The Journal of International Medical Research
2004; 32: 97 – 108 Probiotics, Prebiotics and Child Health: Where Are We Going?
F S ALVINI, L
G RANIERI, L G EMMELLARO AND M G IOVANNINI
Department of Paediatrics, University of Milan, San Paolo Hospital, Milan, Italy
Changes in gastrointestinal (GI) bacteria caused by diet, antibiotics or other factors could alter enteric and systemic immune functions; changing the gut microflora composition by diet supplementation with specific live microbiota (probiotics) may be beneficial. The ‘natural’ target of ingested probiotics is the intestine, its microflora and associated immune system. M ost published data concern use of probiotics to prevent and treat GI infections. Evidence for possible beneficial effects on mucosal barrier dysfunctions, including food allergy, inflammatory bowel disease, and respiratory and urinary tract infections, is emerging. The role of prebiotics (non-digestible oligosaccharides that reduce the growth or virulence of pathogens and induce systemic effects) is being investigated. Preliminary studies indicate that prebiotics may be useful dietary adjuncts for managing GI infections. Prebiotic and probiotic use in infants is attempting to modify a complex microbial ecosystem. Better understanding of the long-term effects of these interventions on infant gut microflora is an important goal.
KEY WORDS: P ROBIOTICS; P REBIOTICS; R EVIEW; C HILDREN; C HILD HEALTH; A LLERGY; I MMUNITY
Introduction
There is an increasing awareness that immune function and health are responsive to the bacteria resident in the gastro-intestinal (G I) tract, and it has been suggested that changes in the GI bacterial population caused by diet, antibiotics or other factors can alter enteric and systemic immune function.
More than 400 species of bacteria reside in the human G tract, forming a complex human intestinal microflora.1The intestinal microflora contribute to the development and function of the mucosal barrier (and colonization resistance in the human intestine) through competition for nutrients and adhesion receptors, production of antimicrobial substances, and stimulation of gut-associated lymphoid tissue (GALT).2 Altering the gut microflora composition by supplementation with specific live microbiota may be beneficial. Such supplements are known as probiotics, and were initially defined as ‘a live microbial feed supplement, which beneficially affects the host by improving its microbial balance’.3n 1998 a European group of scientists suggested that a probiotic is better defined as ‘a live microbial food ingredient that is beneficial to health’.4,5More recently, de Vrese and Schrezenmeir6defined probiotic food as ‘a preparation or product containing viable, defined micro-organisms in sufficient numbers which alters the microflora in a compartment of the host and exerts health
effects in this host’. This definition emphasizes that other compartments of the body apart from the intestine may be targets for probiotics.
Human origin, safe for human consumption and resistance to acid and bile are essential characteristics of microbiota used as probiotics. Furthermore, temporary colonization of the gut mucosa, as measured by faecal counts or mucosal biopsies, must be demonstrated after supplementation.7 The ‘natural’ target of ingested probiotics is the intestine, together with its microflora and associated immune system. The intestine is the largest ‘immunological’organ in the body; it contains 70 – 80% of all the immunoglobulin (I g) A-producing cells, and the amount of IgA produced exceeds the total production of all the other immuno-globulin classes in the body. Attachment to the epithelium and mucosa is thought to be a key mechanism whereby probiotics interact with GALT.8
An important factor for local immunity is the migration of specific activated B- and T-cells from the Peyer’s patches (PPs; the inductive sites) to distant mucosal sites such as the respiratory tract, genitourinary tract and various secretory glands, and also back to the intestinal lamina propria and epi-thelium. Migration of these specific immune cells is the basis of what has been termed the common mucosal immune system.9
To achieve an effective immune response to an ingested antigen, the participation of almost all the different types of immune cells associated with the gut is necessary. Macrophages, regulatory T-cells and effector B- and T-lymphocytes induce the protective gA secretion associated with the mucosal surfaces. This process involves the inductive sites (PPs), where the antigen is encountered and initial responses are induced, and effector sites, where production of secretory gA antibodies by plasma cells results in local immune protection. Although physically separated, they are functionally interconnected (Figs 1 and 2).10IgA inductive
sites in GALT, represented by the PP, have been extensively studied. The PP contains a ‘dome’region enriched by lymphocytes, macrophages and some plasma cells. This area is covered by a unique epithelium containing specialized microfold (M) cells, which have short microvilli, small amounts of cytoplasm and few lysosomes. The M-cells are responsible for the uptake and transport of luminal antigens and small parasites, delivering them intact into the underlying lymphoid tissue.11 The PPs act as germinal centres in which B-cells switch from producing I gM to producing I gA and undergo affinity maturation in response to the presence of an antigen. Accessory cells such as dendritic cells and macrophages in the I gA inductive sites act as antigen-presenting cells (APCs) and are engaged in the regulation of the humoral and cellular immune responses involved in mucosal protection.
