48 Probiotics in Atopic Eczema C. Schnopp

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48 Probiotics in Atopic Eczema

C. Schnopp

48.1

The Hygiene Hypothesis

Atopic eczema (AE) is a chronic relapsing inflammatory skin disorder with a peak prevalence in infancy [34].

In the majority of patients, atopic eczema is the first clinical presentation of atopy, substantial numbers of those with childhood atopic eczema will go on to asthma and rhinitis. Although genetic predisposition remains the single most important risk factor, the increase in prevalence of atopic eczema in industrial- ized countries [11, 35] has been attributed to environmental factors including microbial exposure and nutrition [32].

The hygiene hypothesis postulates that decreasing stimulation of the immune system due to diminished exposure to microbial antigens in Western countries leads to higher susceptibility for atopic disease [42]. It is based on epidemiological data showing an inverse relationship between low socioeconomic status, large number of siblings, early infections, farming environment, and manifestation of atopic diseases [8, 37, 41].

Some microbial compounds are believed to play an important role in enhancing a mature Th1-like immune response, counteracting the persistence of the neonatal Th2-type immune response pattern [22].

However, there is little data on which type of microbial antigen at what time point induces a favorable shift in the immune response pattern. Secondly, most studies supporting the hygiene hypothesis so far have looked at asthmatic symptoms and/or specific sensitization; there is less evidence regarding atopic eczema.

Whereas early infections are assumed to promote Th1-dominated immune response and have an protective effect with regard to allergic disease, increased fre- quency of asthma has been reported in children hospi-

talized for RSV (respiratory syncytial virus) bronchiolitis in early infancy, especially when they had an atopic background [36]. A recent study by Williams and co- workers [41] showed an inverse association of febrile illness during the 1st year of life and allergic sensitization at age 6 – 7 years. Diverging effects of RSV infection on T-mediated immune response depending on the time of infection have been observed in a murine model [4, 9]. In contrast, children growing up on farms were exposed to higher concentrations of bacterial lipopolysaccharides in their daily living environment compared to their nonfarming neighbors. In these children, higher expression of CD14 and Toll-like receptor (TLR) 2 genes, as measured by quantitative PCR [21], has been found. The authors speculate that amplified expression of CD14 and TLR-2, as natural binding sites for microbial components such as bacterial lipopolysaccharides in the innate immune system, reflects increased microbial antigen exposure and might be responsible for an enhanced Th1-type response or modified Th2-type response accounting for lower incidence of atopic disease.

48.2

Primary Prevention Strategies in Atopic Eczema

In view of high prevalence rates, increasing awareness and high economic burden of allergic diseases, primary prevention of atopic eczema as the most common initial presentation of atopic disease has become a focus of interest.

Promotion of breast-feeding has been the mainstay of public health programs for primary prevention [43].

Breast-feeding is considered to fulfill most appropri- ately the needs of infant nutrition concerning nutrients, growth factors, immunoglobulins, and other

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immunologically active compounds. However, breast- fed infants are exposed to a range of food allergens that are secreted into breast milk. Antigen exposure through breast milk has been shown to be an important trigger factor in infants with atopic eczema and food allergy. Hypoallergenic diets, mainly based on extensively hydrolyzd formulas, are essential in the treatment of these children. Maternal diet in breast-fed children is an alternative, but has to be very closely monitored as there is considerable risk of inadequate intake of (micro-) nutrients. Recent studies on breastfeeding and atopy showed conflicting results with regard to effective prevention of the disease [2, 18, 19].

Comparing studies on breast-feeding in the prevention of atopic disease, we have to look carefully at study population (birth cohort vs high-risk children) and study design (observation vs intervention study). To complicate the issue further, it is impossible to ran- domize a study population to breast-feeding and there might be considerable publication bias. However, it is tempting to speculate that prolonged low-dose exposure to food antigens might be a relevant risk factor for early-onset atopic eczema in predisposed children.

