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The effects of a synbiotic in infants with atopic dermatitis
van der Aa, L.B.
Publication date
2010
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Final published version
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Citation for published version (APA):
van der Aa, L. B. (2010). The effects of a synbiotic in infants with atopic dermatitis.
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Leontien van der Aa
The effects of a synbiotic
in infants with atopic dermatitis
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atopic dermatitis
The effects of a synbiotic in infants with atopic dermatitis
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof.dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde
commissie, in het openbaar te verdedigen in de Aula der Universiteit
op vrijdag 15 oktober 2010, te 11.00 uur
door
Leontien Barbara van der Aa
Promotores:
Prof.dr. H.S.A. Heymans
Prof.dr. W.M.C. van Aalderen
Co-promotor:
Dr. A.B. Sprikkelman
Overige leden:
Dr. M.A. Benninga
Prof.dr. R. Gerth van Wijk
Prof.dr. M.L. Kapsenberg
Prof.dr. T.W. Kuijpers
Prof.dr. E.E.S. Nieuwenhuis
Prof.dr. Z. Szépfalusi
Colofon
The effects of a synbiotic in infants with atopic dermatitis Thesis, University of Amsterdam, the Netherlands Copywright © 2010 L.B. van der Aa
All rights are reserved. No part of this thesis may be reproduced, stored or transmitted in any form or by any means, without prior permission of the author.
Lay-out: GVO drukkers & vormgevers B.V. | Ponsen & Looijen Printed by: GVO drukkers & vormgevers B.V. | Ponsen & Looijen Cover: Tobias Bruijn
ISBN: 978-90-6464-416-0
The printing of this thesis was financially supported by: Danone Research-Centre for Specialised Nutrition, ABN-AMRO, het Astma Fonds en Abbott B.V.
Chapter 1 General introduction and outline of the thesis
Pediatric Allergy and Immunology 2010 Mar;21(2 Pt 2):e355-67
Chapter 2 Effect of a new synbiotic mixture on atopic dermatitis in infants: a randomized controlled trial
Clinical & Experimental Allergy 2010 May;40(5):795-804
Chapter 3 No detectable beneficial systemic immunomodulatory effects of a specific synbiotic mixture in infants with atopic dermatitis
Submitted
Chapter 4 Immunological differences between infants with IgE-associated and non-IgE-associated atopic dermatitis
Submitted
Chapter 5 Synbiotics prevent asthma-like symptoms in infants with atopic dermatitis
Allergy 2010 June 17 (Epub ahead of print)
Chapter 6 Impact of maternal atopy and probiotic supplementation during pregnancy on infant sensitization- letter to the editor
Clinical & Experimental Allergy 2008 Oct;38(10):1698
Chapter 7 Infant feeding and allergy prevention: a review of current knowledge and recommendations. A EuroPrevall state of the art paper.
Allergy 2009 Oct;64(10):1407-16
Chapter 8 Summary and general discussion
Appendices Chapter 7
Nederlande samenvatting voor de niet-medisch geschoolde Dankwoord Curriculum Vitae 13 37 57 73 87 103 107 125 137 151 159 165
General introduction and
Outline of the thesis
L.B. van der Aa H.S.A. Heymans W.M.C. van Aalderen A.B. Sprikkelman
Chapter
1
Pediatric Allergy and Immunology 2010 Mar;21(2 Pt 2):e355-67
Adapted from:
Probiotics and prebiotics in atopic dermatitis:
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increase in the prevalence of Th1-mediated autoimmune diseases in the Western world does not concur with the Th1/Th2 paradigm.
These inconsistencies have lead to the proposal of a revised hygiene hypothesis, which considers changes in the intestinal colonisation pattern during infancy, also caused by an overly hygienic lifestyle, as an important reason for the increased allergy prevalence and proposes a lack of activity of regulatory T cells (Tregs), causing overactive Th2 as well as Th1 responses, as the underlying mechanism (18-21).
The intestinal microbiota
The human intestines are inhabited by at least 400 different bacterial species, with the greatest density in the large intestine, where concentrations of 1011-1012 cells/g of luminal contents can
be found (22). Approximately 55% of faecal mass consists of bacteria (23). After birth, bacteria originating from the mother and the environment start colonizing the infant gut. Factors influencing the colonization pattern are mode of delivery (vaginal delivery versus caesarean section), prematurity, hospitalization or antibiotic use after birth, type of feeding (breastfeeding versus formula feeding) and exposure to older siblings (24).
Although some intestinal bacteria are potential pathogens, the relation between the intestinal microbiota and the human host is mostly symbiotic. The microbiota has several important nutritional functions, such as degradation of indigestible dietary carbohydrates, production of short chain fatty acids (SCFA), amino acid synthesis and vitamin synthesis (25). In addition, the intestinal flora appears to be crucial for the development of the mucosal and systemic immune system.
The intestinal microbiota and the immune system
The largest mass of lymphoid tissue of the body is found in the gastrointestinal tract and is called the gut-associated lymphoid tissue (GALT). The GALT interacts with intestinal bacteria, which are sampled by dendritic cells and intestinal epithelial cells, through two classes of pattern recognition receptors, Toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD) molecules (26;27).