Following stimulation of the PP by the antigen and its presentation to B- and T-cells, the antigen-induced B- and T-cells migrate out of the PP via the efferent lymphatics and the mesenteric node. They reach the systemic circulation through the thoracic duct and repopulate not only the lamina propria of the intestine but other distant mucosal sites such as the respiratory tract, the urogenital tract, and the mammary and salivary glands. I n this way stimulation produced by orally ingested antigens leads to the repopulation of distant mucosal sites with IgA-producing cells, to protect these surfaces as part of the common mucosal immune system.12
Probiotics
PROBIOTICS AND DIARRHOEA
The principal use for probiotics is in the treatment and prevention of GI infections. The rationale for using probiotics in acute diarrhoea is based on the assumption that they act against intestinal pathogens through various mechanisms, including the synthesis of antimicrobial substances,13,14 competition for nutrients required for growth,15competitive inhibition of pathogen adhesion,16,17modification of toxins or toxin receptors,18,19and non-specific and specific immune response stimulation.20,21Mack et al.22showed that Lactobacillus species
(particularly L. rhamnosus strain GG and L. plantarum strain 299v) inhibit, in a dose-dependent manner, the binding of Escher i ch i a col i strains to intestine-derived epithelial cells grown in tissue culture by stimulating the synthesis and secretion of mucins.
The clinical efficacy of probiotics in the treatment and prevention of acute infectious diarrhoea has not yet been fully established.
I n 2001, Szajewska and Mrukowicz23 attempted to assess and quantify the evidence from published randomized controlled trials through a systematic review of the literature. Data from eight trials involving 731 children showed that probiotic supplementation is associated with a significantly reduced risk of diarrhoea for a period of 3 days. Lactobacillus GG also showed a consistent effect in reducing the duration of diarrhoea. Two recent randomized controlled studies showed a consistent reduction in the incidence and duration of diarrhoea in children attending day-care centres who were given L. case i (strain DN-114 001) supplements.24,25Other probiotic strains may also be effective, but further research is needed.
The heterogeneity of the clinical and statistical data concerning use of probiotics as prophylaxis for acute gastroenteritis precludes the drawing of firm conclusions. No obvious adverse effects due to probiotic use in this setting were observed.23
Lactobacillus GG has been shown to promote the recovery of children from rotavirus diarrhoea, increasing the local and systemic immune responses.20The mechanisms by which some lactic acid bacteria prevents enteric infections are not fully known. I n viral infections of the GI tract, probiotics may stimulate immuno-modulatory mechanisms, inducing a high level of specific secretory I gA, which is a major immunological barrier against viruses. Yasui et al.26demonstrated that B i f i dobacter i um breve provided passive protection against rotavirus. Similar mechanisms may be responsible for protection against bacterial infections. Another possibility is that probiotics stimulate cytokine production and enhance secretory gA.27n human studies, Link-Amster et al.28demonstrated an enhanced specific serum I gA response against attenuated Salmonella typhi Ty21a given to volunteers who had consumed L. acidophilus. Priming the GALT may be an important part of this protective mechanism. I n addition, competition for binding sites on epithelial cells resulting in competitive exclusion could be another way in which Lactobacillus increases the host’s resistance to infection.
PROBIOTICS AND INFLAMMATORY BOWEL DISEASE
I n a review of clinical studies, Vanderhoof and Young29concluded that probiotics can be useful in the treatment of gut mucosal barrier dysfunctions, including food allergy and inflammatory bowel disease, in addition to their traditional utilization for diarrhoea. In a recent pilot study, Gupta et al.30showed that Lactobacillus GG was effective in improving the clinical status of children with Crohn’s disease.
