confidenceinterval p Value

ARP infection

Yes 2.3 1.7–3.1 !0.001

No 1 (reference) —

Vitamin D level

Low (<50 nmol/L) 1.9 1.1–3.2 0.013a

Medium (50–100 nmol/L) 1.4 0.8–2.2 0.235a

High (>100 nmol/L) 1 (reference) —

Abbreviation: 25-OH-D # 25-hydroxy-vitamin D; ARP infection # at risk period for infection.a p Value for trend: 0.012.

264 Neurology 79 July 17, 2012

Vitamin D: risk factor for disease progression

1. Vitamin D

Vitamin D levels and Brain MRI

Longitudinal cohort study of 469 subjects with MS Vitamin D levels are inversely associated with the risk of new lesion

Ann Neurol. 2012;72(2):234. 1. Vitamin D

What do we measure?

Vitamin D in practice in Geneva University Hospital

25(OH)D (calcidiol) in the serum

Optimal ≥ 75 nmol/L Sub-optimal: ≥50 nmol/L et < 75 nmol/L Moderate deficit: >25 nmol/L et <50nmol/L Severe deficit : < 25 nmol/l

1. Vitamin D

Vitamin D in practice in Geneva University Hospital

1.  Before starting a substitution exclude a: Hypercalcemia Subclinical hyperparathyroidism Measure : Calcium and PTH

2. If PTH and calcium normal but 25(OH)D decreased Severe deficit : < 25 nmol/l

Cholecalciférol 300 000UI per os (vitamine D3 Streuli) then 1000 IU cholecalciferol/jour per os: Vi-De3

Moderate deficit: >25 nmol/L et <50nmol/L Sub-optimal: ≥50 nmol/L et < 75 nmol/L

1000 IU cholecalciferol/day per os (Vi-De3)

1. Vitamin D

Mul0ple Sclerosis

Smoking?

Smoking and Multiple Sclerosis: risk of developing MS

2. Smoking

Cross-sectional study 22312 subjects in the general population of Hordaland County in Norway

years. Exclusion of these cases from the analyses did notchange the results.

The RR estimated by the Cox model comparing ever-smokers with never-smokers was 1.81 (p ! 0.014) (table).The RR was 2.75 for men and 1.61 for women. An analysisexcluding the patients who started to smoke less than 4years prior to the onset of disease gave an RR of 1.74 (p !0.024). Further, an analysis including educational levelgave an RR of 1.75 (p ! 0.023). The RR was significantlyincreased also for myocardial infarction, angina, andasthma.

Discussion. This study of a large general popula-tion found that the risk of developing MS amongindividuals who smoked was nearly twice as high asin never-smokers. Taken together with the signifi-cantly increased risk of MS among smokers found inthe recent prospective study of female nurses in theUnited States2 and the similar (albeit nonsignificant)increases found in two prospective studies in theUnited Kingdom,4,5 this result strongly suggests thatcigarette smoking is a risk factor for MS. The resultsin the current study showed that the excess riskamong men who smoke is at least as high as thatamong women who smoke. These findings add MS tothe list of diseases, including various types of cancer,cardiovascular diseases, and rheumatoid arthritis,for which tobacco smoking represents a risk factor.

The diagnosis of MS for the patients in this studywas based on self-report. Nevertheless, patients whohave been diagnosed as having MS are well aware oftheir diagnosis, and individuals who have not beengiven this diagnosis will probably not report havingMS. A large study of female nurses in the UnitedStates found that as many as 93% of the respondentswho reported having MS were confirmed by hospitalfiles.2 A similar validity of the disease status is ex-pected in the current study population, which repre-sents a high-risk area that has been extensivelystudied and where the community is familiar withthe disease.6-8 The age-specific prevalence rate, in-cluding patients with neurologist-based diagnosisonly, in a study of the same county in 1994 was 338

per 100,000 people 40 to 49 years old.8 This isslightly lower than the rate found in the currentstudy, assuming the same frequency of nonresponseamong patients with MS as the rest of the popula-tion (390 per 100,000). This could reflect a smallincrease in the prevalence rate during these 4 yearsor that the patients with MS in the current studyhad a slightly higher response rate than did the totalstudy population. In any case, any misclassificationof disease status introduced by self-report is notlikely to have resulted in the increased risk of MSfound among smokers. Further, there is little reasonto believe that patients with MS who smoke wouldbe more likely to participate in the study comparedwith subjects without MS who smoke.

The validity of the smoking data was supported bya clear relationship with other smoking-related dis-eases such as myocardial infarction and angina pec-toris. Further, because the questions on smokingwere included in a large questionnaire with manyquestions and a possible relationship between smok-ing and MS is not well known, there is little reasonto believe that the patients with MS would reporttheir smoking history differently from the rest of thestudy population. Interpreting the level of educationas an indirect measure of socioeconomic status, theresults indicated that smoking was not a confound-ing factor for social-economic status.

Several biologic models could explain the in-creased risk of MS among smokers. These includeeffects of smoking on the immune system, direct ef-fects of smoking on the blood–brain barrier, andtoxic effects of smoking on the CNS.2 The relevanceof these mechanisms and the role of specific compo-nents of cigarette smoke such as nicotine or cyanidecould be explored in experimental animal studies.

References1. Compston DAS. Exogenous factors and multiple sclerosis. In: McAlpine’s

multiple sclerosis. 3rd ed. London: Churchill Livingstone, 1998.2. Hernan MA, Olek MJ, Ascherio A. Cigarette smoking and incidence of

multiple sclerosis. Am J Epidemiol 2001;154:69–74.

Table The number of smokers and risk estimate (rate ratio) for six common diseases among 22,312 subjects in the general population ofHordaland County, Norway

Disease No. of patients* Never smoker, n (%) Current or past smoker, n (%) Ratio ratio† (95% CI)

Multiple sclerosis 86 21 (24.4) 65 (75.6) 1.81 (1.13–2.92)

Myocardial infarction 76 9 (11.8) 67 (88.2) 4.53 (2.26–9.01)

Angina pectoris 108 17 (15.7) 91 (84.3) 3.30 (1.96–5.55)

Stroke 93 27 (29.0) 66 (71.0) 1.48 (0.94–2.35)

Asthma 1,350 446 (33.0) 904 (67.0) 1.21 (1.05–1.39)

Diabetes 216 85 (39.4) 131 (60.6) 0.86 (0.65–1.13)

Total population 22,240 8,239 (37.0) 7,892 (35.5) 1.00

* Information on smoking was missing for 72 individuals including one patient with multiple sclerosis.† Rate ratio estimated in a Cox proportional hazard regression model with smoking as a time-dependent covariate. Smoking individuals

are being compared with nonsmoking individuals at the same age for the risk of developing the disease. All analyses were performedstratified by sex.

October (2 of 2) 2003 NEUROLOGY 61 1123

Neurology 2003; 61: 1122-1124

Smoking and risk of MS: popula0on-‐based studies

anese17 but not in Canadian18 patients. Ongoingwork associating vitamin D exposure, ultraviolet ir-radiation, and MS is under way in Australia, as isresearch examining dietary vitamin D supplementa-tion as a possible preventive treatment strategy forthe disease.

An association between solvent exposure and MShas been reported in some studies, but more re-cently, no increase in MS incidence was found follow-ing organic solvent exposure.19,20 The risk ofdeveloping MS is not established to be affected byother exogenous factors such as physical trauma,vaccinations, or stressful life events.

The environmental factors of most interest to peo-ple with MS are modifiable factors that may triggerdisease exacerbations. The role of environmental fac-tors in the prognosis of MS has been largely limitedto investigations of viral infections and physicaltrauma as triggers of disease exacerbations. Prospec-tive studies among patients with MS have consis-tently shown a relationship between viral infectionsand exacerbations of MS, including a recent studyshowing a twofold increased risk of clinical relapsesfollowing symptomatic upper respiratory tract infec-tions and a 3.4-fold increased risk of exacerbationamong those with raised antiviral antibody titres.21

Despite speculation, credible epidemiologic studieshave not shown that physical trauma is a diseasetrigger.22

The quality of life for persons with MS and otherchronic diseases is dependent on their perceived abil-ity to maintain autonomy and control.23 Unprovenexpensive (removal of mercury amalgam), potentiallyharmful (e.g., some herbs, large doses of vitamins Aor D), or burdensome (gluten-free diet) treatmentsshould be avoided. Smoking, on the other hand, hasbeen associated with transient symptomatic worsen-ing,24 and randomized trial data have suggested abeneficial effect of polyunsaturated fatty acid intakeon rate of MS exacerbations.25,26 Smoking cessationand eating a diet low in animal fat and rich in poly-

unsaturated fatty acids and vitamin D foods may beencouraged.

AcknowledgmentThe authors thank Dr. Stephen Reingold, National MS Society,and Dr. Annette-Langer-Gould, Stanford University, for theirinput.

References1. Oksenberg JR, Barcellos LF, Hauser SL. Genetic aspects of multiple

sclerosis. Semin Neurol 1999;19:281–288.2. Sinha AA, Lopez MT, McDevitt HO. Autoimmune diseases: the failure

of self tolerance. Science 1990;248:1380–1388.3. Riise T, Nortvedt MW, Ascherio A. Smoking is a risk factor for multiple

sclerosis. Neurology 2003;61:1122–1124.4. Villard-Mackintosh L, Vessey MD. Oral contraceptives and reproduc-

tive factors in multiple sclerosis incidence. Contraception 1993;47:161–168.

5. Thorogood M, Hannaford PC. The influence of oral contraceptives onthe risk of multiple sclerosis. Br J Obstet Gynecol 1998;105:1296–1299.

6. Ghadirian P, Jain M, Ducic S, et al. Nutritional factors in the aetiologyof multiple sclerosis: a case-control study in Montreal, Canada. Int JEpidemiol 1998;27:845–852.