The intestinal microbiota appears to be important for the development of the GALT. Studies have shown that mice without microbiota, i.e. germ-free mice, have an underdeveloped GALT with low numbers of ab- intestinal intraepithelial lymphocytes (28), hypoplastic Peyer’s patches containing few germinal centres and greatly reduced numbers of IgA-producing plasma cells and lamina propria CD4+ T cells (29).
Furthermore, the intestinal microbiota also seems to be involved in oral tolerance induction. Oral tolerance is the establishment of peripheral tolerance to a specific antigen after ingestion of that antigen. A pivotal role in the induction and maintenance of peripheral tolerance is played by Tregs. The importance of the intestinal microbiota in tolerance induction was demonstrated by Sudo and coworkers, who showed that germ-free mice do not develop oral tolerance after ingestion of ovalbumin and maintain a Th2 response with IgE production (30). Inoculation with Introduction
Atopic dermatitis (AD) is a chronic, itching, inflammatory skin disease that often presents in infancy (1). The prevalence of AD has risen over the past decades, especially in western societies where it varies in primary school children between 5% and 20% (2). The disease is caused by a combination of genetic and environmental factors. Severe AD in children is often associated with food allergy (3;4). The majority of patients have elevated serum IgE levels and peripheral eosinophilia, although in up to 57% of infants with AD, IgE-sensitization can not (yet) be detected (5).
The disease has a significant impact on children and parents, mainly due to itching, scratching and sleep disruption (6). The prognosis is reasonable with a recovery rate of 40% at age two (7) and 65% in adolescence (8). However, AD can be the starting point of the ‘allergic march’, the natural progression of allergic disorders such as asthma and allergic rhinitis. Children with AD have a chance of approximately 40% to develop asthma (7).
Besides avoiding irritants and moisturizing the skin with emollients, local anti-inflammatory treatment with topical corticosteroids is the mainstay treatment for infants with AD. In children with a proven food allergy this is combined with avoidance of the specific allergen. However, flares occur despite treatment and, although rare, topical corticosteroids can have local side effects, such as skin atrophy and telangiectasia, and possibly also systemic side effects, such as growth retardation or osteoporosis. Parents often fear these side effects and this may lead to non-compliance (9). Innovative prevention and treatment strategies for AD, aiming to manipulate the intestinal flora with pro-, pre- or synbiotics are now focus of interest. This review provides an overview of the theoretical basis for using probiotics and prebiotics in AD and presents the current evidence from randomized controlled trials regarding prevention and treatment of AD in children with pro-, pre- and synbiotics.
Hygiene hypothesis
Several hypotheses have been proposed to explain the rise in allergic disease, including atopic dermatitis. The hygiene hypothesis (10) has gained the most attention. This hypothesis was based on the finding that the prevalence of allergic rhinitis and eczema is inversely related to the number of older children in a household, which lead to the general hypothesis that early childhood infections, caused for example by unhygienic contact with older siblings, could prevent the development of allergic disease (10;11). Later, the T helper 1/ T helper 2 lymfocyte paradigm was added to the theory. It was argued that a lack of early childhood infections results in a decreased Th1 response, which disturbs the Th1/Th2 balance and leads to an abundant Th2 response, causing allergic diseases (12).
However, several facts are not consistent with the hygiene hypothesis as it was first proposed. Although the protective effect of multiple siblings, as well as other “unhygienic” circumstances like exposure to farming or day care (13;14), has been a consistent finding, a number of cross-sectional and prospective studies showed no protective effect of childhood infections (15;16). Moreover, Th2-dominated helminthic infections are not associated with allergy (17) and the coinciding
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17 General introduction and Outline of the thesisof bifidobacteria. Recently, a Dutch multicenter trial reported an increased mortality risk in adults with severe acute pancreatitis who received probiotic prophylaxis (44). However, in children with AD no adverse events are reported.
Immunomodulatory effects of probiotics
Many different effects of probiotics have been described in animal, human and in vitro studies, most of which are strain-specific. In general, effects include stabilizing of intestinal barrier function (45;46), stimulation of intestinal IgA-production (47) and, most importantly, modulation of cytokine production. In vitro studies show that probiotics are able to down-regulate Th2 cytokine production by stimulation of Th1 cytokines, such as IL-12 and IFN-g (48) or regulatory cytokines, such as IL-10. The latter is accomplished by either increasing production of these cytokines by antigen presenting cells or by driving the development of IL-10 or TGF-b producing regulatory T cells (49-51).
However, ex vivo immunomodulatory effects of probiotics in children with AD are inconclusive (table 1). There are studies that show up-regulation of IL-10 (52-54) or IFN-g (55;56), although often without accompanying down-regulation of Th2 cytokines, but several studies do not show any effect on cytokine production (57-62). Also, one study showed down regulation of the Th2 cytokine IL-5, combined with down-regulation of IL-10 and TGF-b instead of the expected up-regulation of these cytokines (59). These conflicting outcomes could be caused by the use of different probiotic strains and different ways of stimulating cytokine production.
Prebiotics
Prebiotics are nondigestible food ingredients that beneficially affect the host by stimulating the growth and/or activity of a limited number of bacterial species in the colon (63). A food ingredient must fulfil three criteria to be considered a prebiotic: it should not be hydrolyzed or absorbed in the upper part of the gastrointestinal tract, it has to be a selective substrate for beneficial commensal bacteria in the colon, for example bifidobacteria, and it must be able to alter the intestinal microbiota towards a healthier composition (64).