Ishikawa et al.31conducted a randomized clinical trial of supplementation with B. breve, B. b f dum and L. ac doph lus YIT 0168 at a dose of at least 10 billion per 100 ml bottle of bifidobacteria-fermented milk each day for 1 year in patients with ulcerative colitis. They concluded that supplementation may be effective in the prevention of ulcerative colitis exacerbations, possibly as a result of normalization of the intestinal flora. I t has been reported that the number of Bacteroides
vulgatus strains in the colonic mucosa and blood levels of antibodies against B. vulgatus are increased in patients with ulcerative colitis.32,33Bifidobacteria-fermented milk supplementation led to a significant decrease in the relative proportion of B. vulgatus in the faecal Bacteroidaceae population.31
Further research is needed on the mechanisms of action and safety of probiotics in these target populations, particularly since distinct immunological effects have been detected after probiotic therapy in patients with inflammatory bowel disease compared with healthy subjects.34
PROBIOTICS AND ALLERGIC DISEASES
Probiotic preparations have also been proposed for the treatment or prevention of allergic diseases in children.35I n several in vitro and in vivo studies, probiotics have been reported to cause a wide array of immune effects (Fig. 3), some of them potentially in contrast to those characteristic of the atopic constitution.36,37The underlying denomin-ators of allergic sensitization include genetic susceptibility, aberrant barrier functions of skin epithelium and gut mucosa, and dysregulation of antigen-specific I gE production. gE responses are under the control of cytokines produced by T-helper 1 (Th1) cells that secrete interleukin (IL)-2 and interferon (I FN)-γand T-helper 2 (Th2) cells that produce I L-4 and I L-5 but not I FN-γ. Patients with atopic dermatitis manifest high levels of I L-4 and little or no I FN-γin the peripheral blood. L-4 promotes gE production, whereas FN-γdownregulates both I L-4 and allergen-induced I gE production.
At birth there is a Th2-dominant immunity, which is followed by a shift to Th1 immunity during infancy; however, in atopic children Th2 immunity remains dominant. Specific microbes in the commensal gut microflora are known to promote certain anti-allergenic processes, including Th1-type immunity, generation of transforming
growth factor-β, which has an essential role in the suppression of Th2-induced allergic inflammation and the induction of oral tolerance (defined as the immunological hyporesponsiveness to antigens encountered through the enteric route), and I gA production, an essential component of the mucosal immune defences that is often defective in children with food allergy.
The use of probiotics in the primary prevention of atopic disease is based on their ability to reverse increased intestinal permeability, a characteristic of children with atopic eczema and food allergy. They also help to improve gut barrier function and restore a healthier gut microecology, alterations which have been shown to occur in allergic individuals. Specific strains of indigenous gut microflora have been shown to have profound effects on the physiology and immunology of the host. It has been documented by Sepp et al.38 that Swedish infants, who have a high prevalence of allergy, have an abundance of clostridia in their gut microflora, while Estonian infants, who have a low prevalence of allergy, had high counts of lactobacilli and eubacteria.
The first double-blind placebo-controlled trial on food allergies and probiotics appeared in 1997:39Lactobacillus GG was administered to breastfeeding mothers of 10 infants with atopic eczema/dermatitis syndrome (AEDS) due to milk allergy, and hydrolysed milk formula containing Lactobacillus GG was given to 15 bottle-fed infants with AEDS. I t was concluded that probiotic bacteria may be effective in the treatment of food allergy, possibly by promoting endogenous barrier mechanisms and alleviating intestinal inflammation.
t has been shown that degradation of food antigens by intestinal bacteria-derived enzymes modifies their immunomodulatory activity. I n one study the effect of caseins degraded by Lactobacillus GG-derived enzymes on the cytokines of 14 atopic patients aged 5 – 29 months was investigated.40Without hydrolysation, casein increased the production of I L-4 in lymphocyte cultures from patients with atopic dermatitis, whereas Lactobacillus GG-hydrolysed casein reduced the production of I L-4. Furthermore, I solauri et al.41 demonstrated that atopic eczema improved in terms of the SCORAD score, which reflects the extent and severity of the eczema, in infants given probiotic-supplemented formula (Lactobacillus GG) compared with an unsupplemented group. They also demonstrated a reduction in serum soluble CD4 (a marker of T-cell activation) and in urinary eosinophilic protein (a marker of eosinophilic inflammatory activity),showing how probiotics may counteract inflamma-tory responses beyond the intestinal milieu.
I n a double-blind, randomized, placebo-controlled trial, Kalliomaki et al.42showed how dietary supplementation with Lactobacillus GG of mothers with at least one first-degree relative with atopic eczema reduced the incidence of atopic eczema in their infants at 2 years of age.