7. Hernan M, Olek M, Ascherio A. Cigarette smoking and incidence ofmultiple sclerosis. Am J Epidemiol 2001;154:69–74.

8. Sopori ML, Kozak W. Immunomodulatory effects of cigarette smoke.J Neuroimmunol 1998;83:148–156.

9. Kalra R, Singh SP, Savage SM, et al. Effects of cigarette smoke onimmune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T-cells and depletes IP3-sensitive Ca (2!) stores.J Pharmacol Exp Ther 2000;293:166–171.

10. Silman AJ, Newman J, MacGregor A. Cigarette smoking increases therisk of rheumatoid arthritis. Results from a nationwide study of diseasediscordant twins. Arthritis Rheum 1996;39:732–735.

11. Swank RL, Lerstad O, Strom A, Backer J. Multiple sclerosis in ruralNorway. N Engl J Med 1952;246:721–728.

12. Alter M, Yamoor M, Harshe M. Multiple sclerosis and nutrition. ArchNeurol 1974;31:267–272.

13. Zhang SM, Willett WC, Hernan MA, et al. Dietary fat in relation to riskof multiple sclerosis among two large cohorts of women. Am J Epide-miol 2000;152:1056–1064.

14. Meade CJ, Mertin J, Sheena J, Hunt R. Reduction by linoleic acid of theseverity of experimental allergic encephalomyelitis in the guinea pig.J Neurol Sci 1978;35:291–308.

15. van der Mei IA, Ponsonby AL, Blizzard L, Dwyer T. Regional variationin multiple sclerosis prevalence in Australia and its association withambient ultraviolet radiation. Neuroepidemiology 2001;20:168–174.

16. Hayes CE. Vitamin D: a natural inhibitor of multiple sclerosis. ProcNutr Soc 2000;59:531–535.

17. Fukazawa T, Yabe I, Kikuchi S, et al. Association of vitamin D receptorgene polymorphisms with multiple sclerosis in Japanese. J Neurol Sci1999;166:47–52.

18. Steckley JL, Dyment DA, Sadovnick AD, et al. Genetic analysis ofvitamin D related genes in Canadian multiple sclerosis patients. Neu-rology 2000;54:729–732.

Table Smoking and the risk of MS: Findings from population-based studies

Study Design, location Smoking measureOdds ratio(95% CI)

Reference 4 Prospective cohort study of incident cases amongBritish women

" 15 cigarettes/day* 1.4 (0.9–2.2)

Reference 5 Prospective cohort study of incident cases amongBritish women

"15 cigarettes/day* 1.4 (0.9–2.2)

Reference 6 Case-control study of incident cases in Montreal ever vs never smoked 1.6 (1.0–2.4)

20–40 cigarettes/day* 1.9 (1.2–3.2)

Reference 7 Prospective cohort study among U.S. women ever vs never smoked 1.6 (1.2–2.1)

!25 pack-years* 1.7 (1.2–2.4)

Reference 3 Case-control study of prevalent cases inHordaland, Norway

ever vs never smoked(dose response not reported)

1.8 (1.1–2.9)

* Comparison group for cigarette dosage categories is never smokers.

October (2 of 2) 2003 NEUROLOGY 61 1033

Smoking and Multiple Sclerosis: risk of developing MS

2. Smoking

Neurology 2003; 61: 1032-1034

ard ratio, 2.08; 95% CI, 1.15-3.77 for current smokersvs never-smokers; adjusted hazard ratio, 0.95; 95% CI,0.54-1.68 for ex-smokers vs never-smokers). In con-trast, we found no association between smoking statusand EDSS progression at the end of 2 years; the percent-age in whom disease progressed was 23.3% in smokers,30.8% in ex-smokers, and 26.0% in never-smokers (P=.57,adjusted for baseline age, sex, disease duration, and treat-ment). The probability of sustained progression also wasnot significantly different across the groups (adjustedP=.53). Similar results were found for progression at 5years. Results were similar in analyses restricted to pa-tients with RRMS at baseline.

Progression in terms of BPF and T2-weighted lesionvolume was investigated (Figure 2). Current smokerscompared with never-smokers had a significantly greaterincrease in T2-weighted lesion volume (P = .02, ad-justed for baseline age, sex, disease duration, and dis-ease course) and a significantly greater decrease in theBPF (adjusted P=.02); however, ex-smokers and never-smokers demonstrated no significant difference on eithermeasure.

COMMENT

Aggravation of MS symptoms soon after smoking has beenreported in several early studies12-18; however, only 2 pre-vious investigations have examined whether smoking ad-versely affects MS progression. Hernan et al,3 in a case-control investigation nested within the United KingdomGeneral Practice Research Database, reported a 3-foldhigher rate of conversion in ever-smokers compared withnever-smokers. Those results, however, were based ona small sample of 179 patients with RRMS, of whom only20 demonstrated disease conversion to SPMS during fol-low-up. Further, no MRI results were available. Koch et

al4 examined the relationship between smoking and MSprogression within a database comprising clinical infor-mation collected prospectively since 1985 for 672 pa-tients attending the MS clinic of the University MedicalCenter Gröningen, Gröningen, the Netherlands. Smok-ing information was collected by mailed questionnairesor telephone interviews in 2006 and was available for 364patients. In this population, smoking was not associ-ated with the rate of conversion from RRMS to SPMS orwith time from disease onset to EDSS scores of 4.0 or 6.0.The reasons for the conflicting results between the 2 stud-ies are not entirely clear; however, a potential source ofbias in the Gröningen study is that it was conducted more

Table 2. Baseline Characteristics of Patients Based on Smoking Duration (Smokers Only)

Variable

Pack-Years P Value

!3(n=163)

3-20(n=327)

"20(n=164)

UnivariateAnalysisa Adjustedb

Age, mean (SD), y 38.9 (11.2) 42.6 (10.7) 50.5 (9.0) #.001 . . .Disease duration from first symptom, mean (SD), y 8.60 (8.68) 10.03 (9.73) 12.50 (10.73) .001 . . .Sex, No. of patients .01 . . .

Female 124 255 108Male 39 72 56

BPF, mean (SD) 0.87 (0.05) 0.86 (0.05) 0.85 (0.05) #.001 .53T2 lesion volume, mean (SD), cm3 4.64 (3.75) 5.02 (4.65) 5.10 (4.55) .72 .84EDSS score, median (IQR) 1.5 (1.0-3.0) 2 (1.0-3.5) 2 (3.0-6.0) #.001 .01MSSS, mean (SD) 3.31 (2.76) 3.77 (2.76) 4.57 (2.76) .002 .02Type of MS, No. of patients .04 . . .

Relapsing-remitting 119 211 101Secondary progressive 16 59 32Primary progressive 8 17 16Other 20 40 15

Abbreviations: BPF, brain parenchymal fraction; EDSS, Expanded Disability Status Scale score; ellipses, no data available; IQR, interquartile range; MS, multiplesclerosis; MSSS, Multiple Sclerosis Severity Score.

aP values are for 2 df comparisons of 3 groups.bP values are for 2 df comparisons of 3 groups adjusting for age, sex, and disease duration as appropriate. Twenty-five patients had missing number of

cigarettes smoked per day, and an additional 6 patients stopped smoking before the study but were missing age at smoking initiation; data for these patients arenot included in this table.

.70

.75

.80

.85

.90

.95

.99

Study Time, y

Surv

ival

Pro

babi

lity

0 2 4 6 8

Figure 1. Kaplan-Meier curve for time to conversion from relapsing-remittingto secondary progressive multiple sclerosis. Smoking status was defined atstudy entry. Disease in current smokers progressed significantly faster thanin never-smokers (P=.002). Red line indicates current smokers; green line,ex-smokers; and black line, never-smokers.

(REPRINTED) ARCH NEUROL / VOL 66 (NO. 7), JULY 2009 WWW.ARCHNEUROL.COM861

©2009 American Medical Association. All rights reserved.Downloaded From: http://archneur.jamanetwork.com/ by a Universite de Geneve User on 12/18/2013

Smoking as risk factor for disease progression

2. Smoking

Arch Neurol. 2009; 66 (7): 858-864

Current smokers

Ex-‐smokers

Never-‐smokers

Time to conversion from RR-‐MS to SP-‐MS

Neurology 2009; 73;504-510

Smoking as risk factor for disease progression

2. Smoking

Smoking in practice in Geneva University Hospital

Question about smoking It is never too late to quit smoking

2. Smoking

Counseling for the family : Avoid exposure to smoke

Mul0ple Sclerosis

Specific Diet?

Diet and MS

?

3. Diet

Diet and MS

3. Diet

No Diet has been proven to alter the evolu0on of the disease

HOWEVER

Obesity and risk of developing Mul0ple sclerosis Body size and risk of MS in two cohorts of US women.

pooled analyses of both cohorts (table 2). However,the test for heterogeneity of the pooled results wassignificant (table 2). While there was no associationbetween baseline BMI and MS risk in NHSII, therisk of MS among NHS participants decreased withincreasing baseline BMI; relative to women with aBMI of 18.5–20.9 kg/m2, the RR of MS was 0.61(95% CI 0.32–1.15, p ! 0.13) among obese women(BMI !30 kg/m2), and 0.46 (95% CI 0.23–0.93,p ! 0.03) among those overweight (BMI ! 27–29.9kg/m2). This unexpected result may be explained bythe fact that many cases of MS in the NHS occurredbefore MRI was widely used, and the diagnosis wastherefore often delayed with respect to the onset ofsymptoms. This decrease in MS risk in the NHS maythus be the spurious result of the decrease in weightthat appears to occur after MS onset (see below). Re-sults were unchanged after excluding women whowere pregnant at baseline (data not shown).