Breast milk contains natural prebiotics, human milk oligosaccharides, which could explain the bifidobacteria-dominated microbiota seen in breast fed infants (65). In formula fed infants a similar effect can be realized with a prebiotic mixture of 90% galacto-oligosaccharides (GOS) and 10% fructo-oligosaccharides (FOS), which stimulates the growth of bifidobacteria (66;67) and, to a lesser extent, lactobacilli (68).
Prebiotics are generally considered to be safe, as they are naturally present in several kinds of food. The main side effects of over-consumption in humans are flatulence, bloating and diarrhoea (69). Increased intestinal tumour formation in mice fed inulin has been reported (70), but on the opposite, reduction of colon tumours in mice, has also been described (71).
Immunomodulatory effects of prebiotics
Several animal studies demonstrated effects of prebiotics on the immune system. Elevation of the
Bifidobacterium infantis restored susceptibility to oral tolerance induction, but only when this
was done in the neonatal stage (30). Others have shown that germ-free mice have impaired regulatory T cell function (31) and that colonization of germ-free rats with Lactobacillus
plantarum increases the number of Tregs (32).
Association between microbiota and atopic disease
Studies investigating the composition of the intestinal microbiota in humans have produced more evidence for the role of commensal bacteria in the development of allergies. Breast fed infants have a relatively simple microbiota dominated by bifidobacteria, while formula fed infants have a more diverse flora with higher counts of other anaerobes, e.g. clostridia, and aerobes (33). The lower prevalence of AD in breast fed infants (34) is possibly linked to this difference in composition of intestinal microbiota.
Studies comparing flora composition of atopic and non-atopic infants also show significant differences, which already exist in the first few weeks of life and therefore precede the development of atopic disease (35-37). Although there are some inconsistencies, generally these studies show that atopic children have less bifidobacteria and more clostridia than non-atopic children. However, in a recent multi-centre birth cohort study no significant relation between colonization pattern in infancy and atopy was found (38). Also, a study comparing the composition of the intestinal microbiota between children with IgE (against food allergens)-associated AD and non-IgE-allergens)-associated AD found no significant differences (39). So up to now, the role of the intestinal microbiota in the development of atopic disease in childhood is still not clarified.
Probiotics
If the revised hygiene hypothesis holds true, then it is feasible that atopic disease can be treated or even prevented by manipulating the microbiota. This can be done with probiotics, prebiotics, or synbiotics.
The term probiotic is derived from the Greek language and means “for life”. Probiotics are defined as live micro organisms which, when administered in adequate amounts, confer a health benefit on the host (40). Probiotic agents are preferably isolated from the human gastrointestinal tract and should be non-pathogenic. They must reach the human intestine alive and be able to adhere to the epithelial surface; therefore they have to be resistant to gastric acid digestion and bile salts (41). The bacterial genera most commonly used as probiotics are Lactobacillus and
Bifidobacterium. In addition, yeasts have also been used as probiotics, especially Saccharomyces boulardii (42).
Probiotics have been safely used in the fermentation of food products for decades. Therefore, the United States Food and Drug Administration has designated probiotics as Generally Recognized as Safe (GRAS). However, there have been reports of sepsis linked to probiotic ingestion, all in patients with underlying medical conditions (43). Sepsis was caused by Lactobacillus species,
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Table 1. Effects of probiotics on cytokine responses in children with AD
Study Probiotic Dose (cfu) Number of subjects Age at inclusion
(mean) Treatment period Cytokines Immunomodulatory effect
Majamaa
1997 (62) LGG
a ATCC 53103 5 x 108 cfu/g
formula 27 (AD and cow’s milk allergy) 2.5-15.7 months (range) 4 weeks PBMCs
f (stimulus conA and cow’s milk protein): no difference in IL-4,
IFN-g and TNF-α between the probiotic and the placebo group No effect Pessi 2000 (52) LGG ATCC 53103 2dd 1x1010 9 21 months 4 weeks Serum: IL-10 at 8 weeks, no effect on IL-6, IL-12, IFN-g and TNF-a
PBMCs (stimulus anti-CD3, 4 patients): IL-10 and ¯ IL-4, IL-2 and IFN-g at 4 weeks
Up-regulation regulatory cytokine, down regulation Th1 and Th2 cytokines (no placebo group) Rosenfeldt
2003 (61) L.