The skin, gut, and lungs are the most important organs interacting with environment. A considerable number of studies are dealing with prevention of atopic disease in genetically predisposed children by reducing exposure to alleged allergens, e.g., milk protein [40], house dust mites, and pets [5, 20] – with conflicting results. Whereas allergen avoidance has been proven effective in secondary prevention, the answer for primary prevention is still being debated [29].

48.3

Probiotics for Primary Prevention in High-Risk Families

48.3.1 Background

Increasing immune stimulation by exposure to harm- less microbial antigens in early infancy might be another strategy to promote a protective Th1-dominated immune response. Erika Isolauri’s group from Tur- ku, Finland, who has been working on the effects of probiotic bacteria for years [13, 30], suggested that commensal microflora of the gut, representing the largest bacterial reservoir in humans, might be a strong regulator of the immune system. The pattern of micro-

bial colonization has changed over the last century, and there are considerable differences between privileged (and more allergy-prone) and underprivileged children. They hypothesized that modulating the commensal gut microflora may represent a key to influence the infantile immune system toward a protective Th1 response. The term “probiotics” is applied to different bacterial strains of healthy gut microflora exerting potentially beneficial effects; most studies have been done with Lactobacillus and Bifidobacterium lactis species. For therapeutic use, probiotic strains are cul- tured in vitro and administered orally. They must be stable to acid and bile and capable of adhering to intestinal mucosa to be effective. In contrast, the term “pre- biotics” has been used for nutritional components that favor the growth of probiotic bacteria in the gastrointestinal system.

The Finnish group found differences in the compo- sition of intestinal microflora between atopic and nonatopic children at 3 weeks of age – prior to the occurrence of atopic disease or sensitization. Atopic children, defined as having manifest atopic eczema and/or positive skin prick test to common allergens at age 12 months, were less often colonized with bifidobacteria and more often with clostridiae at 3 weeks than children who did not show signs of allergic sensitization at 12 months [14]. At age 3 months, fecal microflora was comparable in both groups. These results were confirmed by a Swedish group in nine Estonian and nine Swedish children. They found lower counts of bifidobacteria and enterococci and higher counts of clostridiae during the 1st month of life in children who developed atopic disease later in life (defined as a diagnosis of atopic eczema and/or positive skin prick) [3]. Of note, in this study there was no difference in the amount of lactobacilli at all time points except for at 1 week of age, where the number of lactobacilli was significantly lower (!) in children who remained free of atopic disease till the age of 1 year.

Lactobacillus rhamnosus is a Gram-positive probi- otic bacterial strain found in commensal gut flora. It is available for therapeutic use in children with acute diarrhea. Efficacy and safety of Lactobacillus rhamno- sus strain GG added to standard oral rehydration solu- tion (ORS) have been proven in several studies. Results of a large multicenter study showed that addition of Lactobacillus GG (10¹⁰CFU) to 250 ml standard ORS resulted in earlier resolution of gastrointestinal symptoms and shorter hospital stay in rotavirus-associated 450 48 Probiotics in Atopic Eczema

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diarrhea as well as in acute diarrhea due to other infec- tious agents in children aged 1 month to 3 years [6].

48.3.2

Prevention of atopic eczema

Isolauri’s group used this probiotic bacterial strain (Lactobacillus GG, American Type Culture Collection [ATCC] 53103) for a randomized placebo-controlled trial on primary prevention of atopic eczema in high- risk infants. One hundred fifty-nine pregnant women with a positive family history of atopic disease (i.e., at least one family member including themselves with atopic eczema, allergic asthma, or allergic rhinitis) were included in the study from February 1997 to Janu- ary 1998. They were randomized to placebo or two capsules of 10¹⁰CFU Lactobacillus GG daily for 2 – 4 weeks before delivery and 6 months after birth when breastfeeding placebo. In children who were not breast-fed, the content of the capsules was added to infants’ formula. Sixty-four children in the verum group and 68 children in the placebo group were followed up as per protocol until the age of 2 years (drop-out, 13 and 14 in the verum and placebo group, respectively). At the age of 2, a diagnosis of atopic eczema was made in 15 out of 64 (23 %) children on lactobacilli for the first 6 months of life and in 31 out of 68 (46 %) in the placebo group, thus reducing the incidence of atopic eczema by half (relative risk 0.51; 95 % CI 0.32 – 0.84). There were no statistically significant differences in total IgE, skin prick reactivity, and increased RAST reading at age 3, 12 and 24 months [15].

