Umeå 2008 UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1 Department of Clinical Sciences, Pediatrics Department of Clinical Microbiology, Immunology Umeå University, SE-901 87 Umeå, Sweden Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning Effects on Adaptive Immunity and Gut Microbial Function Christina West
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UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 85 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 85 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 85 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 85 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 87 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1191- ISSN 0346-6612 - ISBN 978-91-7264-585-1
Department of Clinical Sciences, Pediatrics
Department of Clinical Microbiology, Immunology Umeå University, SE-901 87 Umeå, Sweden
Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning
Effects on Adaptive Immunity and Gut Microbial Function
Christina West
Umeå 2008
Detta verk skyddas under lagen om upphovsrätt (URL 1960:729) ISBN 978-91-7264-585-1
Printed by Solfjädern Offset AB Umeå Sweden 2008
Detta verk skyddas under lagen om upphovsrätt (URL 1960:729) ISBN 978-91-7264-585-1
Printed by Solfjädern Offset AB Umeå Sweden 2008
To my family
“I see my path, but I don’t know where it leads. Not knowing where I’m going is what inspires me to travel it.” Rosalia de Castro
To my family
“I see my path, but I don’t know where it leads. Not knowing where I’m going is what inspires me to travel it.” Rosalia de Castro
TABLE OF CONTENTS ABSTRACT ............................................................................................. 7
ORIGINAL PAPERS .............................................................................. 9
ABBREVIATIONS IN SELECTION ................................................. 10
PREFACE .............................................................................................. 11
BACKGROUND ................................................................................... 12 ESTABLISHMENT OF THE GUT MICROBIOTA ........................................................... 12
Effects of exogenous factors .......................................................................... 12 Short-chain fatty acids .................................................................................... 14
GUT MICROBIOTA AND THE IMMUNE SYSTEM ........................................................ 15 The gut immune system .................................................................................. 15
Immune defence ......................................................................................................... 15 Oral tolerance .............................................................................................................. 16 Influences of immunity on gut colonization ....................................................... 17
ALLERGIC DISEASE ................................................................................................... 18 Nomenclature ..................................................................................................... 18 Th1/Th2 concept, regulatory T cells and T cell activation .................... 19 IgE-mediated hypersensitivity reaction ...................................................... 22 The atopic march ............................................................................................... 22 Intestinal permeability in allergic individuals ........................................... 24
ALTERATIONS OF GUT MICROBIOTA AND DISEASE ................................................ 24 The hygiene hypothesis .................................................................................. 24 Gut microbiota and allergy ............................................................................. 24
PROBIOTICS .............................................................................................................. 25 Historical view and definition ....................................................................... 25 Safety and guidelines ...................................................................................... 26 Proposed mechanisms of probiotics ............................................................. 27 Probiotics in the treatment of infectious disease ...................................... 28 Probiotics in the treatment and prevention of allergic disease ............ 29 Lactobacillus F19 .............................................................................................. 29
Isolation, colonization and safety .......................................................................... 29 Immunological effects in vitro and in animal models ..................................... 30
OBJECTIVES ....................................................................................... 31
SUBJECTS AND METHODS .......................................................... 32 STUDY DESIGN .......................................................................................................... 32
RESULTS .............................................................................................. 33 CHARACTERISTICS OF THE PARTICIPANTS .............................................................. 33 EFFECTS OF FEEDING PROBIOTICS DURING WEANING ON THE FUNCTIONAL STATUS OF GUT MICROBIOTA IN INFANTS (PAPER I) .............................................. 34
TABLE OF CONTENTS ABSTRACT ............................................................................................. 7
ORIGINAL PAPERS .............................................................................. 9
ABBREVIATIONS IN SELECTION ................................................. 10
PREFACE .............................................................................................. 11
BACKGROUND ................................................................................... 12 ESTABLISHMENT OF THE GUT MICROBIOTA ........................................................... 12
Effects of exogenous factors .......................................................................... 12 Short-chain fatty acids .................................................................................... 14
GUT MICROBIOTA AND THE IMMUNE SYSTEM ........................................................ 15 The gut immune system .................................................................................. 15
Immune defence ......................................................................................................... 15 Oral tolerance .............................................................................................................. 16 Influences of immunity on gut colonization ....................................................... 17
ALLERGIC DISEASE ................................................................................................... 18 Nomenclature ..................................................................................................... 18 Th1/Th2 concept, regulatory T cells and T cell activation .................... 19 IgE-mediated hypersensitivity reaction ...................................................... 22 The atopic march ............................................................................................... 22 Intestinal permeability in allergic individuals ........................................... 24
ALTERATIONS OF GUT MICROBIOTA AND DISEASE ................................................ 24 The hygiene hypothesis .................................................................................. 24 Gut microbiota and allergy ............................................................................. 24
PROBIOTICS .............................................................................................................. 25 Historical view and definition ....................................................................... 25 Safety and guidelines ...................................................................................... 26 Proposed mechanisms of probiotics ............................................................. 27 Probiotics in the treatment of infectious disease ...................................... 28 Probiotics in the treatment and prevention of allergic disease ............ 29 Lactobacillus F19 .............................................................................................. 29
Isolation, colonization and safety .......................................................................... 29 Immunological effects in vitro and in animal models ..................................... 30
OBJECTIVES ....................................................................................... 31
SUBJECTS AND METHODS .......................................................... 32 STUDY DESIGN .......................................................................................................... 32