b rhamnosus
19070-2 and
L. reuteri DSM 12246
2dd 1 x 1010 43 5.2 years 6 weeks PBMCs (stimulus LPS-PHA): no significant changes in IL-2, IL-4, IL-10
and IFN-g No effect
Pohjavuori
2004 (55) LGG ATCC 53103 or Mixturea 2dd 5 x 10
9 62, of which 38
IgE-associated AD 1.4-11.5 months (range) 4 weeks PBMCs (stimulus: anti-CD3/anti-CD28): IFN-g in LGG group only in IgE-associated AD. No effect on IL-4, IL-5 and IL-12 and in mix group Up-regulation Th1 cytokine Viljanen 2005
(53) LGG ATCC 53103 or Mixturec 2dd 5 x 10 9
2dd c 121, of which 69 IgE-associated AD 1.4-11.5 months (range) 4 weeks Plasma: IL-10 in mix group, IL-6 in LGG group only in IgE-associated AD. No effect on IL-4, TGF-b and IFN-g Up-regulation regulatory cytokine and proinflammatory cytokine
Prescott 2005
(56) L. fermentum VRI-003 PCC 2dd 1 x 10
9 53 10.9 months 8 weeks PBMCs stimulated with PHAg and SEBh. IFN-g response, stimulated with
heat-killed bacteria: TNF-a response and with ovalbumin: ¯IL-13 response in probiotic group
No effect on IL-6, IL-10 and TGF-b responses
Up-regulation pro-inflammatory cytokines and down regulation TH2 cytokine
Between-group analyses: only significant difference in TNF-a Brouwer 2006
(60) L. rhamnosus or LGG 3 x 10
8
(cfu/g powder) 23 5.2 months 12 weeks PBMCs (stimulatus conA and anti-CD3/anti-CD28): no effect on IL-4, Il-5 and IFN-g No effect Taylor 2006
(59) L. acidophilus LAVRI-A1 3 x 10
9 118 infants with
allergic mother Prevention study Infants: 6 months PBMCs stimulated with SEB: ¯IL-5 and ¯TGF-b, tetanus toxoid: ¯IL-10, and house dust mite: ¯ frequent IL-10 and TNF-a responses in probiotic group
Other stimuli (PHA, ovalbumin, b-lactoglobulin, house dust mite): no effect on IL-5, IL-6, IL-10, IL-13, IFN-g, TGF-b and TNF-a response
No significant difference in the proportion of circulating CD4+CD25+CTLA4+ T cells or resting FOXp3 expression
Downregulation of Th2, regulatory and proinflammatory cytokines No increase in thymus derived regulatory
T cells Flinterman
2007 (58) Mixture
d 1 x 109 13 (AD and cow’s milk
allergy) 1.7 years 3 months PBMCs unstimulated and stimulated with peanut extract or anti-CD3: ¯IL-10 and ¯IL-6 and TNF-a in probiotic group, but non-significant compared to placebo (p0.063) Non-significant downregulation of proinflammatory and immunosuppressive cytokines Kopp 2007 (57) LGG ATCC 53103 5 x 10
9 68 infants with atopic
family history Prevention study Mothers: 4-6 weeks before delivery Infants: 6 monthse
CBMCsi and PBMCs stimulated with PHA, LGG or b-lactoglobulin: no
difference in IL-10, IL-13 and IFN-g between the 2 groups No effect Marschan
2008 (54) Mixture
c c 98 infants with atopic
family history Prevention study Mothers: 2-4 weeks before delivery Infants: 6 months
Plasma: IL-10 in synbiotic group
Il-2, IL-4, IL-6, IFN-g and TNF-a values below detection limit in both groups Up-regulation of immunosuppressive cytokine
aLactobacillus rhamnosus GG, bLactobacillus, cLGG ATCC 53103 5x109 cfu, L. rhamnosus LC705 5x109
cfu, Bifidobacterium breve Bbi99 2x108 cfu, Propionibacterium freudenreichii ssp shermanii JS 2x109 cfu,
dL. acidophilus, L. casei, Lactococcus lactis, B. infantis, B. lactis and B. longum, ein case of breast feeding mothers took
the probiotics, fperipheral blood mononuclear cells, gphytohemaglutinin mitogen, hStaphylococcus antigen, icord
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21 General introduction and Outline of the thesisIn contrast, two prevention studies did not show a reduced AD incidence (87;88). Moreover, one of these studies found an increased sensitization rate for at least one out of ten food- or aeroallergens and an increased rate of skin prick test positive (food and/or inhalant allergens)AD in the probiotic group (87).
There are several possible explanations for these inconclusive results. First, different Lactobacillus species were used. Probiotic effects are strain specific, therefore not all probiotic strains might be good candidates for AD prevention. Second, probiotic effects are probably dose-dependent and there was considerable variation in the daily dosage that was given in these studies. A third explanation could be differences in study design, such as number of participants, atopic risk of participants, maternal supplementation during pregnancy, supplementation directly to infants or via breastfeeding and the supplementation period.
Probiotics and treatment of atopic dermatitis
RCTs exploring the role of probiotics, mainly lactobacilli, in the treatment of AD in children also have conflicting results (table 3). Four studies had positive outcomes. The first study showed a significant reduction of SCORAD (SCORing Atopic Dermatitis (89)) score in the probiotic group, but unfortunately the study had few participants and no between-group analysis was performed (62). The second study was also small and the included children had relatively mild AD. Although there was a significant reduction in SCORAD score after two months of probiotic treatment compared to placebo, this difference disappeared after 6 months of treatment, as SCORAD score was 0 by that time in all groups (90). The study of Kirjavainen et al (91) was designed to assess the effect of heat-inactivated probiotics. Although this study was terminated early due to gastrointestinal adverse events in the heat-inactivated group and therefore had few participants, it did show that SCORAD score decreased significantly more in the viable probiotic group than in the placebo group. The fourth study, of Weston et al, had more participants, with more severe AD (92). A significant reduction of SCORAD score was found in the probiotic group and not in the placebo group, but this difference was not significant in between-group analysis.
