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Vaccine (2008) 26, 20562057

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Strain-dependent effects of probiotic lactobacilli on EAE autoimmunity


Catharina B.M. Maassen a, Eric Claassen b,c,
a

Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands Athena Institute Vrije Universiteit, Amsterdam, The Netherlands c ErasmusMC, Rotterdam, The Netherlands
b

Received 20 February 2008; accepted 20 February 2008 Available online 3 March 2008

KEYWORDS
Lactobacilli; EAE; Probiotics; Rat; Multiple sclerosis

Summary In this study we present new data showing strain-specic differences on the effect of commercially available probiotic drinks in an EAE rat autoimmune model for multiple sclerosis. In this particular model, we conclude that these drinks do not enhance but rather suppress the disease. We suggest that conclusions on probiotics are limited to specic strains and models and not generalised. We further suggest that physiological use (normal route, normal dose, normal growth phase, specic strain or substrain/species) is studied in all cases, so as not to overwhelm (high dose) or circumvent natural immune processing. 2008 Elsevier Ltd. All rights reserved.

Lactobacilli are among the most prominent members within a plethora of bacterial species with probiotic properties. The popularity of lactobacilli is based on millennia of use in food and feed and almost a hundred years in commercially available probiotic dairy drinks and yoghurts. Most lactobacilli are formally classied as GRAS (generally recognised as safe) organisms. In a recent study [1], lactobacilli were implied in an increased risk of mortality in severe acute pancreatitis patients. The mixture used in this study was uniquely designed for this specic purpose and not available as food. Furthermore, the normal oral route was not used but the bacteria were given by direct enteral

Corresponding author at: ErasmusMC, Viroventures, Dr. Molewaterplein 50, Room Ee 19.87, 3015 GE Rotterdam, The Netherlands. Tel.: +31 620443098; fax: +31 320264828. E-mail address: eric.claassen@planet.nl (E. Claassen).

drip. Nevertheless the resulting anxiety under consumers, scientists and industry alike resulted in a generalisation of these very specic negative effects. In another recent paper [2] lactobacilli cultured from a product available to the consumer were implicated in (marginal) increase in the duration of autoimmunity in a rat model. In earlier studies we showed that immunological effects of lactobacilli are not only strain but also growth-phase-dependent [3], indicating that effects found after culturing [2] commercially available strains could be different from what one would expect with the intact product. In addition to this we showed straindependent cytokine induction [4], as measured, without culturing artefacts in situ in the gut, after oral administration of these strains. These and other studies led to a set of recombinant lactobacilli, expressing human brain proteins, being exquisitely capable of suppressing EAE (experimental autoimmune encephalomyelitis, a model for multiple sclerosis) in both rats and mice [5]. Recombinant receptor strains

0264-410X/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.02.035

Strain-dependent effects of probiotic lactobacilli on EAE autoimmunity

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Figure 1 EAE was induced, and disease activity (DAS) scored exactly as described before [5] Briey: cumulative DAS over 30 days was determined and control rats were 100%. Rats were fed an average of 1.2 10E8 CFU of either placebo yoghurt Lactobacillus casei393, Danone: Lactobacillus casei DN 114-001, Vit: Lactobacillus rhamnosus Goldin-Gorbach or Yakult: Lactobacillus casei Shirota on alternate days for seven feedings before induction of EAE. Results were coded and randomly represented as 1, 2 and 3.

more, we conclude that physiological doses of approved non-adulterated probiotic yoghurt drinks given through the oral route show no enhancement (but rather suppression) of disease in this model. These ndings t well with the old friends hypothesis [7] in which probiotics signal through gut dendritic cells, which in their turn regulate T-regulator cells that can then suppress by specic immuno regulation. These effects are dose- and organism-dependent as long as the probiotics are recognised by the innate immune system as harmless. This direct interaction, of dendritic cells at the mucosal surface of the intact gut, is an essential element in the regulation and can, in our opinion, not be mimicked by closely related strains, bypassed [1] by duodenal drip or overwhelmed [2] with high doses without consequences.

Acknowledgement
Funding: Research was supported by grant MS93-147 from The Netherlands Foundation for supporting Multiple Sclerosis Research.

References
were selected on: (1) cytokine induction prole in the gut, (2) intrinsic EAE suppressive capabilities of the wild type and (3) permissiveness for recDNA modulation. The unmodied strains described before [6], (Lactobacillus plantarum NCIB 8826 and Lactobacillus murines CNRZ) could statistically signicantly reduce cumulative disease burden by 21% and 55%, respectively. After recDNA modication, myelin basic protein expression and oral ingestion disease suppression rose to almost 80% [5]. In Fig. 1 we show data from studies not published before. Three competing brands, the only ones in The Netherlands, which passed the voluntary behavioural code for health and safety claims by the Dutch Voedingscentrum, were investigated for EAE modulating effects in Lewis rats. We here show that all products are capable of reducing total disease burden (2763%) and found the same reductive effects in max disease score and time of onset but not in duration of disease. When these studies were performed in SJL/J mice no statistical differences were found between control and any of the lactobacilli preparations. It should be duly noted that probiotics used in this study (Fig. 1) were directly off the shelf (i.e. not lab cultured) and given orally in a normal physiological dose for only seven times during a 44-day experiment. Furthermore, at the time of this study (1994) all products were confusingly classied as different strains of Lactobacillus casei. We conclude that the effects of lactobacilli on an EAE autoimmune model can be modulated up or down depending on the specic strain used. We emphasise that these results also show major differences even between closely related strains. Further[1] Besselink MGH, Santvoort van HC, Buskens E, Boermeester MA, Goor van H, Timmerman HM, et al. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomized, double blind, placebo-controlled trial. Lancet 2008;371:6519. [2] Ezendam J, Van Loveren H. Lactobacillus casei Shirota administered during lactation increases the duration of autoimmunity in rats and enhances lung inammation in mice. Br J Nutr 2007;8(99):8390. [3] Maassen CBM, Boersma WJA, Van Holten-Neelen C, Claassen E, Laman JD. Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens: implications for vaccine development. Vaccine 2003;21:27517. [4] Maassen CBM, Holten-Neelen van C, Balk F, Heijne den BakGlashouwer MJ, Leer RJ, Laman JD, et al. Strain-dependent induction of cytokine proles in the gut by orally administered Lactobacillus strains. Vaccine 2000;18:261323. [5] Maassen CBM, Laman JD, Holten-Neelen C, Hoogteijling L, Groenewegen L, Visser L, et al. Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigens. Vaccine 2003;21:468593. [6] Maassen CBM, Van Holten JCAM, Balk F, Heijne den BakGlashouwer MJ, Leer R, Laman JD, et al. Orally administered lactobacillus strains differentially affect the direction and efcacy of the immune response. Gastrointestinal disorders in Juveniles. Vet Q 1998;20(3):813. [7] Guarner F, Bourdet-Sicard R, Brandtzaeg P, Gill HS, McGuirk P, van Eden W, et al. Mechanisms of disease: the hygiene hypothesis revisited. Nat Clin Pract Gastroenterol Hepatol 2006;3(5):27584.