RESULTS .............................................................................................. 33 CHARACTERISTICS OF THE PARTICIPANTS .............................................................. 33 EFFECTS OF FEEDING PROBIOTICS DURING WEANING ON THE FUNCTIONAL STATUS OF GUT MICROBIOTA IN INFANTS (PAPER I) .............................................. 34
EFFECTS OF FEEDING PROBIOTICS DURING WEANING ON INFECTIONS AND ANTIBODY RESPONSES TO DIPHTHERIA, TETANUS AND HIB VACCINES (PAPER II) ............................................................................................................................... 37 MATURATION OF T CELL FUNCTION IN THE HUMAN INFANT AND EFFECTS THEREON OF FEEDING PROBIOTICS DURING WEANING (PAPER III) ..................... 39 PROBIOTICS DURING WEANING REDUCE THE INCIDENCE OF ECZEMA (PAPER IV) .............................................................................................................................. 41
GENERAL DISCUSSION .................................................................. 43 PROBIOTIC EFFECTS ON HOMEOSTASIS OF GUT MICROBIOTA ............................. 43
Dose, compliance and timing ......................................................................... 43 Colonization with lactobacilli ......................................................................... 44
PROBIOTIC EFFECTS ON ADAPTIVE IMMUNITY ....................................................... 47 Effects on infections ......................................................................................... 47 Effects on specific antibody responses to common vaccines ................ 48
PROBIOTIC EFFECTS IN THE PREVENTION OF ALLERGY ........................................ 49 Prevention of eczema and respiratory allergies ....................................... 49 Sensitization and cow’s milk allergy ........................................................... 51 Gut barrier function .......................................................................................... 52 Immune-stimulating effects ............................................................................ 52
Maternal and fetal immune responses ................................................................ 52 Infant immune responses ........................................................................................ 53
STRENGTHS AND WEAKNESSES OF THE STUDY ..................................................... 56 FUTURE ASPECTS ..................................................................................................... 57
CONCLUSIONS ................................................................................... 58
POPULÄRVETENSKAPLIG SAMMANFATTNING ..................... 59
ACKNOWLEDGEMENTS ................................................................. 61
REFERENCES ..................................................................................... 63
EFFECTS OF FEEDING PROBIOTICS DURING WEANING ON INFECTIONS AND ANTIBODY RESPONSES TO DIPHTHERIA, TETANUS AND HIB VACCINES (PAPER II) ............................................................................................................................... 37 MATURATION OF T CELL FUNCTION IN THE HUMAN INFANT AND EFFECTS THEREON OF FEEDING PROBIOTICS DURING WEANING (PAPER III) ..................... 39 PROBIOTICS DURING WEANING REDUCE THE INCIDENCE OF ECZEMA (PAPER IV) .............................................................................................................................. 41
GENERAL DISCUSSION .................................................................. 43 PROBIOTIC EFFECTS ON HOMEOSTASIS OF GUT MICROBIOTA ............................. 43
Dose, compliance and timing ......................................................................... 43 Colonization with lactobacilli ......................................................................... 44
PROBIOTIC EFFECTS ON ADAPTIVE IMMUNITY ....................................................... 47 Effects on infections ......................................................................................... 47 Effects on specific antibody responses to common vaccines ................ 48
PROBIOTIC EFFECTS IN THE PREVENTION OF ALLERGY ........................................ 49 Prevention of eczema and respiratory allergies ....................................... 49 Sensitization and cow’s milk allergy ........................................................... 51 Gut barrier function .......................................................................................... 52 Immune-stimulating effects ............................................................................ 52
Maternal and fetal immune responses ................................................................ 52 Infant immune responses ........................................................................................ 53
STRENGTHS AND WEAKNESSES OF THE STUDY ..................................................... 56 FUTURE ASPECTS ..................................................................................................... 57
CONCLUSIONS ................................................................................... 58
POPULÄRVETENSKAPLIG SAMMANFATTNING ..................... 59
ACKNOWLEDGEMENTS ................................................................. 61
REFERENCES ..................................................................................... 63
7
ABSTRACT Introduction: Gut microbial composition has been associated with immune-mediated diseases. Breastfeeding yields a microbiota rich in bifidobacteria and promotes colonization by lactobacilli. Bifidobacteria and lactobacilli are considered health-promoting and are used as probiotics, i.e. live microbial food supplements which when ingested in adequate amounts confer a beneficial effect on the host. During weaning the developing gut immune system is exposed to an increasing variety of antigens from both foods and gut microbiota. Aims: We aimed to determine if daily feeding of 1x108 colony-forming units (CFU) of the probiotic Lactobacillus paracasei ssp. paracasei strain F19 (LF19) to healthy term infants from 4 to 13 months of age could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, T cell function, Th1/Th2 immune balance and eczema incidence. Study design: Infants were randomized to daily intake of cereals with (n=89) or without LF19 (n=90) from 4-13 months of age. Clinical outcome measures were monitored by diaries and a questionnaire. Stool and blood samples were obtained at 4, 6½, 9, 13 and 5½, 6½, 12 and 13 months of age, respectively. Stool samples were analyzed for lactobacilli counts by conventional culture methods and the presence of LF19 was verified by randomly amplified polymerase chain reaction (RAPD-PCR). Fecal short-chain fatty acid (SCFA) pattern, a proxy for gut microbial function, was determined by gas-liquid chromatography. After polyclonal or specific activation of T cells, the cytokine mRNA expression levels [interleukin 2 (IL2), IFN-γ, IL4 and IL10] were determined on isolated mRNA by quantitative real time reverse transcriptase-PCR. Serum concentrations of total and specific IgE antibodies, Haemophilus influenzae type b, diphtheria and tetanus toxoid specific IgG antibodies were analyzed by enzyme immunoassay. Results: Feeding LF19 maintained high fecal lactobacilli counts during weaning. Persistent colonization with LF19 induced differences in the fecal SCFA pattern. The cumulative incidence of eczema was lower in the probiotic group, in conjunction with a higher IFN-γ/IL4 mRNA ratio in polyclonally activated T cells. Even though there was an effect by LF19 on Th1/Th2 immune balance, there was no effect on IgE sensitization. Infants in both groups increased their capacity to express both Th1 and Th2 cytokines during the second half of infancy but the expression was still lower than that of adults. Infants in the probiotic group had lower IL2 levels after polyclonal T cell activation at 13 months of age compared with infants in the placebo group. Infants fed LF19 did not have fewer infections, but had fewer days with antibiotic prescription compared with infants fed placebo. In addition, compared to placebo, persistent colonization by LF19 enhanced specific vaccine responses to protein antigens during the course of vaccination. Conclusions: We conclude that feeding LF19 was safe, based on no observed adverse effects in our study. Infants in both groups demonstrated maturation of adaptive immune responses during weaning. Adding probiotics in complementary foods during weaning reduced the risk of eczema by 50%, with a concomitant shift towards an enhanced Th1/Th2 ratio. The reduction of eczema might be explained by probiotic effects on both T cell-mediated immune responses and reinforced gut microbial function.