Women who reported having a larger body size, us-ing the silhouettes, at age 20 also had a twofold in-

creased risk of MS as compared to women whoreported a thinner body size (table 3). There was also asuggestion of an increased risk of MS associated withhaving a larger body size during childhood at ages 5 or10 (table 3). However, reported body size silhouette atages 5, 10, and 20 years were highly correlated (NHS:age 5–10 r ! 0.93, 5–20 r ! 0.83, 10–20 r ! 0.87;NHSII: 5–10 r ! 0.80, 5–20 r ! 0.49, 10–20 r !0.59). In multivariate analyses simultaneously adjustingfor body size at all 3 ages, having a larger body size at age20 remained significantly associated with MS risk(RRpooled body size !6 vs 3 ! 1.70, 95% CI 1.11–2.60, p ! 0.02; p trend across all categories ! 0.18),whereas there was no longer a suggestion of an increasedrisk with larger body sizes at ages 5 and 10 (age 5RRpooled body size !6 vs 3 ! 1.17 95% CI 0.57–2.38,p trend ! 0.46; age 10 RRpooled ! 1.15, 95% CI 0.65–2.04, p trend ! 0.15).

Weight change among women with MS. Consistentwith the result that BMI at baseline was not associated

Table 2 Pooled relative risks and 95% confidence intervals (CIs) for body mass index (BMI) at age 18 andbaseline and risk of multiple sclerosis, Nurses’ Health Study (NHS) (1976 –2002), and Nurses’Health Study II (NHSII) (1989 –2003)

NHS, cases/person-years

NHSII, cases/person-years

Pooled relative risks (95% CI)

Age-adjustedAge-, smoking-adjusted

Multivariate*adjusted

BMI age 18 (kg/m2)

<18.5 21/292,421 45/231,006 0.94 (0.71–1.24) 0.94 (0.71–1.24) 0.96 (0.73–1.27)

18.5–<21 77/963,413 134/656,714 1 (ref) 1 (ref) 1 (ref)

21–<23 55/648,273 84/347,962 1.15 (0.92–1.42) 1.13 (0.91–1.40) 1.13 (0.91–1.40)

23–<25 24/281,999 31/161,737 1.00 (0.74–1.35) 0.97 (0.72–1.31) 0.97 (0.72–1.31)

25–<27 20/130,338 17/72,559 1.51 (0.90–2.52) 1.45 (0.86–2.43) 1.44 (0.87–2.39)

27–<30 8/72,619 16/50,889 1.47 (0.96–2.24) 1.40 (0.92–2.14) 1.40 (0.92–2.14)

>30 6/45,583 21/39,839 2.41 (1.61–3.60) 2.26 (1.50–3.38) 2.25 (1.50–3.37)

Total 211/2,434,647 348/1,560,707

p, trend "0.001 "0.001 "0.001

p, het 0.98 0.96 0.997

Baseline BMI (kg/m2)

<18.5 7/74,199 14/53,541 1.11 (0.70–1.75) 1.10 (0.69–1.74) 1.13 (0.71–1.78)

18.5–<21 68/656,533 81/396,203 1 (ref) 1 (ref) 1 (ref)

21–<23 66/816,791 68/377,321 0.87 (0.69–1.10) 0.87 (0.69–1.10) 0.87 (0.69–1.10)

23–<25 45/574,546 59/266,505 1.00 (0.78–1.28) 1.00 (0.78–1.28) 1.00 (0.78–1.29)

25–<27 32/344,643 42/157,999 1.22 (0.92–1.62) 1.22 (0.92–1.61) 1.23 (0.93–1.62)

27–<30 9/263,744 36/134,424 0.78 (0.28–2.19) 0.78 (0.29–2.11) 0.79 (0.30–2.12)

>30 11/245,691 48/179,725 0.88 (0.42–1.85) 0.89 (0.45–1.78) 0.91 (0.46–1.79)

Total 238/2,976,147 348/1,565,718

p, trend 0.82 0.85 0.88

p, het 0.02 0.03 0.03

*Age (in months), latitude age 15 (north, middle, south), ethnicity (S European, Scandinavian, other Caucasian, other), smok-ing (never smoker, 1–9, 10 –24, and !25 pack-years).

1546 Neurology 73 November 10, 2009

“High body mass index before age 20 is associated with increased risk for mul0ple sclerosis in both men and women”

Neurology 2009:73:1543.

3 Diet Obesity Mult. Scler. 2012 Sep 18(9)

Salt intake and animal model of Mul0ple sclerosis

3 Diet Salt

Salt intake and animal model of Mul0ple sclerosis

Figure 4. High-salt diet induces Th17 cells in vivo and exacerbates experimental autoimmuneencephalomyelitisa, Naïve murine CD4 cells were stimulated with radiated APC, anti-CD3, IL-6 and TGF-β1in the presence (NaCl) or absence (none) of additional 40mM NaCl and were analysed byFACS (n=3). b, IL-17A secretion (ELISA) of primary splenocytes, stimulated by anti-CD3in the presence or absence of NaCl (n=6). c, Mean clinical scores of EAE in HSD animals(squares) or controls (dots, pooled data of two independent experiments with 12 animals).Histological analyses show sections of the spinal cord stained with hematoxylin and eosin(HE), anti-CD3 and anti-Mac-3 for control or HSD animals (scale bar=100 μM) and werequantified for CD3 and Mac-3 (bar graphs, n=5–6). d, Spinal cord from EAE animals wasanalysed by qRT-PCR (n=5–6). e, Splenocytes from EAE animals were analysed by qRT-PCR (n=4–7). f, Splenocytes from EAE animals were re-stimulated with MOG for 2 days

Kleinewietfeld et al. Page 14

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Sodium Chloride Drives Autoimmune Disease by the Inductionof Pathogenic Th17 Cells

Markus Kleinewietfeld1,2,*, Arndt Manzel3,4, Jens Titze5,6, Heda Kvakan7,8, Nir Yosef2, RalfA. Linker3, Dominik N. Muller7,9,*,+, and David A. Hafler1,2,*,+

1Departments of Neurology and Immunobiology, Yale School of Medicine, New Haven, CT,United States2Broad Institute of MIT and Harvard, Cambridge, MA, United States3Department of Neurology University of Erlangen-Nuremberg, Germany4International Graduate School of Neuroscience, Ruhr-University Bochum, Germany5Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN, United States6Interdisciplinary Center for Clinical Research and Department for Nephrology and Hypertension,University of Erlangen-Nuremberg, Germany7Experimental and Clinical Research Center, a joint cooperation between the Charité MedicalFaculty and the Max-Delbrück Center for Molecular Medicine Berlin, Germany8Helios Klinikum Berlin-Buch, Germany9Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nuremberg, Germany

AbstractThere has been a marked increase in the incidence of autoimmune diseases in the last half-century.While the underlying genetic basis of this class of diseases has recently been elucidatedimplicating predominantly immune response genes1, changes in environmental factors mustultimately be driving this increase. The newly identified population of interleukin (IL)-17producing CD4+ helper T cells (Th17 cells) plays a pivotal role in autoimmune diseases2.Pathogenic IL-23 dependent Th17 cells have been shown to be critical for the development ofexperimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS),and genetic risk factors associated with MS are related to the IL23/Th17 pathway1, 2. However,little is known regarding the environmental factors that directly influence Th17 cells. Here weshow that increased salt (sodium chloride; NaCl) concentrations found locally under physiologicalconditions in vivo dramatically boost the induction of murine and human Th17 cells. High-saltconditions activate the p38/MAPK pathway involving the tonicity-responsive enhancer binding

*Correspondence and requests for materials should be addressed to M.K ([email protected]) or D.A.H.([email protected]).+these authors contributed equally to the workReprints and permissions information is available at www.nature.com/reprints.The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article at www.nature.com/nature. The microarray data sets aredeposited in Gene Expression Omnibus database under accession number GSE42569.Author contributions. M.K. designed the study, planned and performed experiments, analysed data and wrote the manuscript. A.M.planned and performed experiments, analysed data and wrote the manuscript. J.T. and H.K. interpreted data and supported the workwith key suggestions and editing the manuscript. N.Y. analysed data. R.A.L. planned experiments, analysed data and wrote themanuscript. D.N.M. designed the study, planned experiments, analysed data and wrote the manuscript. D.A.H. designed the study,planned experiments, analysed data, and wrote the manuscript. M.K., D.N.M and D.A.H. co-directed the project.

NIH Public AccessAuthor ManuscriptNature. Author manuscript; available in PMC 2013 August 19.

Published in final edited form as:Nature. 2013 April 25; 496(7446): 518–522. doi:10.1038/nature11868.

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3 Diet Salt

Ectrims 2013; abstract 119

Salt and risk of disease progression

Es0mated daily sodium intake: spot urine collec0on to es0mate daily sodium excre0on Salt intake classified as: high: > 4.8 g/day intermediate: 2 to 4.8 g/day Low: < 2 g/day

Relapse rate were 3.95 0mes greater (95% CI 1.39-‐11.21) in high salt intake than those with intakes less than 2 g/day Par0cipants with daily intake of 2.0 to 4.8 g/day had relapse rates that were 2.75 0mes that of the low-‐intake group (95% CI 1.30-‐5.81). Each increment in intake of 1 g above the cohort average was associated with 3.65-‐lesion increase in T2 count (SD 0.77, P<0.001).

122 RR-‐MS pa0ents

3 Diet Salt

Diet in practice in Geneva University Hospital

3. Diet

Mul0ple Sclerosis

Future promising research direction?