Three other studies showed no effect on AD in general, but did find a significant, although modest, positive effect on SCORAD score in children with IgE-associated AD (61;93;94). This suggests that possibly only children who at a young age already have evidence of IgE sensitization (positive skin prick test and/or elevated total or specific IgE levels) benefit from probiotics. Children in two of these studies (61;94) had a mean age at inclusion of 4 to 5 years, which was considerably older than the age at inclusion in other treatment studies. In the third study (93) children were younger but were only treated for a period of 4 weeks. These factors possibly reduced the beneficial effect of probiotics.
Finally, three studies didn’t show any effect of probiotics on the severity of AD (60;95;96). In the study of Brouwer et al (60) groups were small, since three groups were formed (two probiotic groups and one placebo group) of the 50 children that were included in total. However, the two other studies (95;96) were well-designed, with more participants, and did not show any effect on AD or on IgE-associated AD.
total cell number in Peyer’s patches (72), increased natural killer cells and peritoneal macrophage phagocytic activity (73), increased production of IgA, IL-10 (74), IFN-
g
(75) and TGF-β (76) have all been described. A human study in which FOS was given to 10 patients with Crohn’s disease reported an increase in the number of IL-10 positive dendritic cells (77).There are at least two possible underlying mechanisms that can elicit the immunological effects of prebiotics. First, most prebiotics stimulate the growth and/or activity of lactic acid bacteria, such as bifidobacteria or lactobacilli, which have immunomodulatory qualities as described above. Second, fermentation of prebiotics by lactic acid bacteria enhances SCFA and lactate production (63). SCFA’s primarily act as energy substrate for colonocytes and several other cells in the human body (78). The three main SCFA’s are acetate, propionate and butyrate. In vitro, acetate and propionate are significantly increased after prebiotic fermentation (79). It has been shown that these two SCFA’s stimulate IFN-γ and IL-10 production (80). Receptors for SCFA’s have been identified on leukocytes (81), which could explain their immunological effect but more studies are needed to elucidate this pathway.
Pro-, pre- or synbiotics for prevention and treatment of atopic dermatitis
To identify all randomized controlled trials (RCTs) regarding prevention or treatment of AD in children with probiotics, prebiotics and combinations of both, i.e. synbiotics, the databases of Pubmed, Embase and Cochrane up to February 2008 were searched. The following keywords were used: (probiotics OR prebiotics OR synbiotics) AND (atopic dermatitis OR atopic eczema OR eczema OR food allergy). Only randomized controlled trials considering children were included in this review. Reference lists of the found articles were checked for additional randomized studies.
Probiotics and prevention of atopic dermatitis
Several RCTs have investigated the effect of probiotics on the prevention of AD (table 2). In five trials the preventive effect on the development of AD in infants was investigated. In these trials probiotics (different Lactobacillus species and one Bifidobacterium strain) were given to infants with a high risk of developing allergy, starting immediately after birth. In four of these trials mothers also received probiotics during the last weeks of pregnancy.
The first study reported a 50% reduction of the incidence of AD in the probiotic group compared to the placebo group at the age of 2 years (82). This effect was still evident at the age of 4 and 7 years (83;84). No effect on the incidence of other allergic diseases or sensitization was found. In the study of Abrahamsson and co-workers no reduction of the incidence of AD was found, but subgroup analysis revealed a significant reduction in IgE-associated AD (85). Again, no effect on the incidence of other allergic diseases or sensitization was found. Recently, Wickens et al showed that Lactobacillus rhamnosus, but not Bifidobacterium animalis, significantly reduced the incidence of AD with almost 50% compared to placebo, without any effect on sensitization (86).
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Table 2. Prevention of AD with probiotics, prebiotics and synbiotics
Study Probiotic Dose Study design Number of subjects Treatment
period Follow-up period Level of evidencef Effect
Kalliomaki
2001 (82) LGG
a 1x1010 cfu RDBPCTd 132,
mother, father or sibling with atopic disease
Mothers: 2-4 weeks before delivery Infants: 6 monthse
7 years A2 Reduction of AD incidence at age 2 (23% vs. 46%, p 0.008), reduction still significant at age 4 and 7.
No reduction other allergic diseases or sensitization. Abrahamsson 2007 (85) L. b reuteri (ATCC 55730) 1x10 8 cfu RDBPCT 188, mother, father or sibling with atopic disease
Mothers: week 36 until delivery Infants: 12 months
2 years A2 No reduction of eczema incidence (probiotics: 36%, placebo: 34%). Less IgE-associated eczema at age 2 (8% vs. 20%, p 0.02). No reduction other allergic diseases or sensitization.
If allergic mother: significantly less IgE-associated AD and less sensitization at age 6-24 months.
Taylor
2007 (87) L. acidophilus (LAVRI-A1) 3x10
9 cfu RDBPCT 178,
atopic mother Infants: 6 months 1 year A2 No prevention of AD (probiotics: 26%, placebo: 23%)Probiotic group: higher sensitization rate (40% vs.24%, p0.03) and more IgE-associated AD (26% vs.14%, p0.045) at age 12 months
Kopp
2008 (88) LGG (ATCC 53103) 1x10
10 cfu RDBPCT 94,
mother, father or sibling with atopic disease
Mothers: 4-6 weeks before delivery
Infants: 6 monthse)
2 years A2 No prevention of AD (probiotics: 28%, placebo: 27%).