PROBIOTICS AND URINARY TRACT INFECTIONS
Bacterial and fungal infections of the urogenital tract are also a promising field of application for probiotics. The flora of the vagina and urinary tract consists of a well-balanced system of about 50 bacterial strains. Lactobacilli dominate the healthy flora of pre-menopausal women. They are believed to protect the host against infections by several mechanisms, including occupation of specific adhesion sites at the epithelial surface of the urinary tract, stabilization of a low pH and the production
of antimicrobial substances such as acids, hydrogen peroxide and bacteriocins, degradation of polyamines and the production of surfactants, which have anti-adhesive properties. The balance can be disturbed by the overgrowth of indigenous vaginal bacteria such as Gardenella or Bacterioides, or by the invasion of foreign micro-organisms such as E. coli, Enterococcus faecalis or Candida. An obvious goal is to restore the balance of the urogenital flora by adding protective ‘probiotic’ lactobacilli. Reid and co-workers43in human studies demonstrated that oral L. rhamnosus reduced the risk of urinary tract infections and bacterial vaginosis.
PROBIOTICS AND RESPIRATORY TRACT INFECTIONS
The fact that certain lactic acid bacteria can modulate the immune system suggests the potential use of such micro-organisms as immune modulators in the upper respiratory tract. Gluck and Gebbers44found a significant reduction in the occurrence of nasal potentially pathogenic bacteria (Staphylococcus aureus, Streptococcus pneumoniae and β-haemolytic streptococci) in volunteers who received a probiotic drink but not in those who consumed yoghurt. The mechanisms underlying this result may have involved stimulation of the B-lymphocytes of the GALT, which then migrate to the upper respiratory immune system and lead to the production of the more effective secretory gA, which may have contributed to the elimination of the potentially pathogenic bacteria.
n a study of the effects of long-term consumption of a probiotic milk on respiratory tract infections, Hatakka and co-workers45 showed that Lactobacillus GG may reduce the incidence and severity of respiratory infections in children attending day-care centres.
Expanding fields of application for probiotics include their possible role in ameliorating intestinal dysfunction and malabsorption in children infected with HIV. Cunningham-Rundles et al.46demonstrated the ability of L. plantarum 299v to colonize children with HI V and to elicit a specific systemic immune response after oral supplementation.
Prebiotics
According to a recent report from the European Functional Food Science Working Group,4the criterion for a food ingredient to be classified as prebiotic is its ability to selectively stimulate in humans the growth of potentially beneficial bacteria in the gut. Prebiotics are oligosaccharides (short-chain carbohydrates) that resist digestion in the small intestine and are fermented in the colon. They reduce the growth or virulence of pathogens and induce systemic effects with potential health benefits.
An important natural example of prebiotic-containing food is human milk. After the initial colonization of the aseptic intestine during delivery (when it comes in to contact with the vaginal flora of the mother, leading to an inoculation with a diverse flora of bifidobacteria, enterobacteria, Bacterioides, clostridia and Gram-positive cocci), the flora of the newborn infant changes rapidly, presumably under the influence of diet.47I n this study, Harmsen et al.47demonstrated the differences in the development of the intestinal flora between breast-fed and formula-fed infants: using fluorescence i n s i tu hybridization, bifido-bacteria were shown to be dominant in breast-fed infants, whereas in most formula-fed infants there was a more diverse flora consisting of Bacterioides, enterobacteria, enterococci and clostridia in addition to bifidobacteria.
Oligosaccharides are the third largest solid constituent in human milk, after lactose and fat, comprising 20 g/l in colostrum and 12 g/l in milk. They consist of lactose at the reducing end, with a carbohydrate core that often contains a fructose or sialic acid at the non-reducing end.48Evidence has been found for the existence of oligosaccharides with up to 32 sugars and up to 15 fructoses.49The possibilities for structural isomers suggest that there may be tens of thousands, or perhaps even hundreds of thousands, of potential human milk oligosaccharide structures.