The gut-‐brain axis

4 Gut immunology Nat reviews Neuroscience October 2012, vol 13. 701-‐712

Gut immunology and MS

Nature Reviews | Immunology

BasophilB cell

iNKT cell

Gut lumen

Laminapropria

IgE

Pancreas Lungs

! Firmicutes " Bacteroidetes

TReg cell

IL-1#

CNS Joint

TH17 cell IL-1R

antagonist

TH17 cell

TH17 cell

Peripheral blood

Multiple sclerosisEAE

Type 1 diabetesPancreatitis

AllergicKPȯCOOCVKQP

Arthritis

$GPGȮEKCN�commensal bacteria

SFB

Experimental autoimmune encephalomyelitis(EAE). An experimental model of multiple sclerosis that is induced by immunization of susceptible animals with myelin-derived antigens, such as myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein.

K/BxN transgenic miceMice formed by crossing non-obese diabetic (NOD)/Lt mice with KRN T cell receptor-transgenic mice on the C57BL/6 background. As the KRN receptor on T cells recognizes a peptide from the autoantigen glucose-6-phosphate isomerase, these mice develop an arthritis that is mediated, and transferable, by circulating antibodies against glucose-6- phosphate isomerase.

experimental autoimmune encephalomyelitis (EAE)149. In one model of multiple sclerosis, mice are immunized with the self antigen myelin oligodendrocyte glyco-protein (MOG) in complete Freund’s adjuvant (CFA). Disease symptoms in either MOG–CFA-induced EAE or in a spontaneous EAE mouse model are reduced when the mice are housed under germ-free conditions150,151. Monocolonization of germ-free mice with SFB results in an increase in the number of TH17 cells in both the intestinal lamina propria and the CNS, which results in severe EAE150 (FIG. 4). Thus, SFB-enhanced TH17 cell-mediated inflammation might contribute to EAE exac-erbation. However, it is unclear whether the disease is caused by the migration of SFB-specific TH17 cells into the CNS or by the expansion of pathogenic autoantigen-specific T cells that are promoted by intestinal TH17 cell responses (FIG. 4). By contrast, certain populations of commensal bacteria are capable of attenuating CNS inflammation. For example, PSA+ B. fragilis, which

induces FOXP3+ TReg cell differentiation, can prevent EAE symptoms152 (FIG. 4). Thus, the pathogenesis of CNS disorders might ultimately depend on the balance of different community members in the gut microbiota.

Arthritis. Autoimmune arthritis, such as rheumatoid arthritis, is a systemic inflammatory disease that pri-marily affects the joints but can also affect other parts of the body. The events that trigger the development of autoimmune arthritis remains unknown. However, in mouse models, the gut microbiota contributes to disease symptoms. Arthritis symptoms in K/BxN transgenic mice are reduced in a germ-free environment136,153. As in EAE, TH17 cell responses are implicated in promoting disease, and SFB-mediated enhancement of TH17 cell immunity stimulates autoantibody production by B cells, which leads to arthritic symptoms153 (FIG. 4). TH17 cell immunity is also a key factor in spontaneous rheumatoid arthri-tis in IL-1R antagonist (Il1rn)−/− mice, which exhibit

Figure 4 | Gut microbiota affects extra-intestinal autoimmune diseases. Segmented filamentous bacteria (SFB) colonization induces T helper 17 (T

H17) cell development in the intestine. These T

H17 cells might migrate to the periphery

to affect systemic and central nervous system (CNS) immunity; increased intestinal TH17 cells enhance the expansion

QH�RCVJQIGPKE�CWVQCPVKIGP�URGEKHKE�6|EGNNU�KP�VJG�KPVGUVKPG�CPF�ECWUG�KPHNCOOCVKQP�KP�VJG�%05��$[�EQPVTCUV��nDGPGHKEKCNo�commensal bacteria can attenuate CNS inflammation through the induction of forkhead box P3 (FOXP3)+ regulatory T (T

Reg)

cells. Induced TH��EGNNU�ECP�CNUQ�RTQOQVG�CWVQKOOWPG�CTVJTKVKU�D[�HCEKNKVCVKPI�CWVQCPVKDQF[�RTQFWEVKQP�D[�$|EGNNU�PQV�

shown). In addition, microbiota-induced interleukin-1β (IL-1β) signalling participates in the development of rheumatoid arthritis through the induction of T

H17 cells. The IL-1 receptor (IL-1R) antagonist blocks IL-1β signalling and abrogates

LQKPV�KPHNCOOCVKQP��$CNCPEG�KP�VJG�OKETQDKCN�EQOOWPKV[�CNUQ�FGVGTOKPGU�UWUEGRVKDKNKV[�VQ�V[RG|��FKCDGVGU��#�FGETGCUGF�Firmicutes/Bacteroidetes ratio as a result of a deficiency in myeloid differentiation primary-response protein 88 (MYD88) in non-obese diabetic mice is associated with an attenuated risk of type 1 diabetes. SFB-induced T

H17 cells protect the host

against type 1 diabetes development by an unknown mechanism. Finally, exposure to microorganisms in neonatal, but not adult, life decreases the accumulation of invariant natural killer T (iNKT) cells in the gut, which results in protection against CNNGTIKE�KPHNCOOCVKQP�KP�VJG�NWPIU��+P�CFFKVKQP��OKETQDKCN�EQORQWPFU�UVKOWNCVG�RGTKRJGTCN�$|EGNNU�VJTQWIJ�$|EGNN�KPVTKPUKE�MYD88 signalling and inhibit IgE production. Decreased levels of peripheral IgE result in decreased numbers of basophils, and attenuate the risk of allergic airway inflammation. EAE, experimental autoimmune encephalomyelitis.

REVIEWS

NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 331

© 2013 Macmillan Publishers Limited. All rights reserved

Nat review may 2013, vol 13. 335

Gut microbiota affects extra-‐intes0nal autoimmune diseases

4 Gut immunology

The gut-‐brain axis

Gut immunology : a new risk factor for MS?

4 Gut immunology

resonance imaging (MRI scans) in MS) may contribute to some ex-tent to the observed increased prevalence of allergy and autoim-munity, the widespread, consistent increases of many allergicand autoimmune diseases in the developed world in the last cen-tury implies that the epidemic of these disorders is real and notsimply an artefact of better ascertainment. This assertion is bol-stered by long-term surveys of MS prevalence done in one locationwith consistent ascertainment methods (Koch-Henriksen andSorensen, 2011).

1.3. Current MS synthesis

Taken together, the genetic and epidemiological evidence sug-gests that MS may occur in a genetically-susceptible individualwho is exposed to a critical, at present unknown, environmentalexposure in early life, the combination of which is thought to in-duce autoimmunity. The major targets of the autoimmune re-sponse in MS are CNS myelin and oligodendrocytes. Focal areasor plaques of immune-mediated tissue damage may lead to tran-sient or permanent symptoms and disability. Critical questionstherefore relate to the underlying causes and corrections of theautoimmune process in MS.

2. Hygiene hypothesis

2.1. Autoimmunity

Autoimmune disorders (Anaya, 2012) occur when the activity ofan organism’s immune system produces sustained damage to itsown tissues and cells. General explanations or conditions leading

to autoimmunity include (i) a primary failure of mechanismswhich normally promote immunological tolerance to self (e.g., cen-tral tolerance and peripheral tolerance) (Mackay, 2000; Bluestone,2011), (ii) infections or other exogenous factors which promoteinflammation and thereby override tolerance to self (Kivity et al.,2009; Sener and Afsar, 2012), (iii) an abnormal state of the targettissues themselves (e.g., inappropriate release of danger-stress sig-nals or aberrant antigen presentation within tissues) (Tveita,2010), or (iv) a combination of processes.

In this regard, Dobzhansy observed that ‘‘Nothing in biologymakes sense except in the light of evolution’’ (Ayala, 1977). The hy-giene or microbial deprivation hypothesis is an evolutionarily-based explanation (Pollard, 2008) for the epidemic of allergy andautoimmunity which has been observed in the developed worldduring the last century (Bach, 2002) and which now appears tobe occurring in the developing world (Cabre, 2009; Aguirre-Cruzet al., 2011; Etemadifar and Maghzi, 2011; Inshasi and Thakre,2011). Rook (2012) has summarised how the modern, extremelyhygienic environment in the developed world leads to loss of evo-lutionarily ancient microbes, including parasitic worms, for whichhe coined the term ‘‘Old Friends’’ (Rook et al., 2004). For example,as recently as 1947, it was estimated that 36% of the European pop-ulation was colonised by intestinal helminths, while at presentthese infections are virtually absent in the same region (Stoll,1947; Gale, 2002). It may sound paradoxical to describe parasitesas friends or beneficial, given the major public health hazard theyconstitute, especially in tropical regions (McKay, 2006). Neverthe-less, from an evolutionary perspective, the case:mortality ratio ofmost parasitic diseases is relatively low (e.g., 10,000:1 for hook-worm (Brooker et al., 2004)); the relationship is not without con-flict and morbidity, but over millions of years of co-evolutionmany parasites and their hosts have mostly learnt not to kill eachother. As Rook points out (Rook, 2012), the immune system has co-evolved with Old Friend microbes which were common and there-fore had to be tolerated or at least controlled without provokingsignificant immunopathology. However, in the modern, evolution-arily abnormal environment depleted of Old Friend organisms, thehygiene hypothesis posits that the function of the immune systemis disordered and the risk of allergic and autoimmune diseases isincreased (see Section 2.2).