No reduction in sensitization to inhalant allergens (food allergens were not determined)
Wickens
2008 (85) L. rhamnosus (HN001) or Bifidobacterium
animalis subsp lactis
(HN 019)
6x109 cfu
9x109 cfu
RDBPCT 474, parental atopic
disease Mothers: week 35 until baby was 6 months (in case of BF) Infants: 2 years
2 years A2 Reduction of AD (HRh 0.51, 95% CI 0.30-0.85, p0.01) and
IgE-associated AD (HR 0.51, 95% CI 0.27-0.97, p0.04) incidence in
Lactobacillus group compared to placebo. Bifidobacterium group: no
effect.
Both groups: no effect on sensitization. Moro
2006 (97) Prebiotics 0.8 g/100 ml formula RDBPCT 206,parental atopic disease
Infants: 6 months 6 months Bg Reduction of AD incidence at age 6 months (10% vs.23%, p0.01),
reduction still significant at age 2.
Less recurrent wheezing and allergic urticaria at age 2 (7.6% vs.20.6% and 1.5% vs.10.3%, p<0.05) Kukkonen 2007 (101) Mixture c + prebiotics (synbiotics) c RDBPCT 925, parental atopic disease Mothers: 2-4 weeks before delivery Infants: 6 months
2 years A2 Reduction of AD (ORi 0.74, 95% CI 0.55-0.98, p0.035) and
IgE-associated AD (OR 0.66, 95% CI, 0.46-0.95, p0.025) incidence at age 2 No reduction other allergic diseases or sensitization
aLactobacillus rhamnosus GG, bLactobacillus, cLGG 5x109 cfu, L. rhamnosus LC705 5x109 cfu, Bifidobacterium breve
Bbi99 2x108 cfu, Propionibacterium JS 2x109 cfu, drandomized, double-blind, placebo-controlled trial, ein case of
breast feeding mothers took the probiotics, fLevel of evidence according to the criteria of the Dutch Institute for
Healthcare Improvement (CBO) : A2=RCT of good quality, B=RCT of less quality, greason: <80% follow-up, hhazard
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25 General introduction and Outline of the thesisTable 3. Treatment of AD with probiotics and synbiotics
Study Probiotic Dose Study design Number
of subjects Age at inclusion (mean) MeanSCORAD at inclusion
Treatment
period Level of evidencef Effect
Majamaa
1997 (62) LGG
a (ATCC 53103) 5 x 108 cfu/g formula RDBPCTe 27 (AD and
suspected cow’s milk allergy)
2.5-15.7 months
(no mean reported) 23.4 4 weeks B
g Significant reduction of SCORAD in probiotic (-11 points)
and not in placebo group (-2 points). No between-group analyses performed, follow-up after 2 months: no differences between the groups.
Isolauri
2000 (90) LGG (ATCC 53103) orB.lactis Bb-12 b 3 x 10 8 cfu/g
1 x 109 cfu/g RDBPCT 27 4.6 months 16 6 months B
g Significant reduction of SCORAD in both probiotic groups
compared to placebo after 2 months, after 6 months no difference between the 3 groups (median SCORAD 0 in all groups).
Kirjavainen
2003 (90) viable LGG (ATCC 53103) or heat-inactivated LGG 1 x 10
9 cfu/g formula RDBPCT 35 (AD and
suspected cow’s milk allergy) 5.5 months 16 mean: 7.5 weeks (range 0.4-45.3)
Bg Significant reduction of SCORAD in viable LGG group
compared to placebo (p 0.02), study was prematurely terminated due to adverse gastrointestinal symptoms in the heat-inactivated LGG group Rosenveldt 2003 (61) L. rhamnosus (19070-2) c and L. reuteri (DSM 12246) 2dd 1 x 10 10 cfu RDBPC
cross-over study 43 5.2 years 35 6 weeks B
h Reduction of SCORAD only in IgE-associated AD (-2.4
points vs +3.2, p 0.04) Weston
2005 (92) L. fermentum (VRI-003 PCC) 2dd 1 x 10
9 cfu RDBPCT 53 10.9 months 42.4 8 weeks A2 Significant reduction of SCORAD in probiotic (-18) and not
in placebo group (-10) at 8 wks.
No significant difference in between-group analyses. Viljanen
2005 (93) LGG (ATCC 53103) orMixtured 2dd 5 x 10 9 cfu
d RDBPCT 230 (AD and suspected
cow’s milk allergy)
6.4 months 32.6 4 weeks A2 LGG: reduction of SCORAD only in IgE-associated AD (-26 vs -20, p 0.04) Mixture: no effect
Sistek
2006 (94) L. rhamnosus and B. lactis 2 x 10
10 cfu/g RDBPCT 59 (AD and
suspected cow’s milk allergy)
4.1 years 30.6 12 weeks A2 Marginal effect in food sensitized children: SCORAD ratio 0.73 (95% CI 0.54-1.00, P=0.047) Brouwer
2006 (60) L. rhamnosus orLGG 3 x 10
8 cfu/g powder
(5 x 109 cfu/100 ml formula RDBPCT 50 5.2 months 18.7 12 weeks A2 No effect on SCORAD or sensitization in either probiotic group
Folster-Holst
2006 (95) LGG 2dd 5 x 10
9 cfu RDBPCT 47 18.8 months 42.3 8 weeks A2 No effect on SCORAD in AD or in IgE-associated AD
Grüber
2007 (105) LGG 2dd 5 x 10
9 cfu RDBPCT 102 7.4 months 24.1 12 weeks A2 No effect on SCORAD in AD or in IgE-associated AD or on
sensitization Passeron
2006 (102) synbiotics: L. rhamnosus (Lcr35) and prebiotics (placebogroup: only prebiotics)
3dd 1.2 x 109 cfu RDBT 39 5.9 years 39.7 12 weeks A2 No difference in SCORAD between the 2 groups
aLactobacillus rhamnosus GG, bBifidobacterium, cLactobacillus, d2dd LGG 5x109 cfu, L. rhamnosus LC705 5x109 cfu,
B. Bbi99 2x108 cfu, Propionibacterium freudenreichii ssp. shermanii JS 2x109 cfu, erandomized, double-blind,
placebo-controlled trial, flevel of evidence according to the criteria of the Dutch Institute for Healthcare Improvement (CBO):
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Moreover, the number of participants was small and they were school aged children. It needs to be considered that manipulating the intestinal microbiota probably has more effect in early infancy, when immune programming is initiated (103). To further explore the role of synbiotics in the treatment of AD, it is necessary to conduct larger, well-designed, randomized placebo-controlled trials in a young age group, preferably infants.