7
ABSTRACT Introduction: Gut microbial composition has been associated with immune-mediated diseases. Breastfeeding yields a microbiota rich in bifidobacteria and promotes colonization by lactobacilli. Bifidobacteria and lactobacilli are considered health-promoting and are used as probiotics, i.e. live microbial food supplements which when ingested in adequate amounts confer a beneficial effect on the host. During weaning the developing gut immune system is exposed to an increasing variety of antigens from both foods and gut microbiota. Aims: We aimed to determine if daily feeding of 1x108 colony-forming units (CFU) of the probiotic Lactobacillus paracasei ssp. paracasei strain F19 (LF19) to healthy term infants from 4 to 13 months of age could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, T cell function, Th1/Th2 immune balance and eczema incidence. Study design: Infants were randomized to daily intake of cereals with (n=89) or without LF19 (n=90) from 4-13 months of age. Clinical outcome measures were monitored by diaries and a questionnaire. Stool and blood samples were obtained at 4, 6½, 9, 13 and 5½, 6½, 12 and 13 months of age, respectively. Stool samples were analyzed for lactobacilli counts by conventional culture methods and the presence of LF19 was verified by randomly amplified polymerase chain reaction (RAPD-PCR). Fecal short-chain fatty acid (SCFA) pattern, a proxy for gut microbial function, was determined by gas-liquid chromatography. After polyclonal or specific activation of T cells, the cytokine mRNA expression levels [interleukin 2 (IL2), IFN-γ, IL4 and IL10] were determined on isolated mRNA by quantitative real time reverse transcriptase-PCR. Serum concentrations of total and specific IgE antibodies, Haemophilus influenzae type b, diphtheria and tetanus toxoid specific IgG antibodies were analyzed by enzyme immunoassay. Results: Feeding LF19 maintained high fecal lactobacilli counts during weaning. Persistent colonization with LF19 induced differences in the fecal SCFA pattern. The cumulative incidence of eczema was lower in the probiotic group, in conjunction with a higher IFN-γ/IL4 mRNA ratio in polyclonally activated T cells. Even though there was an effect by LF19 on Th1/Th2 immune balance, there was no effect on IgE sensitization. Infants in both groups increased their capacity to express both Th1 and Th2 cytokines during the second half of infancy but the expression was still lower than that of adults. Infants in the probiotic group had lower IL2 levels after polyclonal T cell activation at 13 months of age compared with infants in the placebo group. Infants fed LF19 did not have fewer infections, but had fewer days with antibiotic prescription compared with infants fed placebo. In addition, compared to placebo, persistent colonization by LF19 enhanced specific vaccine responses to protein antigens during the course of vaccination. Conclusions: We conclude that feeding LF19 was safe, based on no observed adverse effects in our study. Infants in both groups demonstrated maturation of adaptive immune responses during weaning. Adding probiotics in complementary foods during weaning reduced the risk of eczema by 50%, with a concomitant shift towards an enhanced Th1/Th2 ratio. The reduction of eczema might be explained by probiotic effects on both T cell-mediated immune responses and reinforced gut microbial function.
8
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9
ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-IV). I. Christina E West, Elisabeth Norin, Marie-Louise Hammarström, Olle
Hernell. Effects of feeding probiotics during weaning on the functional status of gut microbiota in infants. Submitted.
II. Christina E West, Leif Gothefors, Marta Granström, Helena Käyhty,
Marie-Louise K.C. Hammarström, Olle Hernell. Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines. Pediatr Allergy Immunol 2008;19:53-60.
III. Christina E West, Olle Hernell, Yvonne Andersson, Marianne Sjöstedt,
Marie-Louise Hammarström. Maturation of T cell function in the human infant and effects thereon of probiotic feeding during weaning. In manuscript.
IV. Christina E West, Marie-Louise Hammarström, Olle Hernell. Probiotics
during weaning reduce the incidence of eczema. Pediatr Allergy Immunol, in press.
Paper II and IV are reprinted with permission from the publisher.
9
ORIGINAL PAPERS
This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-IV). I. Christina E West, Elisabeth Norin, Marie-Louise Hammarström, Olle
Hernell. Effects of feeding probiotics during weaning on the functional status of gut microbiota in infants. Submitted.
II. Christina E West, Leif Gothefors, Marta Granström, Helena Käyhty,
Marie-Louise K.C. Hammarström, Olle Hernell. Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines. Pediatr Allergy Immunol 2008;19:53-60.