2.2. Immunoregulation

With regard to the mechanisms underlying the hygiene hypoth-esis and the role of microbial deprivation in autoimmunity, Rook(2012) and others have focused on the central concept of immuno-regulation. Immunoregulation may be defined as the activity of thesystem of integrated controls (Atlan and Cohen, 1998; Cohen andHarel, 2007) which governs and coordinates the individual compo-nents of immunity. Normal immunoregulation promotes healthand homeostasis by assuring that all-over immune responsessimultaneously and optimally (i) resist invasive pathogens while(ii) promoting active tolerance to commensal organisms and, tosome extent, even to pathogens, so that any collateral immunedamage to host issues is minimised (Eberl, 2010; Medzhitovet al., 2012). Thus, the IPEX syndrome (d’Hennezel et al., 2012),characterised by deficient T regulatory (Treg) cell activity andautoimmune pathology in many organs, is considered a primarydisease of immunoregulation, a disorder of the control network it-self; by contrast, hereditary angioedema (Ferraro et al., 2011), inwhich a deficiency of the C1 inhibitor leads to only one immunemanifestation, recurrent subcutaneous oedema, is not considereda disease of immunoregulation in the usual sense. Importantly,per the hygiene hypothesis, Old Friend organisms are active induc-ers of normal immunoregulation (see Section 2.4), and in the ab-sence of these ancient microbes, immunological activity is

0 20 40 60 80

0

20

40

60

80

100

120

TT Prevalence (%)

MS

Prev

alen

ce (p

er 1

00,0

00)

Fig. 2. The prevalence of multiple sclerosis (patients per 100,000 general popula-tion) and the human whipworm, Trichuris trichiura (TT) (percentage of surveyedpopulation infected) by country or region. North America: northern United States(1), southern United States (2), Canada (3). Latin America: Brazil (4), Mexico (5),Paraguay (6), Honduras (7), Cuba (8), Jamaica (9), Panama (10), Argentina (11), Chile(12). Oceania: Australia (13). Africa: Ethiopia (14), Kenya (15), Cameroon (16),Nigeria/Ghana (17), Ivory Coast/Senegal (18). Middle East: Iran/Iraq (19), Lebanon/Jordan (20), Jerusalem–Jewish (21), Jerusalem–Arab (22). Europe: Italy (23), Poland(24), Belgium (25), East Germany (26), United Kingdom (27). Asia: Indonesia (28),Japan (29), Korea (30), Malaysia (31), Taiwan (32), Philippines (33), China (34),Central India (35). Reproduced with permission from Fleming and Cook, 2006.Multiple sclerosis and the hygiene hypothesis. Neurology 67, 2085-2086.

J.O. Fleming / International Journal for Parasitology 43 (2013) 259–274 261

Neurology 2006, 67, 2085-2086

resonance imaging (MRI scans) in MS) may contribute to some ex-tent to the observed increased prevalence of allergy and autoim-munity, the widespread, consistent increases of many allergicand autoimmune diseases in the developed world in the last cen-tury implies that the epidemic of these disorders is real and notsimply an artefact of better ascertainment. This assertion is bol-stered by long-term surveys of MS prevalence done in one locationwith consistent ascertainment methods (Koch-Henriksen andSorensen, 2011).

1.3. Current MS synthesis

Taken together, the genetic and epidemiological evidence sug-gests that MS may occur in a genetically-susceptible individualwho is exposed to a critical, at present unknown, environmentalexposure in early life, the combination of which is thought to in-duce autoimmunity. The major targets of the autoimmune re-sponse in MS are CNS myelin and oligodendrocytes. Focal areasor plaques of immune-mediated tissue damage may lead to tran-sient or permanent symptoms and disability. Critical questionstherefore relate to the underlying causes and corrections of theautoimmune process in MS.

2. Hygiene hypothesis

2.1. Autoimmunity

Autoimmune disorders (Anaya, 2012) occur when the activity ofan organism’s immune system produces sustained damage to itsown tissues and cells. General explanations or conditions leading

to autoimmunity include (i) a primary failure of mechanismswhich normally promote immunological tolerance to self (e.g., cen-tral tolerance and peripheral tolerance) (Mackay, 2000; Bluestone,2011), (ii) infections or other exogenous factors which promoteinflammation and thereby override tolerance to self (Kivity et al.,2009; Sener and Afsar, 2012), (iii) an abnormal state of the targettissues themselves (e.g., inappropriate release of danger-stress sig-nals or aberrant antigen presentation within tissues) (Tveita,2010), or (iv) a combination of processes.

In this regard, Dobzhansy observed that ‘‘Nothing in biologymakes sense except in the light of evolution’’ (Ayala, 1977). The hy-giene or microbial deprivation hypothesis is an evolutionarily-based explanation (Pollard, 2008) for the epidemic of allergy andautoimmunity which has been observed in the developed worldduring the last century (Bach, 2002) and which now appears tobe occurring in the developing world (Cabre, 2009; Aguirre-Cruzet al., 2011; Etemadifar and Maghzi, 2011; Inshasi and Thakre,2011). Rook (2012) has summarised how the modern, extremelyhygienic environment in the developed world leads to loss of evo-lutionarily ancient microbes, including parasitic worms, for whichhe coined the term ‘‘Old Friends’’ (Rook et al., 2004). For example,as recently as 1947, it was estimated that 36% of the European pop-ulation was colonised by intestinal helminths, while at presentthese infections are virtually absent in the same region (Stoll,1947; Gale, 2002). It may sound paradoxical to describe parasitesas friends or beneficial, given the major public health hazard theyconstitute, especially in tropical regions (McKay, 2006). Neverthe-less, from an evolutionary perspective, the case:mortality ratio ofmost parasitic diseases is relatively low (e.g., 10,000:1 for hook-worm (Brooker et al., 2004)); the relationship is not without con-flict and morbidity, but over millions of years of co-evolutionmany parasites and their hosts have mostly learnt not to kill eachother. As Rook points out (Rook, 2012), the immune system has co-evolved with Old Friend microbes which were common and there-fore had to be tolerated or at least controlled without provokingsignificant immunopathology. However, in the modern, evolution-arily abnormal environment depleted of Old Friend organisms, thehygiene hypothesis posits that the function of the immune systemis disordered and the risk of allergic and autoimmune diseases isincreased (see Section 2.2).

2.2. Immunoregulation

With regard to the mechanisms underlying the hygiene hypoth-esis and the role of microbial deprivation in autoimmunity, Rook(2012) and others have focused on the central concept of immuno-regulation. Immunoregulation may be defined as the activity of thesystem of integrated controls (Atlan and Cohen, 1998; Cohen andHarel, 2007) which governs and coordinates the individual compo-nents of immunity. Normal immunoregulation promotes healthand homeostasis by assuring that all-over immune responsessimultaneously and optimally (i) resist invasive pathogens while(ii) promoting active tolerance to commensal organisms and, tosome extent, even to pathogens, so that any collateral immunedamage to host issues is minimised (Eberl, 2010; Medzhitovet al., 2012). Thus, the IPEX syndrome (d’Hennezel et al., 2012),characterised by deficient T regulatory (Treg) cell activity andautoimmune pathology in many organs, is considered a primarydisease of immunoregulation, a disorder of the control network it-self; by contrast, hereditary angioedema (Ferraro et al., 2011), inwhich a deficiency of the C1 inhibitor leads to only one immunemanifestation, recurrent subcutaneous oedema, is not considereda disease of immunoregulation in the usual sense. Importantly,per the hygiene hypothesis, Old Friend organisms are active induc-ers of normal immunoregulation (see Section 2.4), and in the ab-sence of these ancient microbes, immunological activity is

0 20 40 60 80

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120

TT Prevalence (%)

MS

Prev

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00)

Fig. 2. The prevalence of multiple sclerosis (patients per 100,000 general popula-tion) and the human whipworm, Trichuris trichiura (TT) (percentage of surveyedpopulation infected) by country or region. North America: northern United States(1), southern United States (2), Canada (3). Latin America: Brazil (4), Mexico (5),Paraguay (6), Honduras (7), Cuba (8), Jamaica (9), Panama (10), Argentina (11), Chile(12). Oceania: Australia (13). Africa: Ethiopia (14), Kenya (15), Cameroon (16),Nigeria/Ghana (17), Ivory Coast/Senegal (18). Middle East: Iran/Iraq (19), Lebanon/Jordan (20), Jerusalem–Jewish (21), Jerusalem–Arab (22). Europe: Italy (23), Poland(24), Belgium (25), East Germany (26), United Kingdom (27). Asia: Indonesia (28),Japan (29), Korea (30), Malaysia (31), Taiwan (32), Philippines (33), China (34),Central India (35). Reproduced with permission from Fleming and Cook, 2006.Multiple sclerosis and the hygiene hypothesis. Neurology 67, 2085-2086.

J.O. Fleming / International Journal for Parasitology 43 (2013) 259–274 261

Parasites infec0on and prevalence of MS

4 Gut immunology Parasites

Observa0onal study, no immunomodulatory or immunosuppressive treatment

Annals of Neurology Vol 61 No 2 February 2007

All relapses observed in infected MS patients and 48exacerbations (86%) observed in uninfected MS sub-jects were treated with intravenous methylprednisolonefollowed by a short tapering course of oral prednisone.The scant number of exacerbations observed in the in-fected MS group (n ! 3) during this study precludesstatistical analysis of treatment impact on the results ofthis investigation.

In infected MS individuals, the median total IgElevel (1,800IU/ml; range, 525–3,300) was significantlygreater (p ! 0.001) than in healthy subjects (285IU/ml; range, 170–1,508) or in uninfected MS patients(380IU/ml; range, 213–2,100), remaining constantthroughout follow-up. Furthermore, total IgE concen-trations showed positive correlation with the totalnumber of parasite eggs per gram in feces (r ! 0.48;p ! 0.01). Concentrations of parasite-specific IgE werenot investigated. The limited number of patients in-fected with each different type of parasite precludes ap-propriate statistical analysis regarding impact of specifichelminth species of immunological response. Parasiteinfections are associated with increased production ofTGF-" and IL-10 and reduced production of IFN-#and IL-12.