Aim of the thesis
The principal aim of this thesis is to investigate the clinical, microbiological and immunological effects of synbiotics, a combination of the probiotic strain Bifidobacterium breve M-16V and a prebiotic mixture of 90% short chain galactooligosaccharides and 10% long chain fructooligosaccharides (Immunofortis®) in infants with atopic dermatitis.
Outline of the thesis
In Chapter 2 of this thesis we present the results of a randomized controlled multi-centre trial that we performed to evaluate the effects of an infant formula with an added synbiotic, a combination of Bifidobacterium breve M-16V and a specific mixture of 90% short chain galactooligosaccharides and 10% long chain fructooligosaccharides (Immunofortis®), on the severity of atopic dermatitis in infants. Additionally, we investigated the effect of this synbiotic on topical corticosteroid usage, serum total and specific IgE (against inhalant- and foodallergens), serum eosinophil count and on the composition and metabolic activity of the intestinal microbiota.
In Chapter 3 we investigate the immunological effects of this synbiotic on plasma levels of IL-5, IgG1, IgG4 and the AD-associated chemokines cutaneous T cell-attracting chemokine (CTACK) and thymus and activation-regulated chemokine (TARC), on ex vivo cytokine responses of peripheral mononuclear blood cells (PBMCs) to non-specific and allergen-specific stimuli, and on circulating regulatory T cell percentages in these infants.
In Chapter 4, we investigate the differences in atopic markes (eosinophilic granulocytes, IL-5, IgG1, IgG4, CTACK and TARC), ex vivo cytokine responses of PBMCs, and circulating regulatory T cell percentages between infants with IgE-associated atopic dermatitis and non IgE-associated atopic dermatitis.
In Chapter 5, we present the results of a one-year follow-up study of the infants that participated in the trial. The aim of this follow-up study was to determine the prevalence of asthma-like symptoms, use of asthma medication and sensitization against aeroallergens in the infants that had received the synbiotic and the infants that had received placebo.
In Chapter 6 we respond to the paper ‘The impact of maternal atopy and probiotic supplementation during pregnancy on infant sensitization’ of Huurre and colleagues, published in Clinical and Experimental Allergy (104).
There are considerable differences in the probiotic strains and dosages that were used in the treatment studies. Also the number, age and eczema severity of the participants and the treatment period differs between studies. These differences could possibly explain the conflicting results. It should also be mentioned that these trials are complicated by the natural tendency of AD to improve over time, with adequate advice and topical corticosteroid treatment, which explains the substantial improvement that is often seen in the placebo group.
Prebiotics in the prevention and treatment of atopic dermatitis
One double-blind RCT has been performed to investigate the preventive effect of prebiotics in the development of AD (97) (table 2). In this study 259 high risk infants were enrolled. They received a hydrolysed protein formula with either GOS/ FOS mixture or maltodextrine (placebo) for 6 months. The incidence of AD, diagnosed according to clinical criteria, during the study period was significantly lower in the intervention group than in the placebo group (9.8 % compared to 23.1%). At age 2, the cumulative incidence of AD was still significantly reduced in the prebiotic group, as were the cumulative incidence of recurrent wheezing and allergic urticaria (98). Faecal bifidobacteria counts were significantly higher in the intervention group. Although these results seem promising, a limitation of the study was the relatively large percentage (more than 20%) of infants that were lost to follow-up during the intervention period. Up until now there are no studies that explore the role of prebiotics in the treatment of AD.
Synbiotics
Combinations of pro- and prebiotics are called synbiotics. This term should be used to describe products in which the prebiotic compound selectively stimulates the probiotic compound, thus creating a synergetic effect (99). Theoretically, optimal synbiotic preparations can be expected to obtain better results in AD prevention or treatment than either pro- or prebiotics alone. This was confirmed by an animal study that shows that severity of AD lesions and total immunoglobulin E levels were significantly reduced in mice fed Lactobacillus casei subsp. casei together with its prebiotic, dextran, compared to placebo. This effect was also seen in mice that were fed either the probiotic or the prebiotic compound alone, but to a lesser extent than when the two compounds were given together (100).