III. Christina E West, Olle Hernell, Yvonne Andersson, Marianne Sjöstedt,
Marie-Louise Hammarström. Maturation of T cell function in the human infant and effects thereon of probiotic feeding during weaning. In manuscript.
IV. Christina E West, Marie-Louise Hammarström, Olle Hernell. Probiotics
during weaning reduce the incidence of eczema. Pediatr Allergy Immunol, in press.
Paper II and IV are reprinted with permission from the publisher.
10
ABBREVIATIONS IN SELECTION APC Antigen-presenting cell BCR B cell receptor CMA Cow’s milk allergy CTL Cytotoxic T cell DC Dendritic cell EIA Enzyme immunoassay FOS Fructo-oligosaccharides GALT Gut-associated lymphoid tissue GI Gastrointestinal GOS Galacto-oligosaccharides HibPS Haemophilus influenzae type b capsular polysaccharide IEL Intraepithelial lymphocyte IFN-γ Interferon-gamma Ig Immunoglobulin IL Interleukin LF19 Lactobacillus paracasei ssp. paracasei strain F19 LPL Lamina propria lymphocyte LPS Lipopolysaccharide LTA Lipoteic acid mAb Monoclonal antibody MHC Major histocompatibility complex MLN Mesenteric lymph node NFкB Nuclear factor kappa beta PAMP Pathogen-associated molecular patterns PBMC Peripheral blood mononuclear cells PP Peyer’s plaque RAPD-PCR Randomly amplified polymerase chain reaction qRT-PCR Quantitative real time-polymerase chain reaction SCFA Short-chain fatty acid SCORAD Scoring atopic dermatitis SPT Skin prick test TGF-β Transforming growth factor beta Th T helper cell TLR Toll-like receptor TNF-α Tumor necrosis factor α Treg T regulatory cell Tr1 T regulatory cell type 1 TCR T cell receptor TT Tetanus toxoid
10
ABBREVIATIONS IN SELECTION APC Antigen-presenting cell BCR B cell receptor CMA Cow’s milk allergy CTL Cytotoxic T cell DC Dendritic cell EIA Enzyme immunoassay FOS Fructo-oligosaccharides GALT Gut-associated lymphoid tissue GI Gastrointestinal GOS Galacto-oligosaccharides HibPS Haemophilus influenzae type b capsular polysaccharide IEL Intraepithelial lymphocyte IFN-γ Interferon-gamma Ig Immunoglobulin IL Interleukin LF19 Lactobacillus paracasei ssp. paracasei strain F19 LPL Lamina propria lymphocyte LPS Lipopolysaccharide LTA Lipoteic acid mAb Monoclonal antibody MHC Major histocompatibility complex MLN Mesenteric lymph node NFкB Nuclear factor kappa beta PAMP Pathogen-associated molecular patterns PBMC Peripheral blood mononuclear cells PP Peyer’s plaque RAPD-PCR Randomly amplified polymerase chain reaction qRT-PCR Quantitative real time-polymerase chain reaction SCFA Short-chain fatty acid SCORAD Scoring atopic dermatitis SPT Skin prick test TGF-β Transforming growth factor beta Th T helper cell TLR Toll-like receptor TNF-α Tumor necrosis factor α Treg T regulatory cell Tr1 T regulatory cell type 1 TCR T cell receptor TT Tetanus toxoid
Preface
11
PREFACE The gut microbiota consists of a complex mixture of microorganisms contributing to human health. Some of the gut microbial functions act in collaboration with epithelial and immune cells of the intestinal mucosa. Under normal circumstances the host and gut microbiota thrive in symbiosis, i.e. in close mutuality. It takes several years to develop an adult type gut microbiota with over 500 different species of mostly harmless bacteria - the commensal microbiota. Once the commensal microbiota is established, it remains rather stable. A human adult carries about 1 kg of bacteria in the gut, predominantly in the large bowel (colon), and the numbers of bacteria in the gastrointestinal tract are approximately 1014 thus outnumbering the number of cells in our body by a factor of 10. The importance of the gut microbiota and its composition in health and disease have gained much attention during the last decade. A current hypothesis is that a disturbed gut microbial composition can be linked to immune-mediated diseases. This thesis focuses on possible health effects by modulating the gut microbiota during a critical period for its establishment, i.e. the weaning period. Probiotic means “for life” and refers to bacteria associated with beneficial effects for humans and animals. By adding a probiotic in weaning foods, we studied the effects on health, gut microbial function and development of adaptive immunity during the second half of infancy.
Preface
11
PREFACE The gut microbiota consists of a complex mixture of microorganisms contributing to human health. Some of the gut microbial functions act in collaboration with epithelial and immune cells of the intestinal mucosa. Under normal circumstances the host and gut microbiota thrive in symbiosis, i.e. in close mutuality. It takes several years to develop an adult type gut microbiota with over 500 different species of mostly harmless bacteria - the commensal microbiota. Once the commensal microbiota is established, it remains rather stable. A human adult carries about 1 kg of bacteria in the gut, predominantly in the large bowel (colon), and the numbers of bacteria in the gastrointestinal tract are approximately 1014 thus outnumbering the number of cells in our body by a factor of 10. The importance of the gut microbiota and its composition in health and disease have gained much attention during the last decade. A current hypothesis is that a disturbed gut microbial composition can be linked to immune-mediated diseases. This thesis focuses on possible health effects by modulating the gut microbiota during a critical period for its establishment, i.e. the weaning period. Probiotic means “for life” and refers to bacteria associated with beneficial effects for humans and animals. By adding a probiotic in weaning foods, we studied the effects on health, gut microbial function and development of adaptive immunity during the second half of infancy.