Of the children who completed the 2-year study, 107 out of 132 were available for follow-up at the age of 4 years. At this time point, 14 out of 53 (26 %) children receiving lactobacilli in the first 6 months of life and 25 out of 54 (46 %) on placebo had developed atopic eczema (relative risk 0.57; 95 % CI 0.33 – 0.97), ten children in the lactobacillus group and five in the placebo group had developed seasonal allergic rhinitis, three children in the lactobacillus group and one in the placebo group were diagnosed of having asthma. Skin prick test reactivity did not differ between the two groups. In summa- ry, the 4-year follow-up showed a sustained effect of the intervention regarding the incidence of atopic eczema, whereas there was a nonsignificant trend toward higher incidence of respiratory allergic symptoms in the lactobacillus group. No adverse effects were noted [16].

In a subgroup analysis (n = 62), the authors looked

at those infants who had been breast-fed for at least 3 months (30 in the Lactobacillus GG group, 32 in the placebo group), of whom 57 completed the study. In this subgroup of breast-fed infants, the effect of probiotic supplementation on the incidence of atopic eczema was even more pronounced, with a relative risk of 0.32 (95 % CI 0.12 – 0.85). Four of 27 (15 %) children in the lactobacillus group were diagnosed of having atopic eczema at 2 years of age vs 14 of 30 (47 %) in the placebo group (RR 0.32; 95 % CI 0.12 – 0.85). More children in the placebo group showed elevated IgE anti- bodies at the age of 2 years compared to the lactobacillus group (37 % vs 28 %), but more children in the lactobacillus group were allergic to cow’s milk (21 %) than in the placebo group (10 %); these values did not reach statistical significance. Of note, there was a higher incidence of maternal atopic disease in the placebo group (75 %) compared to the lactobacillus group (60 %), although this was not statistically significant [31].

The data on primary prevention of atopic disease by early exposure to probiotic bacteria has attracted considerable interest. Experimental data and theoretical considerations suggest that probiotics will work most effectively in an immature immune system and possi- bly immature gut.

48.3.3

Probiotics in the Treatment of Atopic Eczema

Only a few studies have so far addressed the question of probiotics in treating manifest atopic eczema. The Finnish group [12] added Bifidobacterium lactis Bb-12 (10⁹CFU) or Lactobacillus GG (ATCC 53103) (3×10⁸ CFU) or placebo to an extensively hydrolyzed whey formula. Twenty-seven children with a diagnosis of atopic eczema were randomized to one of the preparations at a mean age of 4.6 months, having been exclusively breast-fed up to then. Severity of atopic eczema was evaluated clinically (by SCORAD) before introducing the formula, after 2 months and 6 months of formula feeding. A range of immunological markers has been studied prior to introduction of formula and 2 months thereafter. Median SCORAD before weaning was 16 (7 – 25). After 2 months of treatment with extensively hydrolyzed formula, median SCORAD had dropped significantly in both supplemented groups (with Lac- tobacillus GG to a median of 1 (0.1 – 8.7), in the Bifido- bacterium lactis group to 0 (0 – 3.8), whereas the me- dian SCORAD in the formula-only group was 13.4

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(4.5 – 18.2). Of note, numbers were very small: of 27 children who entered the study, at the end of the study there were nine in each of the supplemented groups and only four in the formula-only group. After 6 months, the median SCORAD was 0 (0 – 6.6) in all groups. In conclusion, skin symptoms disappeared more rapidly in the probiotic-supplemented groups compared to the unsupplemented group; 6 months later, no difference was detectable clinically. Looking at immunological parameters, the authors reported significantly decreased levels of soluble CD4 lymphocytes and decreased urinary eosinophilic protein X (EPX) in supplemented vs control infants after 2 months, but clinical relevance of these findings remains to be estab- lished.

