Centre for Infectious Disease, ICMS, Barts and the London School of Medicine and Dentistry, London, UK
Received March 9, 2006; accepted March 29, 2006.
Address correspondence and reprint requests to Professor Thomas T. MacDonald, Centre for Infectious Disease, ICMS, Barts and the London School of Medicine and Dentistry, Whitechapel, London E1 2AB, UK. (e-mail: firstname.lastname@example.org).
At birth, the immune system, especially in the gut, is exposed to the largest single antigenic insult it is ever likely to encounter. The sterile gut rapidly becomes colonized with maternal fecal flora, whereas at the same time, large amounts of foreign protein (cow's milk) may be given as a source of nourishment. Even the breast-fed baby is exposed to food proteins via maternal milk. Gut bacteria and food antigens do not interact independently with the immune system; instead, bacteria and their products can activate pattern recognition receptors on immune cells, particularly dendritic cells, which then condition or control the type of adaptive immune response made to a nominal antigen such as cow milk. This complex interplay is only beginning to be appreciated and understood mechanistically (1).
If gut bacteria could be generically important in controlling immune responses, then it is not a great leap of faith to consider that variations in the constituent members of the flora might also be important in the development of inappropriate responses to food proteins in children, such as allergies. Now that detailed molecular analysis of the bacterial flora can be achieved, it is clear that the flora is complex and varies from individual to individual (2). However, traditional culture-based methods have suggested that differences in the flora might somehow be related to the development of inappropriate responses to food in children. Thus, there are differences in the flora of atopic versus nonatopic children, with fewer bifidobacteria and more clostridia in the atopic children (3,4).
Probiotics have been proposed as tools to influence the composition and thus, the immunomodulatory action of intestinal microflora in early childhood (5). Lactobacillus rhamnosus GG is one of the most commonly used probiotics. It has been reported to transiently colonize the gut of new born infants when administered to mothers during pregnancy, presumably via fecal contamination, (6) so it is possible to treat the newborn by treating the mother. The potential of Lactobacillus rhamnosus GG to prevent atopic disease was demonstrated in landmark double-blind, randomized, placebo-controlled studies showing that prenatal and postnatal (breast-feeding mothers) administration of this strain to mothers and their children at high risk of atopic disease reduced the prevalence of atopic eczema by half (23%) in comparison with infants receiving placebo (46%) at the age of 2 and then 4 years (7,8). Probiotic therapy did not alter any laboratory markers, and so the biological effects may be due to any one or a combination of the known effects of probiotics. These include their ability to increase epithelial barrier function (9), reverse the increased intestinal permeability characteristic of children with atopic eczema and food allergy (10), enhance gut-specific IgA responses (11), enhance the in vivo production of the immunosuppressive cytokine interleukin 10 (12), or promote the amounts of the immunosuppressive cytokine transforming growth factor β in breast milk (13).
In this issue, Gueimonde and colleagues (14) now report on the presence or absence of Lactobacillus rhamnosus GG in the stools of the children examined in their seminal earlier studies (7,8). Two time points were examined, at 6 months when the infants or their mothers had just completed probiotic treatment and at 12 months. Positivity was assessed by direct culture (with colonies identified as Lactobacillus rhamnosus GG by polymerase chain reaction [PCR]) and direct PCR of stools. At 6 months, viable Lactobacillus rhamnosus GG were isolated from 29 of 50 infants receiving the probiotic but were detected by PCR from 39 of 50. At 12 months, 24% were culture positive, and only 20% were positive by PCR. The authors therefore suggest that Lactobacillus rhamnosus GG only transiently colonizes the gut.
However, the interesting aspect of the study lies with the control group who did not receive any probiotic, either directly or from their mother. At 6 months, 13 of 47 the infants were positive by culture; and 20 of 47, by PCR, although only 3 had evidence of intake of probiotics by dietary records. At 12 months, 14% were positive by culture; and 20%, by PCR, which was not different from the group given lactobacilli.
The authors state that if a comparison was made between infants who were PCR positive and PCR negative, the incidence of atopic eczema was 28% in the former and 51% in the latter. This is remarkably similar to the results of the previous study for active treatment versus placebo cited above. Unfortunately, the authors do not explicitly state whether the comparison between PCR positive and negative children was made regardless of whether the child or their mother was on active treatment or placebo. The critical information is whether, if the placebo group was examined separately, a protective effect may have been seen in those who were PCR positive. Likewise, were PCR negative children on active treatment protected? The study would not have been powered for these analyses, but some details would have been welcome.
These results have clear implications for the earlier studies from this group where probiotic therapy halved the incidence of atopic eczema (7,8). If 42% of the placebo group had organisms in stools and this parameter protects regardless of whether the child was in the control or placebo group, it makes it rather difficult to ascribe protection from atopic eczema to active therapy with probiotics alone. It also makes it rather difficult to explain the very high incidence of eczema in the control group (46%) when 42% had Lactobacillus rhamnosus GG in their stools, unless they only had trace amounts and were colonized much later. The critical information which is needed to fully interpret these data and which should be further investigated is when does Lactobacillus rhamnosus GG first appear in the stools of controls? Sensitive methodologies for detecting and quantifying Lactobacillus rhamnosus GG in stool should also be used because it is hard to see how 9 of 32 infants receiving 20 billion Lactobacillus rhamnosus GG per day for 6 months could be PCR negative.
To explain the high incidence of PCR positivity in controls, the authors suggest environmental contamination because Lactobacillus rhamnosus GG may have been present in a wide range of food in Finland. However, if real, it does raise the issue of why atopic eczema is so very high in placebo-treated children in published works from Finland when the organism is so widespread in the environment. A plausible explanation is that the critical immunomodulatory effects of probiotics may be very early in life, at the time the virgin immune system is first exposed to luminal bacteria and food antigens. An equally plausible explanation is that it is a dose effect because there is now evidence that probiotics can be used therapeutically in children with atopic dermatitis (15,16) but which would argue against any effect being confined to the immediate postnatal period.
This study however does highlight one of the main flaws in probiotic research. It would be inconceivable for a new drug-based therapy to be tested on patients without pharmacodynamics and dose responses. It would also be inconceivable for a product to go forward into human without a clear mechanism of action for the "class" of therapeutics. With probiotics, however, dose responses are rarely, if ever done in humans, persistence is rarely studied, and a bewildering array of probiotics, either on their own or in a random combination with other organisms, are administered in a large variety of conditions. Mechanisms of action in the human gut are still unclear. Investigators are also slow to publish negative studies. Given the importance of the seminal observations on atopic eczema and probiotics shown in Finland, of which this present study is an important part, it would seem sensible for investigators to try to reproduce this observation in another cohort, in another country, using the same protocol and same probiotic. In this way, progress on the therapeutic use of probiotics can then proceed on a solid evidence base. A quick look at a trials register (www.controlled-trials.com) and searching on probiotics reveals the problem. There is a study virtually identical to the Finnish work ongoing in Wales in the UK, except a cocktail of probiotics are being used (ISRCTN26287422), and there is a study which has finished recruiting in Norway using Lactobacillus rhamnosus GG, similar to the Finnish study, except it is only being given to the mothers (NCT00159523). Let us hope that in these studies and the many others ongoing, the investigators look for the administered organisms in the placebo group.
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