The glycosyltransferases that are involved in the production of human milk oligosaccharides are similar to the glycosyltransferases that synthesize the oligosaccharide moieties of mammalian cell surface receptors. Thus, milk oligosaccharides are likely to have some structural motifs similar to those found at the cell surface. These human milk oligosaccharides can therefore act as soluble homologues to the cell surface receptors that are the targets of specific pathogens. Serving as decoys, they bind to the pathogens and inhibit their ability to bind to the host cells, which is an essential first step in pathogenesis. Moreover, fermentation of inulin and oligofructose increases the production of short-chain fatty acids, mainly acetate, butyrate and propionate, which have immunomodulatory and anti-inflammatory properties. They lower the gut pH to levels at which pathogens such as E. coli are not able to compete effectively, and butyrate may also reduce the requirement of epithelial cells for glutamine, sparing it for other cells such as those of the immune system,50enhance intestinal defence functions and stimulate mucosal defences by increasing the proliferation of enterocytes.51Oligosaccharides in human milk have been found to inhibit the adherence of S. pneumon i ae to target cells i n v i tro,52 infection by invasive strains of Campylobacter jejuni53and the toxicity of the heat-stable toxin of E. coli.54
In pre-term infants, the establishment of a normal intestinal flora dominated by bifidobacteria increases the tolerance to enteral feeding and may prevent the development of necrotizing enterocolitis; however, due to the artificial microbiological environment in an intensive care unit, this is much more difficult than in breast-fed term infants. To mimic the molecule size distribution of neutral oligosaccharides in human milk, Boehm et al.55designed a mixture of neutral oligosaccharides consisting of galacto-oligosaccharides and fructo-oligosaccharides. Stool frequency and consistency in a pre-term group fed with this supplemented formula were closer to those of the breast-fed reference infants than were those of the control group. The same authors also demonstrated that the mixture of galacto-oligosaccharides and fructo-oligosaccharides had a dose-dependent bifidogenic effect.56 Recently, preliminary studies have indicated that prebiotics may be a useful dietary adjunct in the management of minor G disorders during infancy (infantile colic, regurgitation and constipation).57n a 16-week study in children aged between 10 and 24 months it was demonstrated that prophylactic feeding with fructo-oligosaccharides reduced the duration and number of episodes of recurrent diarrhoea.58
One particular human milk prebiotic is lactadherin, a 46 kDa glycoprotein produced by breast epithelial cells during lactation. It acts as a water-soluble cell surface analogue that can inhibit enteropathogen binding to host-cell receptors, thereby serving as an additional tier of defence. Newburg59showed
that lactadherin is a potent inhibitor of rotavirus in children and is representative of
a class of non-antibody glycoconjugates in human milk that have inhibitory activity against specific pathogens. Lactadherin probably prevents infection by binding to the rotavirus, thereby blocking viral binding
to the host-cell receptors, functioning as a competitive homologue to the host-cell rotavirus receptors.
Rather than human milk being a food that contains some antibodies, perhaps it is more accurate to regard it as a collection of biologically active protective agents that also provides nutritional support.59Human milk may protect the infant from harmful pathogens during the first year of life, before the immune system is fully mature and before a stable microflora has been established, whilst providing an environment that stimulates the growth of commensal bacteria.
Symbiotics
Apart from probiotics and prebiotics, a third approach is the use of symbiotics, a mixture
of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the G I tract, and by selectively stimulating the growth or activating the metabolism of one or a limited number of health-promoting bacteria, and thus improving the welfare of the host.60 Symbiotics seems to offer interesting possibilities in the prevention of infections. A study in malnourished children aged 3 – 5 years (with at least one sick episode) reported a lower number of sick days in the group fed with symbiotics.61
Conclusions
The body of evidence for a strong inter-relationship between the gut microflora and the immune response has grown considerably during the past 5 years, and the amount of published data on human clinical trials is increasing. Lactic acid bacteria and fibre have been an important part of the human diet for centuries, and it is only during the past 100 years that their consumption has dramatically reduced. I t is possible to hypothesize a negative effect of this dietary change on the development of the immune system and defences against infections.
During the past decade several studies have shown a specific immunological effect of different strains of lactic acid bacteria and bifidobacteria, suggesting the possibility of modulating the immune response in a favourable way to protect against infections and allergies. Further studies are needed to understand the best way to alter the composition of the gut microflora; at present, the published studies cover a wide range of strains, fibres and prebiotic mixtures.
n addition, although the absence of adverse effects due to the consumption of probiotics and prebiotics has been well documented in adults, the short- and long-term effects of their use during infancy, when the immune system is still developing, are not known. When using prebiotics and probiotics in infants, we are attempting to modify an extremely complex microbial ecosystem and are changing the type of intestinal microflora, and the timing and kind of colonization. A better understanding of the long-term effects of this artificial intervention on the infant gut microflora is an important future goal. Until then, clinicians should use a formulation of oligosaccharides as similar as possible to those contained in human milk, the best prebiotic-containing natural food.
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Address for correspondence
Dr F Salvini
Department of Paediatrics, University of Milan, San Paolo Hospital, via Di Rudinì, 8, 20142,
Milan, Italy.
E-mail: pediatria@hspsanpaolo.mi.it