Background
12
BACKGROUND
Establishment of the gut microbiota Colonization of the previously germfree gut starts immediately after birth and is dependent on the microorganisms derived from the mother’s intestinal, vaginal and skin microbiotas together with some environmental species. It is a complex and multifactorial process that is shaped by interactions between the environment, diet, microbe-associated and host-related factors. The gut of the neonate is not anaerobic (strictly oxygen-free) and consequently the numbers of aerobic bacteria are higher than later in life. Aerobic bacteria are part of the normal gut microbiota but may cause infections if they reach other parts of the body. Colonization of the gut during the first weeks of life is dominated by aerobic and facultative anaerobic bacteria. As oxygen is consumed, anaerobes can establish. Many anaerobic bacteria are harmless and fight for space and nutrients and as a result they restrain the number of aerobic and facultative anaerobic bacteria. Escherichia coli (E. coli) is typically found in feces and is one of the most common facultative anaerobic bacteria in the gut. Decades ago, enteric bacteria e.g. E. coli, and enterococci appeared as initial gut colonizers, followed by anaerobic bacteria e.g. bifidobacteria, Bacteroides and streptococci (1). Recent data from a prospective multicenter European birth cohort study, the AllergyFlora study, demonstrated that among facultative anaerobic bacteria, coagulase-negative staphylococci were the earliest colonizers, followed by enterococci. E. coli and other enteric bacteria traditionally viewed upon as early colonizers appeared late, and it was not before 6 months of age that the majority of infants were colonized by E. coli. In fact, Staphylococcus aureus (S. aureus) was almost as frequent in fecal samples as E. coli during the first two months of life. Among anaerobic bacteria, bifidobacteria appeared first, followed by clostridia and Bacteroides. This indicates that colonization by typical fecal bacteria e.g. E. coli is slow in contemporary western societies, suggesting a very limited spread of bacteria. In their absence, skin bacteria like staphylococci and other bacteria that are normally not dominant in the gut microbiota become the first colonizers, indicating a reduced competition by other gut bacteria (2, 3).
Effects of exogenous factors Exogenous factors e.g. environmental factors, hygienic measures, preterm delivery, route of delivery, antibiotics to the mother prior to birth or to the neonate and diet modulate the dynamics and outcome of colonization (4). In economically disadvantaged areas of the world, gut colonization is earlier and the microbiota comprises a more diverse range of bacteria with a faster strain turnover compared with that in industrialized countries (5). Colonization with E. coli, bifidobacteria and Bacteroides appeared later in caesarean-delivered infants with increased colonization by clostridia, Klebsiella and enteric bacteria other than E. coli (2, 6). Antibiotic administration to the mother during pregnancy or the infant during the first 6 months of life was linked to a lower ratio of strict to facultative anaerobes (2). The effects of diet on early gut microbial composition have been extensively studied. Although not unambiguously found, breastfed infants are considered to
Background
12
BACKGROUND
Establishment of the gut microbiota Colonization of the previously germfree gut starts immediately after birth and is dependent on the microorganisms derived from the mother’s intestinal, vaginal and skin microbiotas together with some environmental species. It is a complex and multifactorial process that is shaped by interactions between the environment, diet, microbe-associated and host-related factors. The gut of the neonate is not anaerobic (strictly oxygen-free) and consequently the numbers of aerobic bacteria are higher than later in life. Aerobic bacteria are part of the normal gut microbiota but may cause infections if they reach other parts of the body. Colonization of the gut during the first weeks of life is dominated by aerobic and facultative anaerobic bacteria. As oxygen is consumed, anaerobes can establish. Many anaerobic bacteria are harmless and fight for space and nutrients and as a result they restrain the number of aerobic and facultative anaerobic bacteria. Escherichia coli (E. coli) is typically found in feces and is one of the most common facultative anaerobic bacteria in the gut. Decades ago, enteric bacteria e.g. E. coli, and enterococci appeared as initial gut colonizers, followed by anaerobic bacteria e.g. bifidobacteria, Bacteroides and streptococci (1). Recent data from a prospective multicenter European birth cohort study, the AllergyFlora study, demonstrated that among facultative anaerobic bacteria, coagulase-negative staphylococci were the earliest colonizers, followed by enterococci. E. coli and other enteric bacteria traditionally viewed upon as early colonizers appeared late, and it was not before 6 months of age that the majority of infants were colonized by E. coli. In fact, Staphylococcus aureus (S. aureus) was almost as frequent in fecal samples as E. coli during the first two months of life. Among anaerobic bacteria, bifidobacteria appeared first, followed by clostridia and Bacteroides. This indicates that colonization by typical fecal bacteria e.g. E. coli is slow in contemporary western societies, suggesting a very limited spread of bacteria. In their absence, skin bacteria like staphylococci and other bacteria that are normally not dominant in the gut microbiota become the first colonizers, indicating a reduced competition by other gut bacteria (2, 3).