A randomized placebo-controlled cross-over study for treatment of manifest eczema comes from Den- mark. Forty-three pediatric patients (mean age 5.2 years, range 1 – 13 years) with moderate to severe atopic eczema (median SCORAD 40, range 25 – 51) were treated in a cross-over design for 6 weeks with a combination of two probiotic strains (Lactobacillus rhamnosus 19070-2 and Lactobacillus reuteri DSM 122460) and placebo, with a wash-out interval of 6 weeks between the two intervention periods. For evaluation of clinical severity, SCORAD was used as an objective parameter and patients were asked for their subjective evaluation of effectiveness. As immunologic parameters, serum eosinophil cationic protein (ECP) as well as IL-2, IL-4, IL-10, and IFN-* production of PBMCs were determined by ELISA. In order to detect differences in efficacy between atopic and nonatopic eczema, participating patients were classified as atopic when they had at least one positive skin prick test or elevated IgE (> 150 kU/l). There was no significant difference in the change in clinical severity as measured by total SCORAD during active and placebo treatment.

Analyzing single SCORAD items, the extent of disease was significantly more reduced during the active treatment period compared to the placebo period. This effect was more pronounced in the atopic subgroup (n = 27). Immunological parameters did not change on either treatment regimen. Of note, there was no corre- lation of extent and activity of eczema to serum ECP levels. Of all parameters studied, only IL-4 production decreased significantly with clinical improvement in more severely affected children. Interestingly, patients and parents subjectively preferred the active treatment to the placebo and gave a better overall score on a scale

of better/worse/unchanged during active treatment.

The Danish study group concluded that more studies are needed to select patients who may benefit from intervention with probiotics [33].

48.4

Suggested Mechanisms

Several hypothetical concepts help to explain the mechanism of action of probiotic strategies (for review, see [25]).

48.4.1

Shift of Th2 to Th1 Immune Response Pattern

In the activation of the innate immune system, so- called Toll-like receptors (TLR) – first described in Drosophila – play a critical role. TLR2, TLR4, and TLR9 bind different microbial compounds. Recognition of microbial structure proteins on the bacterial cell sur- face or DNA sequences will lead to induction of Th1 cytokines (dependent on NF-κB). TLRs have been identified on enterocytes and various immune cells, suggesting a possible effect of gastrointestinal microflora on T helper cell modulation [1, 7, 24]. In vitro experiments have shown activation of NF-κB via CD 14 and TLR2 in response to Lactobacillus lipoteichoic acid [23]. This was confirmed by a Swedish group who analyzed the cytokine pattern from cord blood relative to adult mononuclear cells after stimulation with different bacterial strains from the normal flora. Stimulation with Lactobacillus plantarum resulted in strong signals via TLR2, TLR4, and CD14 [17].

In view of the hygiene hypothesis, a Japanese group used BALB/c mice, genetically biased toward Th2- dominant immunity, and C57BL/6 mice, genetically biased toward Th1, to study the influence of early antibiotic use on Th1/Th2 balance. It was shown that neonatal administration of kanamycin (600 µg/die p.o. for 7 consecutive days) results in a Th2-skewed immunity in adolescence with elevated IgE/IgG2a ratio as marker for Th2/Th1 imbalance in the Th2-prone BALB/c mice, but not in C57BL/6 mice [27]. To see whether or not such an effect could be reversed by probiotics, they repeated the experiment adding 5×10⁸CFU/d of three different types of probiotic bacteria (Enterococcus fae- calis, Lactobacillus acidophilus, and Bacteroides vulga- tus) for 5 consecutive days after kanamycin treatment.