Helminth infections have been shown to promote

cytokine production associated with a Th2-type re-sponse.8 Conversely, MS is characterized by an in-flammatory response associated with the productionof Th1-type cytokines, such as IFN-#.2 To testwhether parasite infection influences antigen-specificT-cell phenotype during the course of MS, we char-acterized cytokine production in PBMC using ELIS-POT assays. As shown in Figures 2A and 2B, thenumber of MBP83-102 peptide–specific IL-10 andTGF-" secreting cells was significantly greater insamples collected from parasite-infected MS patients,compared with those obtained from uninfected MSpatients or control subjects ( p $ 0.0001). At thesame time, as illustrated in Figures 2C and 2D,parasite-infected MS patients showed significantly re-duced numbers of IL-12– and IFN-#–secreting cellscompared with uninfected MS patients or controlsubjects ( p ! 0.0001). No significant difference inthe number of IL-4 –secreting cells was observed be-tween patient groups.

Regulatory T-Cell Isolation and CharacterizationTo further examine potential mechanisms explainingimmune response changes in parasite-infected MS pa-tients, we obtained MBP peptide–specific TCCs with

Fig 1. Number of exacerbations (A) and changes in extended disability status scale (EDSS; B) and magnetic resonance imaging (Cand D) parameters observed over time in parasite infected (squares) and uninfected (diamonds) multiple sclerosis (MS) patients.Gd ! gadolinium.

Correale and Farez: Parasite Infections and MS 101






















Helminths infec0on decreases MS progression


of Iowa, USA in 2002; subsequently a research grant was submittedto the US National Multiple Sclerosis Society in 2004, preclinicalanimal studies were conducted in 2005–2007, and an Investiga-tional New Drug application for TSO administration in MS was sub-mitted to the US Food and Drug Administration IND in 2008. In2009, the first part of the clinical trial (HINT 1), a short safety studyin which five RRMS subjects were treated with TSO for 3 months,was initiated (Fleming et al., 2011). The purpose of the first-useHINT 1 study was to assess any immediate safety concerns, e.g.,unexpected but possible increases in MS MRI activity or new at-tacks of neurological disability. After review of the results of HINT1 by regulatory authorities, permission was granted to initiate afollow up clinical trial (HINT 2) with 18 RRMS subjects studiedfor 20 months in a baseline versus treatment design (HINT 2).The final results of the HINT 2 study are expected in late 2013.The history of HINT 1 and HINT 2 illustrates the prolonged, butnecessary, period required when introducing a novel, yet poten-tially dangerous (Erb, 2009) treatment, such as live parasites, inMS. In this regard, it should be noted that several putative treat-ments which initially were thought to have strong promise basedon results from animal models or clinical trials in other diseaseswere subsequently found to have anti-therapeutic or toxic effectsin MS (Wiendl and Hohlfeld, 2002; Ziemssen, 2008).

In the HINT 1 study, five newly-diagnosed, treatment-naïveRRMS subjects underwent brain MRI investigations (i) at presenta-tion before treatment (baseline phase); (ii) during each of3 months in which they were given 2,500 live, good manufacturingproduct (GMP) certified, TSO orally every 2 weeks (treatmentphase); and (iii) at 2 months after the end of TSO treatment(post-treatment phase). In addition to microbiological determina-tions done by the producer of GMP TSO (OvaMed, Hamburg Ger-many), extensive retesting of TSO upon receipt was performedfor all porcine adventitious agents and other microbiological

contaminants at the Waisman Clinical Biomanufacturing Facilityat the University of Wisconsin. For the group of five subjects inHINT 1, before TSO treatment the mean number of new activebrain MS lesions was 6.6 at baseline; this value fell to a mean of2.0 after 3 months of TSO administration; and it rose again to amean of 5.8 at 2 months after TSO was stopped. Although theMRI results in this exploratory study are promising, particularlyas they speak against an early, major anti-therapeutic effect ofTSO in MS, they should be interpreted with caution, given the smallnumber of subjects and the short period of observation. Thefavourable MRI results could be due to chance or regression tothe mean; on the other hand, the ‘‘rebound’’ or return to the levelof baseline activities after TSO administration was stopped sug-gests that a significant biological effect may be operative.

In terms of safety, no major adverse clinical effects were notedin the HINT 1 subjects. However, in three of the five subjects, tran-sient (lasting 3–5 days) mild gastrointestinal symptoms werenoted approximately 30 days after TSO initiation, likely represent-ing a first-dose or adjustment phenomenon, similar to that notedby Bager and colleagues in a study of TSO for allergic rhinitis (Bageret al., 2011). Preliminary evidence suggests that the early gastroin-testinal symptoms may occur at a time when T. suis larval coloni-sation is sufficient to evoke an inflammatory innate immuneresponse, but prior to the period in which a controlling and immu-nomodulatory adaptive immune response appears. Treatment-re-lated gastrointestinal symptoms were not detected in priorstudies of TSO in inflammatory bowel disease (Summers et al.,2003, 2005a,b), possibly due to pre-existing gastrointestinal symp-toms in those subjects. It is important to note that gastrointestinaladverse effects after TSO have been limited to symptoms, not signsor serious adverse events; were transient; and did not interferewith activities of daily living such as school or work. Nevertheless,the potential for serious gastrointestinal events remains (Hsu et al.,

Table 2Clinical studies of helminth therapy in multiple sclerosis: completed, in progress or anticipated.

Type ofinvestigationa

Studyb Statusc Subjectsd Helminthe ClinicalTrials.govf Results Publicationsg

ObservationalCorrealeandFarez

C 12 RRMS Natural gastrointestinalinfections with humanhelminths, see text

Dramatic reduction in clinical, MRI, andimmunological measures of MS activityfound

Correale andFarez (2007,2011)

ExploratoryHINT 1 C 5 RRMS Trichuris suis ova 2,500 q

2 weeks ! 12 weeks orallyNCT00645749 Treatment was safe; MRI and

immunological outcomes favourableFleming et al.(2011)

Charite C 4 SPMS T. suis ova 2,500 q2 weeks ! 24 weeks orally

Treatment was safe; moderate positiveimmunomodulatory impact

Benzel et al.(2012)

TRIMS A C 10 RRMS T. suis ova 2,500 q2 weeks ! 12 weeks orally

NCT01006941 Treatment was safe; no clinical, MRI,immunological benefit

AAN (2012)

Phase 1/2HINT 2 P 15 RRMS T. suis ova 2,500 q

2 weeks ! 10 months orallyNCT00645749 Safety confirmed; interim MRI and

immunological measures positive; finalresults expected 2013

AAN (2012)ACTRIMS

(2012)TRIOMS P 50 RRMS T. suis ova 2,500 q

2 weeks ! 12 months orallyNCT01413243 Trial enrolling

WIRMS A 36 RRMS 25 live Necator americanusdermally

NCT01470521 Trial initiation anticipated in near future Edwards andConstantinescu(2009)

MRI, magnetic resonance imaging.a Studies are classified as observational (field study, naturally-acquired infections), exploratory (preliminary pilot first-use safety clinical trials), or early phase 1/2 (follow

up clinical trials).b Each study is designated by location, investigators or acronym.c Study status is indicated by C (completed), P (in progress or enroling), or A (initiation anticipated in near future).d The number and type of subjects are noted (RRMS = relapsing-remitting MS; SPMS = secondary progressive MS); only the number of subjects with MS are shown,

exclusive of subjects in placebo or observational arms.e The heminth infection or treatment indicated by agent, dose, duration and route.f Clinical trial listings are provided by study number on the clinicaltrials.gov website; these listings provide details of study design and periodic updates on study progress.g Publications or meeting presentations (AAN, American Academy of Neurology, 2012; ACTRIMS, Americas Committee for Treatment and Research in Multiple Sclerosis,

2012) are indicated.

266 J.O. Fleming / International Journal for Parasitology 43 (2013) 259–274

Invited Review

Helminth therapy and multiple sclerosis

J.O. Fleming !

Department of Neurology, University of Wisconsin School of Medicine and Public Health, 7124 MFCB, 1685 Highland Avenue, Madison, WI 53705, USA

a r t i c l e i n f o

Article history:Received 15 September 2012Received in revised form 16 October 2012Accepted 17 October 2012Available online 5 January 2013

Keywords:Multiple sclerosisHygiene hypothesisAutoimmunityImmunoregulationHelminth therapyTrichurisNecatorGut-systemic-CNS axis

a b s t r a c t

Multiple sclerosis is a common and frequently disabling neurological disease of young adults. It is char-acterised by recurrent areas of focal inflammation (plaques) in the CNS which give rise to episodic neu-rological signs and symptoms. According to the hygiene (microbial deprivation) hypothesis,evolutionarily abnormal high levels of sanitation in the environment of the developed world may contrib-ute to disordered immunoregulation in this and other putative autoimmune disorders. Helminths havebeen shown to augment immunoregulation. On this basis, the possibility of treating multiple sclerosiswith live helminths or helminth products has been explored in animal models, natural human infectionsand phase 1 clinical trials. To date helminth therapy appears safe and preliminary clinical, magnetic res-onance imaging and immunological outcomes have generally been favourable. Nevertheless, seriousadverse effects are always possible, particularly with live parasitic administration. Follow up studies withsafety monitoring, regulatory oversight and objective outcome measures will be required to definitivelyassess safety and efficacy for this novel class of potential immunological therapies in multiple sclerosis.

! 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Multiple sclerosis (MS)

1.1. What is MS?

Multiple sclerosis (MS) is the most common non-traumaticcause of neurological disability in young adults in developed coun-tries (Noseworthy et al., 2000; Goldenberg, 2012; Nylander andHafler, 2012). World-wide, more than two million individuals areaffected, with a mean onset of approximately 25 years of age (Sel-mi et al., 2012). MS affects the CNS in a pattern characterised bydissemination in time and space. The disease usually begins withattacks of neurological dysfunction, such as loss of vision in oneeye (involvement of an optic nerve), weakness (corticospinal tractsof brain or spinal cord), numbness (sensory tracts), double vision(brainstem), or incoordination (cerebellum). These episodes typi-cally progress for several days or weeks and are usually followedby partial or complete recovery, a pattern that is characteristic ofthe relapsing-remitting subtype of MS (RRMS). After a decade ormore of relapses and remissions, in some patients the pace of dis-ease may change to one of steady progression of disability, consti-tuting the secondary progressive subtype of MS (SPMS). A minorityof MS patients will present with a form of the disease characterisedby relentless progression from onset, that is, primary progressiveMS (PPMS). MS may be mild initially and partially effective medi-

cations are available for treatment. Nevertheless, with time manypatients will experience substantial and tragic neurological disabil-ity related to their disease or serious complications due to immu-nosuppressive therapy. Better MS treatments are needed.