Effects of exogenous factors Exogenous factors e.g. environmental factors, hygienic measures, preterm delivery, route of delivery, antibiotics to the mother prior to birth or to the neonate and diet modulate the dynamics and outcome of colonization (4). In economically disadvantaged areas of the world, gut colonization is earlier and the microbiota comprises a more diverse range of bacteria with a faster strain turnover compared with that in industrialized countries (5). Colonization with E. coli, bifidobacteria and Bacteroides appeared later in caesarean-delivered infants with increased colonization by clostridia, Klebsiella and enteric bacteria other than E. coli (2, 6). Antibiotic administration to the mother during pregnancy or the infant during the first 6 months of life was linked to a lower ratio of strict to facultative anaerobes (2). The effects of diet on early gut microbial composition have been extensively studied. Although not unambiguously found, breastfed infants are considered to
Background
13
have a microbiota dominated by bifidobacteria, and its composition is less diverse compared with that of formula-fed infants (1, 4, 7). Bifidobacteria are considered health-promoting and may inhibit growth of pathogenic bacteria in vitro (8). Breast milk has a lower buffering capacity compared with formula, is rich in oligosaccharides and may promote the growth of bifidobacteria (9). It has been suggested that breast milk might even provide bifidobacteria and lactobacilli (10, 11). Formula-fed infants appear to develop a more complex microbiota, although it depends on the composition of the formula. Higher levels and frequency of facultative anaerobes, Bacteroides and clostridia have been observed in formula-fed compared with breastfed infants (1, 7, 12). Lactobacilli appear and disappear from birth until weaning, suggesting transient colonization (1, 2). Many infants are mixed-fed (breast+ formula-feeding), but little is known about the impact of a mixed feeding on gut microbial composition (13). One small longitudinal study comprising 11 initially breastfed infants weaned to formula, demonstrated high inter-individual variability of gut microbial composition, maintenance of high bifidobacterial counts throughout weaning and maturation of fecal gut microbiota (14). Around the time of introduction of complementary foods, i.e. solid foods, the gut microbial composition changes, the change being more pronounced in the breastfed infant. Successively the gut microbiota becomes more diverse, and resembles that of adults by the age of two years, (Fig. 1) (12, 15). However, surprisingly little is known about the development of the gut microbiota during the period of cessation of breastfeeding and introduction of complementary foods, and the effects of which foods are introduced at which time (16).
Aerobes and facultative anaerobes
Anaerobes
Bifidobacteria
Unculturable bacteria
Increase in microbial diversity
Establishedgut microbiota
Breastfeeding
Weaning
Complementary foods
0 Age (years) 2
Figure 1. Schematic illustration of the development of gut microbiota during the first 2 years of life. Adapted from S Salminen et al, 2005 (15), and printed with permission from the publisher.
Background
13
have a microbiota dominated by bifidobacteria, and its composition is less diverse compared with that of formula-fed infants (1, 4, 7). Bifidobacteria are considered health-promoting and may inhibit growth of pathogenic bacteria in vitro (8). Breast milk has a lower buffering capacity compared with formula, is rich in oligosaccharides and may promote the growth of bifidobacteria (9). It has been suggested that breast milk might even provide bifidobacteria and lactobacilli (10, 11). Formula-fed infants appear to develop a more complex microbiota, although it depends on the composition of the formula. Higher levels and frequency of facultative anaerobes, Bacteroides and clostridia have been observed in formula-fed compared with breastfed infants (1, 7, 12). Lactobacilli appear and disappear from birth until weaning, suggesting transient colonization (1, 2). Many infants are mixed-fed (breast+ formula-feeding), but little is known about the impact of a mixed feeding on gut microbial composition (13). One small longitudinal study comprising 11 initially breastfed infants weaned to formula, demonstrated high inter-individual variability of gut microbial composition, maintenance of high bifidobacterial counts throughout weaning and maturation of fecal gut microbiota (14). Around the time of introduction of complementary foods, i.e. solid foods, the gut microbial composition changes, the change being more pronounced in the breastfed infant. Successively the gut microbiota becomes more diverse, and resembles that of adults by the age of two years, (Fig. 1) (12, 15). However, surprisingly little is known about the development of the gut microbiota during the period of cessation of breastfeeding and introduction of complementary foods, and the effects of which foods are introduced at which time (16).
Aerobes and facultative anaerobes
Anaerobes
Bifidobacteria
Unculturable bacteria
Increase in microbial diversity
Establishedgut microbiota
Breastfeeding
Weaning
Complementary foods
0 Age (years) 2
Figure 1. Schematic illustration of the development of gut microbiota during the first 2 years of life. Adapted from S Salminen et al, 2005 (15), and printed with permission from the publisher.
Background
14
Short-chain fatty acids To study the intestinal microbiota requires compound methodology. One method is to study the metabolic products of the microbial ecosystem. The metabolic activity of the intestinal microbiota is complex and its biochemical activities may be more important to the host organism than the numbers of defined microbes at any particular compartment of the intestine. Bacteria metabolize unabsorbed carbohydrates to short-chain fatty acids (SCFA), CO2 and H2 in the colon.. SCFA are intermediate or end products of carbohydrate fermentation by groups of bacteria in the colon. They are monocarboxylic acids with a chain-length up to 6 carbon atoms i.e. acetic, propionic, butyric, iso-valeric, valeric, iso-caproic and caproic acids. The fecal pattern of SCFA reflects the functional status of the gut microbiota. Thus, analysis of SCFA is a complementary method to other established methods for the study of gut microbial composition (17). Evidently, the two key prerequisites for formation of SCFA are the presence of substrates and a gut microbiota capable of fermenting them. The fermentation of polysaccharides yields acetic, propionic and butyric acids, whereas branched-chain acids, (the iso-acids), and other minor acids are likely to be products of the digestion of proteins and lipids. Microbially produced SCFA is an important fuel for the colonocytes, and can also contribute to the overall energy balance. The three major SCFA, acetic, propionic and butyric acids, are important for epithelial cell proliferation and differentiation (18). Furthermore, SCFA may be important in establishing a balanced ecosystem in the gut. As mentioned, the intestine is sterile at birth and consequently there is no production of SCFA. In early infancy the production of acetic acid becomes predominant, followed by propionic- and butyric acid. The proportion of acetic acid decreases while those of the other SCFA increase with age, reflecting the development of a more complex microbiota. Breastfed infants have a SCFA pattern dominated by lactic and acetic acids with little butyric acid, whereas formula-fed infants have a pattern dominated by acetic and propionic acids with some butyric acid (19). During the period of introduction of complementary foods, more non-digestible complex carbohydrates are introduced to the infant diet and the fecal SCFA profile changes. The ability to ferment these complex carbohydrates may be slow, predominantly in breastfed infants. The change in SCFA profile differs between exclusively breastfed and formula-fed infants. In the former, propionic acid increases at the start of weaning and butyric acid concentrations increase at a slow pace. The proportion of lactic acid decreases over the first year of life. In formula-fed infants the change in fecal SCFA profile is less marked, since they are faster in developing their capacity to ferment complex carbohydrates, due to the more diverse microbiota from early on in life. In formula-fed infants there is a slow and gradual increase in butyric acid during the period of introduction of complementary foods (13, 16), (Fig. 2).