452 48 Probiotics in Atopic Eczema

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As in the previous study, serum IgE was significantly increased in the BALB/c mice 10 weeks after kanamy- cin administration. Supplementation of Enterococcus faecalis significantly suppressed elevation of IgE in kanamycin-treated BALB/c mice, whereas Lactobacil- lus acidophilus had some, though not significant, effect, and inoculation of Bacteroides vulgatus showed the opposite effect [38].

48.4.2

Induction of Oral Tolerance

Immunological tolerance to dietary antigens as well as to commensal microflora can develop as anergy or as an active mechanism of lymphocyte subpopulations. A study in transgenic mice suggests that commensal gastrointestinal microflora plays an important role in the generation of regulatory T lymphocytes (Th3 and Tr1), downregulating mucosal inflammatory response via prostaglandin E2 from macrophages (induced by IL-10 and transforming growth factor beta, [TGFq ]), thus inducing oral tolerance [26, 28]. In another murine study, a Japanese group showed that induction of oral tolerance is severely impaired when stimuli from commensal microflora from the gut are lacking in early infancy [38]. In a subgroup analysis of the initial prevention study, breast milk samples were analyzed for transforming growth factor (TGF)-q 1 and TGF- q 2 when the infant was 3 months old. As determined by ELISA, it was shown that the amount of anti-inflammatory TGF-q 2 was significantly higher (p=0.018) in lac- tating mothers on probiotics (2,885 pg/ml, 95 % CI 1,624 – 4,146) compared to placebo (1,340 pg/ml, 95 % CI 978 – 1,702), which might be one of the mechanisms of action [31].

48.4.3

Privilege by Early Colonization

Early administration of probiotics could lead to a sustained effect as the first bacteria colonizing the gut might be privileged and establish a permanent niche (by glycosylation of the glycocalix). It has been further suggested that probiotics contribute to normalization of increased gut permeability in allergy-prone infants [10, 29]. Besides, some of the effect might be due to increased concordance between maternal and infant flora, when probiotics are administered during late pregnancy to the mother.

48.5

Remaining Questions

In spite of the promising results in the above-men- tioned studies, some questions regarding the use of probiotics in atopic eczema remain:

) Is the effect of Lactobacillus GG, as shown in the Finnish study population, reproducible in other countries? The incidence of atopic eczema in the Finnish study was very high (23 % in the intervention group, 46 % in the placebo group), even when considering a positive family history. The expected incidence of atopic eczema in central Europe ranges within that of the intervention group.

) Are probiotics effective for the prevention of other allergic diseases apart from atopic eczema (respiratory, gastrointestinal)? In the 4-year follow- up study, children in the lactobacillus group tend- ed to have more asthma and rhinitis than children in the placebo group. Although these differences were not statistically significant, further studies should elucidate this question by means of a long follow-up.

) Are there any side effects? Although probiotics seem to be rather safe (experiences from many studies on diarrhea), there is a theoretical concern of potentially hazardous effects of a possible Th1 shift, e.g., higher susceptibility to autoimmune disease with early treatment. Moreover, septicemia and liver abscess formation have been described sporadically in immunocompromised children.

) Who is going to benefit? Identification of patient- related factors (e.g., extrinsic vs intrinsic type) will help to increase the cost-benefit ratio.

) Which bacterial strains are most effective? Existing data show that not all probiotic bacteria have the same efficacy. To identify the most effective strains or possible combinations, a reliable animal model would be helpful.

48.6 Conclusion

Intestinal microflora has been an area of alternative medicine for a long time. Since Isolauri’s first publication on prevention of atopic disease by supplementation with probiotics, supplementation with commensal living bacteria has attracted considerable interest in

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health professionals, scientists, pharmaceutical indus- try, and patients.

The results with Lactobacillus GG (ATCC 53103) in primary prevention of atopic eczema are very impres- sive and should soon be reproduced by other groups.

Before probiotics can become part of evidence-based public health recommendations, larger studies are needed to evaluate effectiveness, usefulness of different strains, dose and timing, the cost-benefit ratio and the potential risks of probiotics.

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