1.2. MS: Pathology, genetics, epidemiology

Classically, MS is considered a disease of CNS white matter inwhich there is an autoimmune attack, usually considered to be ini-tiated by dysregulated T cells, on myelin and the myelin-producingcell, the oligodendrocyte. Recently, however, there has been an in-creased appreciation of wider aspects of MS pathology, includinginvolvement of grey matter, loss of axons, participation of B cellsand activated microglia, subtle histological changes in the grosslynormal appearing white matter and subtypes of immunopathology(Kipp et al., 2012; Popescu and Lucchinetti, 2012). The major histo-logical abnormality or lesion of MS is the plaque (Fig. 1), a focalarea of CNS inflammation and tissue damage (Frohman et al.,2006). In RRMS, new plaques occur at an average rate of approxi-mately 5–10 per year, although there is considerable variation be-tween individuals. Only in about one in 10 plaques is there a ‘‘hit’’on a critical area of the CNS which gives rise to obvious symptomsand deficits. MS occurs in all ethnic groups; however, surveys haveshown that MS is most common in persons of northern Europeandescent. At a finer level, twin studies have shown that, given onetwin with MS, the rate of concordance for MS is approximately2% in dizygotes and 30% in monozygotes, indicating the strong,

0020-7519/$36.00 ! 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.ijpara.2012.10.025

! Tel.: +1 608 263 2593; fax: +1 608 263 0412.E-mail address: [email protected]

International Journal for Parasitology 43 (2013) 259–274

Contents lists available at SciVerse ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

Helminth therapy trials


Gut immunology and Mul0ple sclerosis

LETTERdoi:10.1038/nature10554

Commensal microbiota and myelin autoantigencooperate to trigger autoimmune demyelinationKerstin Berer1, Marsilius Mues1, Michail Koutrolos1, Zakeya Al Rasbi1, Marina Boziki1, Caroline Johner2, Hartmut Wekerle1

& Gurumoorthy Krishnamoorthy1

Active multiple sclerosis lesions show inflammatory changes sug-gestive of a combined attack by autoreactive T and B lymphocytesagainst brain white matter1. These pathogenic immune cells derivefrom progenitors that are normal, innocuous components of thehealthy immune repertoire but become autoaggressive upon patho-logical activation. The stimuli triggering this autoimmune conver-sion have been commonly attributed to environmental factors, inparticular microbial infection2. However, using the relapsing–remitting mouse model of spontaneously developing experimentalautoimmune encephalomyelitis3, here we show that the commensalgut flora—in the absence of pathogenic agents—is essential in trig-gering immune processes, leading to a relapsing–remitting auto-immune disease driven by myelin-specific CD41 T cells. We showfurther that recruitment and activation of autoantibody-producingB cells from the endogenous immune repertoire depends on avail-ability of the target autoantigen, myelin oligodendrocyte glyco-protein (MOG), and commensal microbiota. Our observationsidentify a sequence of events triggering organ-specific autoimmunedisease and these processes may offer novel therapeutic targets.

The relapsing–remitting (RR) mouse model uses transgenic SJL/Jmice expressing, in a large proportion of their CD41 T cells, a trans-genic T-cell antigen receptor (TCR) recognizing MOG peptide 92–106in the context of MHC class II, I-As. These mice spontaneously developexperimental autoimmune encephalomyelitis (EAE) with successivedisease bouts that often affect different central nervous system (CNS)tissues. The disease is initiated by the transgenic CD41 T cells, whichfirst infiltrate the CNS, and by MOG-autoantibody-producing B cellsrecruited from the natural immune repertoire3.

Whereas in our facility close to 80% of RR mice developed spon-taneous EAE within 3–8 months of age, the rate was variable in otherinstitutions, with spontaneous EAE incidences ranging from 35–90%(unpublished data). This recalled previous investigations that alsoobserved that the frequency of spontaneous EAE in myelin-specificTCR transgenic mice varied in different breeding centres4. Because ourmice were reared under specific pathogen-free (SPF) conditions, wetested the possible contributions of the non-pathogenic commensalflora to the triggering of a spontaneous CNS-specific autoimmunedisease.

We first compared the incidence of spontaneous EAE between RRmice housed under SPF and completely germ-free conditions. Thedifferences were marked. Whereas, as reported before, most SPF-bredRR mice came down with EAE within 3–8 months3, germ-free RR miceremained fully protected throughout their life (Fig. 1a). As the com-mensal microbiota have a central function in driving the correctdevelopment of the immune system5, the absence of spontaneousEAE in germ-free RR mice may have reflected a general immune defi-ciency due to missing microbial stimuli. However, two observationsargue against a profound and irreversible non-reactivity. First, RR mice,which had been germ free (and disease free) for 6–12 weeks, promptlydeveloped EAE when re-colonized with conventional commensalmicrobiota (Fig. 1b). Mono-colonization with segmented filamentous

bacteria (SFB), which restored autoimmunity in another mouse model,was of low efficiency (unpublished data). This suggests that theimmune system of germ-free mice had grown efficient enough tomount a full autoimmune attack within a relatively brief period of time.Second, the basic immune competence of germ-free animals was con-firmed by active immunization of germ-free wild-type SJL/J mice withrecombinant MOG (rMOG) in complete Freund’s adjuvant (CFA). Inaccord with one previous report6, although not with another morerecent one7, all immunized germ-free mice developed EAE like theirSPF counterparts, although with some delay (Fig. 1c), and transfer ofpre-activated T cells induced comparable EAE in both germ-free andSPF mice (Supplementary Table 1). Moreover, germ-free and SPF SJL/Jmice immunized with rMOG produced comparable levels of anti-MOG antibodies in their serum (Fig. 1d).

Recent studies established that components of the commensal micro-biota profoundly shape the gut-associated lymphatic tissue (GALT),

1Department of Neuroimmunology, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany. 2Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany.

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Figure 1 | Commensal microbiota are required for the development ofspontaneous EAE. a, Incidence of spontaneous EAE in a cohort of RR micehoused in germ-free (GF; n 5 35) or SPF (n 5 41) conditions. b, Incidence ofspontaneous EAE in germ-free RR mice (n 5 10) re-colonized withconventional flora from SPF mice. c, Delayed EAE onset in germ-free wild-type(GF WT) SJL/J mice immunized with rMOG/CFA. Mean EAE scores (6 s.e.m.)of germ-free (n 5 7) and SPF (n 5 8) SJL/J mice are shown. *P , 0.05;**P , 0.01 (two-way ANOVA). d, Germ-free and SPF wild-type SJL/J miceproduce similar levels of anti-MOG antibodies after immunization. Each circlerepresents an individual mouse and bars depict mean 6 s.e.m. Panels c andd represent two individual experiments.

5 3 8 | N A T U R E | V O L 4 7 9 | 2 4 N O V E M B E R 2 0 1 1

Macmillan Publishers Limited. All rights reserved©2011

Nature 2011 Nov 24, 279, 538-542

LETTERdoi:10.1038/nature10554

Commensal microbiota and myelin autoantigencooperate to trigger autoimmune demyelinationKerstin Berer1, Marsilius Mues1, Michail Koutrolos1, Zakeya Al Rasbi1, Marina Boziki1, Caroline Johner2, Hartmut Wekerle1

& Gurumoorthy Krishnamoorthy1

Active multiple sclerosis lesions show inflammatory changes sug-gestive of a combined attack by autoreactive T and B lymphocytesagainst brain white matter1. These pathogenic immune cells derivefrom progenitors that are normal, innocuous components of thehealthy immune repertoire but become autoaggressive upon patho-logical activation. The stimuli triggering this autoimmune conver-sion have been commonly attributed to environmental factors, inparticular microbial infection2. However, using the relapsing–remitting mouse model of spontaneously developing experimentalautoimmune encephalomyelitis3, here we show that the commensalgut flora—in the absence of pathogenic agents—is essential in trig-gering immune processes, leading to a relapsing–remitting auto-immune disease driven by myelin-specific CD41 T cells. We showfurther that recruitment and activation of autoantibody-producingB cells from the endogenous immune repertoire depends on avail-ability of the target autoantigen, myelin oligodendrocyte glyco-protein (MOG), and commensal microbiota. Our observationsidentify a sequence of events triggering organ-specific autoimmunedisease and these processes may offer novel therapeutic targets.

The relapsing–remitting (RR) mouse model uses transgenic SJL/Jmice expressing, in a large proportion of their CD41 T cells, a trans-genic T-cell antigen receptor (TCR) recognizing MOG peptide 92–106in the context of MHC class II, I-As. These mice spontaneously developexperimental autoimmune encephalomyelitis (EAE) with successivedisease bouts that often affect different central nervous system (CNS)tissues. The disease is initiated by the transgenic CD41 T cells, whichfirst infiltrate the CNS, and by MOG-autoantibody-producing B cellsrecruited from the natural immune repertoire3.

Whereas in our facility close to 80% of RR mice developed spon-taneous EAE within 3–8 months of age, the rate was variable in otherinstitutions, with spontaneous EAE incidences ranging from 35–90%(unpublished data). This recalled previous investigations that alsoobserved that the frequency of spontaneous EAE in myelin-specificTCR transgenic mice varied in different breeding centres4. Because ourmice were reared under specific pathogen-free (SPF) conditions, wetested the possible contributions of the non-pathogenic commensalflora to the triggering of a spontaneous CNS-specific autoimmunedisease.