Background
14
Short-chain fatty acids To study the intestinal microbiota requires compound methodology. One method is to study the metabolic products of the microbial ecosystem. The metabolic activity of the intestinal microbiota is complex and its biochemical activities may be more important to the host organism than the numbers of defined microbes at any particular compartment of the intestine. Bacteria metabolize unabsorbed carbohydrates to short-chain fatty acids (SCFA), CO2 and H2 in the colon.. SCFA are intermediate or end products of carbohydrate fermentation by groups of bacteria in the colon. They are monocarboxylic acids with a chain-length up to 6 carbon atoms i.e. acetic, propionic, butyric, iso-valeric, valeric, iso-caproic and caproic acids. The fecal pattern of SCFA reflects the functional status of the gut microbiota. Thus, analysis of SCFA is a complementary method to other established methods for the study of gut microbial composition (17). Evidently, the two key prerequisites for formation of SCFA are the presence of substrates and a gut microbiota capable of fermenting them. The fermentation of polysaccharides yields acetic, propionic and butyric acids, whereas branched-chain acids, (the iso-acids), and other minor acids are likely to be products of the digestion of proteins and lipids. Microbially produced SCFA is an important fuel for the colonocytes, and can also contribute to the overall energy balance. The three major SCFA, acetic, propionic and butyric acids, are important for epithelial cell proliferation and differentiation (18). Furthermore, SCFA may be important in establishing a balanced ecosystem in the gut. As mentioned, the intestine is sterile at birth and consequently there is no production of SCFA. In early infancy the production of acetic acid becomes predominant, followed by propionic- and butyric acid. The proportion of acetic acid decreases while those of the other SCFA increase with age, reflecting the development of a more complex microbiota. Breastfed infants have a SCFA pattern dominated by lactic and acetic acids with little butyric acid, whereas formula-fed infants have a pattern dominated by acetic and propionic acids with some butyric acid (19). During the period of introduction of complementary foods, more non-digestible complex carbohydrates are introduced to the infant diet and the fecal SCFA profile changes. The ability to ferment these complex carbohydrates may be slow, predominantly in breastfed infants. The change in SCFA profile differs between exclusively breastfed and formula-fed infants. In the former, propionic acid increases at the start of weaning and butyric acid concentrations increase at a slow pace. The proportion of lactic acid decreases over the first year of life. In formula-fed infants the change in fecal SCFA profile is less marked, since they are faster in developing their capacity to ferment complex carbohydrates, due to the more diverse microbiota from early on in life. In formula-fed infants there is a slow and gradual increase in butyric acid during the period of introduction of complementary foods (13, 16), (Fig. 2).
Background
15
Birth
Breastfed Mixed-fed Formula-fedBifidobacteria/lactobacilli Data are lacking More diverse biota
Lactic and acetic acid Acetic and propionic acid
WeaningIncrease in microbial diversity
Increase in propionic acid and butyric acid
Adult>500 species in dominant microbiota
Acetic/propionic/butyric acid
Figure 2. Schematic description of the development of gut microbiota and production ofSCFA depending on feeding mode. Adapted from C Edwards, 2006 (16) and printed withpermission from the publisher.
Gut microbiota and the immune system Intestinal bacteria are mandatory for the activation of the host immune system, and have been proposed to contribute to appropriate balancing of immune responses later in life. Gnotobiotic (microbe-free) animal models have demonstrated that intestinal bacteria are indispensible for the development of gut and systemic immune responses (20-22). The establishment of the intestinal microbiota is considered to be a prerequisite for establishing immune balance also in humans (23, 24).
The gut immune system
Immune defence The gut immune system has a dual role in mounting immune responses to pathogens and yet not reacting to harmless bacteria and food antigens. Since many offending infections enter the human body at mucosal sites, efficient immune responses are needed for defence. However, active immune responses to harmless food antigens can be harmful, as in hypersensitivity reactions causing food allergies and in celiac disease (25, 26). The immune defence in the intestine primarily acts to prevent adhesion and invasion of pathogens by peristalsis and mucus lining the mucosal epithelium of the gastrointestinal tract. A superficial layer of secreted non-specific, e.g. mucins and defensins, and specific secretory components i.e. secretory IgA (sIgA), also protect
Background
15
Birth
Breastfed Mixed-fed Formula-fedBifidobacteria/lactobacilli Data are lacking More diverse biota
Lactic and acetic acid Acetic and propionic acid
WeaningIncrease in microbial diversity
Increase in propionic acid and butyric acid
Adult>500 species in dominant microbiota
Acetic/propionic/butyric acid
Figure 2. Schematic description of the development of gut microbiota and production ofSCFA depending on feeding mode. Adapted from C Edwards, 2006 (16) and printed withpermission from the publisher.