A large human prevention trial showed that a preparation of 4 probiotic strains combined with prebiotics significantly reduced the incidence of eczema (26% versus 32%, p 0.04) and IgE-associated eczema (12% versus 18%, p 0.03) in high risk children compared to placebo (101) (table 2).
Only one clinical trial investigated the efficacy of synbiotics as treatment for AD (102) (table 3). Thirty-nine children, with a mean age of 6 years, were included and randomized to receive either synbiotics or prebiotics. A significant improvement of AD was found in both study groups, but synbiotics did not appear to be superior to prebiotics alone. However, the study design, without a placebo group, makes it impossible to draw conclusions whether synbiotics and prebiotics improve AD since the improvement in both groups may also be due to the natural course of the disease.
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29 General introduction and Outline of the thesisReference List
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Effect of a new synbiotic mixture
on atopic dermatitis in infants:
a randomized controlled trial
L.B. van der Aa H.S.A. Heymans W.M.C. van Aalderen J.H. Sillevis Smitt J. Knol K. Ben Amor D.A. Goossens A.B. Sprikkelman Synbad Study Group
Chapter
2
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ter
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ABSTRACT
Background: Clinical trials investigating the therapeutic effect of probiotics on atopic dermatitis
(AD) show inconsistent results. Better results can possibly be achieved by combining probiotics with prebiotics, i.e. synbiotics.
Objective: To investigate the therapeutic effect of a synbiotic mixture on the severity of AD in
infants.
Methods: In a double-blind, placebo-controlled multicenter trial, ninety infants with AD
(SCORAD score ≥ 15), aged < 7 months and exclusively formula fed, were randomly assigned to receive either an extensively hydrolyzed formula with Bifidobacterium breve M-16V and a galacto-/ fructooligosaccharide mixture (Immunofortis®), or the same formula without synbiotics during 12 weeks. The primary outcome was severity of AD, assessed with the SCORAD index. A secondary outcome measure was intestinal microbiota composition.
Results: There was no difference in SCORAD score improvement between the synbiotic and the
placebo group. The synbiotic group did have a significantly higher percentage of bifidobacteria (54.7% vs. 30.1%, P<0.001) and significantly lower percentages of Clostridium lituseburense/
Clostridium histolyticum (0.5 vs. 1.8, P = 0.02) and Eubacterium rectale/Clostridium coccoides (7.5
vs. 38.1, P < 0.001) after intervention than the placebo group. In the subgroup of infants with IgE-associated AD (n=48), SCORAD score improvement was significantly greater in the synbiotic than in the placebo group at week 12 (-18.1 versus -13.5 points, P = 0.04).
Conclusions: This synbiotic mixture does not have a beneficial effect on AD severity in infants,
although it does successfully modulate their intestinal microbiota. Further randomized controlled trials should explore a possible beneficial effect in IgE-associated AD.
INTRODUCTION
Atopic dermatitis (AD) is a highly prevalent, chronic, itching skin disease that often presents in infancy and greatly affects the quality of life of children and their families (1). Currently, topical corticosteroids are the mainstay treatment of this disease; however, relapses are common and parents often fear possible side effects, leading to non-compliance.
There is increasing evidence that the intestinal microbiota plays an important role in the development of allergic diseases (2;3). Indeed, microbiota composition has been shown to differ between children with and without AD (4). Therefore, innovative treatment strategies aiming to modulate the intestinal microbiota with probiotics, living micro-organisms with immunomodulatory effects, or prebiotics, nondigestible food ingredients that stimulate the growth and/or activity of one or a limited number of beneficial gut bacteria (5), are now foci of interest.
Animal studies show that AD severity can be reduced by certain probiotic strains (6;7). Yet, clinical trials in children have conflicting outcomes (8-13), although for IgE-associated AD modest improvement was demonstrated (14-16). A systematic review of all these clinical trials concluded that the probiotic strains studied up to now are not an effective treatment for AD; however other strains might have a greater effect (17). In most trials lactobacilli were used, while it can be argued that bifidobacteria, which are also considered to be safe for use as probiotics, might be better candidates for AD treatment. Low bifidobacteria levels appear to be associated with AD, as children with AD have lower Bifidobacterium percentages than their healthy peers and percentages are lower in severe than in mild AD (4). Recently, it has been shown that the specific strain Bifidobacterium breve M-16V reduces allergic symptoms in ovalbumin-sensitized mice more effectively than lactobacilli (18). Moreover, in a small study this strain has been shown to decrease AD severity in children (19). The only study that investigated the efficacy of prebiotics as AD treatment showed a beneficial effect on AD severity (20).
Theoretically, optimal synergetic combinations of pro- and prebiotics, so called synbiotics, are most promising for treating AD. In whey-sensitized mice, a synbiotic combination of
B. breve M-16V and 90% short chain galactooligosaccharide (scGOS) and 10% long chain
fructooligosaccharide (lcFOS) mixture (Immunofortis®) reduced the acute allergic skin response more effectively than either the pre- or the probiotic component alone (21).
We performed a randomized, double-blind, placebo-controlled multicenter trial to investigate the effect of an infant formula with added synbiotics, B. breve M-16V and a scGOS/lcFOS mixture (Immunofortis®), on the severity of AD in infants. Additionally, we investigated the effect of this synbiotic formula on the composition and the metabolic activity of the intestinal microbiota.