We first compared the incidence of spontaneous EAE between RRmice housed under SPF and completely germ-free conditions. Thedifferences were marked. Whereas, as reported before, most SPF-bredRR mice came down with EAE within 3–8 months3, germ-free RR miceremained fully protected throughout their life (Fig. 1a). As the com-mensal microbiota have a central function in driving the correctdevelopment of the immune system5, the absence of spontaneousEAE in germ-free RR mice may have reflected a general immune defi-ciency due to missing microbial stimuli. However, two observationsargue against a profound and irreversible non-reactivity. First, RR mice,which had been germ free (and disease free) for 6–12 weeks, promptlydeveloped EAE when re-colonized with conventional commensalmicrobiota (Fig. 1b). Mono-colonization with segmented filamentous

bacteria (SFB), which restored autoimmunity in another mouse model,was of low efficiency (unpublished data). This suggests that theimmune system of germ-free mice had grown efficient enough tomount a full autoimmune attack within a relatively brief period of time.Second, the basic immune competence of germ-free animals was con-firmed by active immunization of germ-free wild-type SJL/J mice withrecombinant MOG (rMOG) in complete Freund’s adjuvant (CFA). Inaccord with one previous report6, although not with another morerecent one7, all immunized germ-free mice developed EAE like theirSPF counterparts, although with some delay (Fig. 1c), and transfer ofpre-activated T cells induced comparable EAE in both germ-free andSPF mice (Supplementary Table 1). Moreover, germ-free and SPF SJL/Jmice immunized with rMOG produced comparable levels of anti-MOG antibodies in their serum (Fig. 1d).

Recent studies established that components of the commensal micro-biota profoundly shape the gut-associated lymphatic tissue (GALT),

1Department of Neuroimmunology, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany. 2Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany.

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Figure 1 | Commensal microbiota are required for the development ofspontaneous EAE. a, Incidence of spontaneous EAE in a cohort of RR micehoused in germ-free (GF; n 5 35) or SPF (n 5 41) conditions. b, Incidence ofspontaneous EAE in germ-free RR mice (n 5 10) re-colonized withconventional flora from SPF mice. c, Delayed EAE onset in germ-free wild-type(GF WT) SJL/J mice immunized with rMOG/CFA. Mean EAE scores (6 s.e.m.)of germ-free (n 5 7) and SPF (n 5 8) SJL/J mice are shown. *P , 0.05;**P , 0.01 (two-way ANOVA). d, Germ-free and SPF wild-type SJL/J miceproduce similar levels of anti-MOG antibodies after immunization. Each circlerepresents an individual mouse and bars depict mean 6 s.e.m. Panels c andd represent two individual experiments.

5 3 8 | N A T U R E | V O L 4 7 9 | 2 4 N O V E M B E R 2 0 1 1

Macmillan Publishers Limited. All rights reserved©2011

4 Gut immunology Gut flora

From Mouse to Man (MS >RR)

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State of the art Luzern 26.01.2013 Presenta0on from Prof Wekerle

Fecal Microbiota Transplanta0on and Mul0ple sclerosis


n engl j med 368;5 nejm.org january 31, 2013 407

The new england journal of medicineestablished in 1812 january 31, 2013 vol. 368 no. 5

Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile

Els van Nood, M.D., Anne Vrieze, M.D., Max Nieuwdorp, M.D., Ph.D., Susana Fuentes, Ph.D., Erwin G. Zoetendal, Ph.D., Willem M. de Vos, Ph.D., Caroline E. Visser, M.D., Ph.D., Ed J. Kuijper, M.D., Ph.D.,

Joep F.W.M. Bartelsman, M.D., Jan G.P. Tijssen, Ph.D., Peter Speelman, M.D., Ph.D., Marcel G.W. Dijkgraaf, Ph.D., and Josbert J. Keller, M.D., Ph.D.

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From the Departments of Internal Medi-cine (E.N., A.V., M.N., P.S.), Microbiology (C.E.V.), Gastroenterology (J.F.W.M.B., J.J.K.), and Cardiology (J.G.P.T.) and the Clinical Research Unit (M.G.W.D.), Aca-demic Medical Center, University of Am-sterdam, Amsterdam; the Laboratory of Microbiology, Wageningen University, Wageningen (S.F., E.G.Z., W.M.V.); the Department of Experimental and Medical Microbiology, Leiden University Medical Center, Leiden (E.J.K.); and the Department of Gastroenterology, Hagaziekenhuis, The Hague (J.J.K.) — all in the Netherlands; and the Department of Bacteriology and Immunology, Medical Faculty, University of Helsinki, Helsinki (W.M.V.). Address reprint requests to Dr. Keller at the Aca-demic Medical Center, Department of Gastroenterology, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands, or at [email protected].

This article was published on January 16, 2013, at NEJM.org.

N Engl J Med 2013;368:407-15.DOI: 10.1056/NEJMoa1205037Copyright © 2013 Massachusetts Medical Society.

BackgroundRecurrent Clostridium difficile infection is difficult to treat, and failure rates for anti-biotic therapy are high. We studied the effect of duodenal infusion of donor feces in patients with recurrent C. difficile infection.

MethodsWe randomly assigned patients to receive one of three therapies: an initial vanco-mycin regimen (500 mg orally four times per day for 4 days), followed by bowel lavage and subsequent infusion of a solution of donor feces through a nasoduode-nal tube; a standard vancomycin regimen (500 mg orally four times per day for 14 days); or a standard vancomycin regimen with bowel lavage. The primary end point was the resolution of diarrhea associated with C. difficile infection without relapse after 10 weeks.

ResultsThe study was stopped after an interim analysis. Of 16 patients in the infusion group, 13 (81%) had resolution of C. difficile–associated diarrhea after the first infu-sion. The 3 remaining patients received a second infusion with feces from a differ-ent donor, with resolution in 2 patients. Resolution of C. difficile infection occurred in 4 of 13 patients (31%) receiving vancomycin alone and in 3 of 13 patients (23%) receiving vancomycin with bowel lavage (P<0.001 for both comparisons with the infusion group). No significant differences in adverse events among the three study groups were observed except for mild diarrhea and abdominal cramping in the in-fusion group on the infusion day. After donor-feces infusion, patients showed in-creased fecal bacterial diversity, similar to that in healthy donors, with an increase in Bacteroidetes species and clostridium clusters IV and XIVa and a decrease in Proteobacteria species.

ConclusionsThe infusion of donor feces was significantly more effective for the treatment of recurrent C. difficile infection than the use of vancomycin. (Funded by the Nether-lands Organization for Health Research and Development and the Netherlands Organization for Scientific Research; Netherlands Trial Register number, NTR1177.)

The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITE DE GENEVE on December 23, 2013. For personal use only. No other uses without permission.

Copyright © 2013 Massachusetts Medical Society. All rights reserved.

Fecal Microbiota Transplanta0on


Risk factors and multiple sclerosis

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Conclusion : risk factors and mul0ple sclerosis

Gene$c

Environment

Pathogens (viral exposi0on)

Vitamin (D) Smoking Diet

Commensal flora

Epigene$c

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Dietary Salt Associated With MSActivityPublished: Oct 4, 2013 | Updated: Oct 4, 2013

By John Gever, Deputy Managing Editor, MedPageTodayReviewed by Zalman S. Agus, MD; Emeritus Professor,Perelman School of Medicine at the University of

Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner

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Note that this study was published asan abstract and presented at aconference. These data andconclusions should be considered to bepreliminary until published in a peer-reviewed journal.

This study suggests that higher sodiumintake is associated with increasedclinical activity and MRI lesions inpatients with multiple sclerosis.

COPENHAGEN -- Sodium intake waspositively correlated with risk of increaseddisease activity in patients with multiplesclerosis, according to a small studyreported here.

Each gram of estimated daily sodiumintake above the average in a 52-patientsample was associated with an increase of3.65 in MRI lesion counts, said MauricioFarez, MD, PhD, of Fundación para laLucha contra las EnfermedadesNeurológicas de la Infancia in BuenosAires.

Also, patients with estimated salt intakeclassified as high -- more than 4.8 g daily --showed relapse rates that were 3.95 timesgreater (95% CI 1.39-11.21) than thosewith intakes less than 2 g/day, he toldattendees at the European Committee forTreatment and Research in MultipleSclerosis annual meeting.

Farez emphasized repeatedly that thefindings did not prove that high salt intakecaused the increased disease activity. Heacknowledged that, if there is a causal relationship, it possibly could go in the reversedirection -- that patients with highly active MS may increase their salt intake as a result. Buthe said he did not view that as very likely.

The study in a total of 122 patients with relapsing-remitting MS grew out of previousresearch connecting salt intake with vitamin D levels and body mass index, he said.Numerous studies have indicated an association between vitamin D status and MS risk --including one reported minutes earlier at the Copenhagen meeting -- and it seemed logical toexamine whether sodium may share a similar association, Farez explained.

He and colleagues initially recruited 70 patients for a first phase of the observational study.They underwent a baseline MRI scan in November 2010, followed by MRI scans and analysisof urinary sodium excretion as a means of estimating sodium intake 1 year later. Finally, inNovember 2013, relapse rates for the preceding 2 years were calculated.

During this first phase, the MRI analyses included "combined unique activity" counts -- thetotal of new T2 lesions and new gadolinium-enhancing T1 lesions since the baseline scan.

A second group of 52 patients was examined in June 2013 with MRI scans and urinarysodium testing to provide replication data for the association between sodium intake andMRI lesion activity. Because this group had only a single scan and no follow-up, Farez and

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Risk factors in Multiple Sclerosis: Detection and Treatment in Daily Life

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