Gut microbiota and the immune system Intestinal bacteria are mandatory for the activation of the host immune system, and have been proposed to contribute to appropriate balancing of immune responses later in life. Gnotobiotic (microbe-free) animal models have demonstrated that intestinal bacteria are indispensible for the development of gut and systemic immune responses (20-22). The establishment of the intestinal microbiota is considered to be a prerequisite for establishing immune balance also in humans (23, 24).
The gut immune system
Immune defence The gut immune system has a dual role in mounting immune responses to pathogens and yet not reacting to harmless bacteria and food antigens. Since many offending infections enter the human body at mucosal sites, efficient immune responses are needed for defence. However, active immune responses to harmless food antigens can be harmful, as in hypersensitivity reactions causing food allergies and in celiac disease (25, 26). The immune defence in the intestine primarily acts to prevent adhesion and invasion of pathogens by peristalsis and mucus lining the mucosal epithelium of the gastrointestinal tract. A superficial layer of secreted non-specific, e.g. mucins and defensins, and specific secretory components i.e. secretory IgA (sIgA), also protect
Background
16
against pathogen adhesion and invasion. Acidity and proteolytic enzymes break down ingested proteins to peptides, thus destroying immunogenic epitopes, i.e. the immunologically reactive region of a complex antigen. However, if antigens reach contact with the epithelium, they are met by the gut-associated lymphoid tissue (GALT). In humans the GALT accounts for two thirds of the immune system of the body which reflects the enormous immunological challenge conferred by the intestinal luminal contents. Lymphocytes are found all along the intestine, both in organized tissue, i.e. in the solitary follicles in the mucosa, more numerous in the colon, appendix and in the aggregates of lymphoid follicles in the small intestine called Peyer’s patches (PP). Lymphocytes are also distributed within the epithelium (IEL) and in underlying connective tissue (LPL). The IEL are mainly T lymphocytes (T cells) whereas LPL are both T and B cells. There are numerous antibody-producing plasma cells in the intestine. Antigen can enter the follicles through specialized epithelial cells, the microfold cells (M-cells) and interact with antigen-presenting cells (APC), B and T cells. Dendritic cells (DC) are professional APC located in the PP and the lamina propria. Professional APC are specialized for uptake, processing and presenting of antigen to T cells. It has been suggested that DC located in the lamina propria may sample dietary antigens and present them to T cells. Intestinal IEL and T cell LPL are activated under normal physiological conditions and produce both down-regulatory and anti-inflammatory cytokines, leading to a state that can be referred to as controlled inflammation. Cytotoxic T cells (CTL) are also present in the small intestine, thus able to eliminate invading pathogens by means of cytotoxicity (27). When lymphocytes encounter antigen in the lymphoid tissue, they become activated. Then they leave the lymphoid organ as effector cells and enter the blood stream and in time migrate to the site where the initial antigen was encountered. Afferent lymphatics drain the villus lamina propria and PP into the mesenteric lymph nodes (MLN). The adhesion molecule L-selectin need to be expressed on the lymphocyte in order to enter peripheral tissues whereas α4β7 integrin need to be expressed on the lymphocyte in order to enter mucosal tissues, respectively, (25, 28-30). However, the entry of lymphocytes into the MLN requires both adhesion molecules to be expressed (31). Thus, MLN are a meeting point for peripheral and mucosal recirculation pathways.
Oral tolerance The feeding of soluble dietary proteins typically directs the immune response to a state of specific and active unresponsiveness, termed oral tolerance. Data on oral tolerance induction come mainly from rodent models, and the mechanisms behind oral tolerance remain undecided. There appears to be a very complex interaction of genetics, age, dose and timing of postnatal feeding, as well as antigenic structure and composition of the food protein, mucosal barrier mechanisms and local immune activation. It is an antigen-driven process and both microbes in the gut and food antigens are driving forces. Although data are lacking, it is believed that oral tolerance also operates in humans. Schematically, hyporesponsiveness to harmless antigens entering the GALT via the M-cells or through the intestinal surface epithelium may be mediated by 1) T cell anergy, which means that when the T cell is presented to antigens without co-stimulatory signals, the T cell becomes refractory to
Background
16
against pathogen adhesion and invasion. Acidity and proteolytic enzymes break down ingested proteins to peptides, thus destroying immunogenic epitopes, i.e. the immunologically reactive region of a complex antigen. However, if antigens reach contact with the epithelium, they are met by the gut-associated lymphoid tissue (GALT). In humans the GALT accounts for two thirds of the immune system of the body which reflects the enormous immunological challenge conferred by the intestinal luminal contents. Lymphocytes are found all along the intestine, both in organized tissue, i.e. in the solitary follicles in the mucosa, more numerous in the colon, appendix and in the aggregates of lymphoid follicles in the small intestine called Peyer’s patches (PP). Lymphocytes are also distributed within the epithelium (IEL) and in underlying connective tissue (LPL). The IEL are mainly T lymphocytes (T cells) whereas LPL are both T and B cells. There are numerous antibody-producing plasma cells in the intestine. Antigen can enter the follicles through specialized epithelial cells, the microfold cells (M-cells) and interact with antigen-presenting cells (APC), B and T cells. Dendritic cells (DC) are professional APC located in the PP and the lamina propria. Professional APC are specialized for uptake, processing and presenting of antigen to T cells. It has been suggested that DC located in the lamina propria may sample dietary antigens and present them to T cells. Intestinal IEL and T cell LPL are activated under normal physiological conditions and produce both down-regulatory and anti-inflammatory cytokines, leading to a state that can be referred to as controlled inflammation. Cytotoxic T cells (CTL) are also present in the small intestine, thus able to eliminate invading pathogens by means of cytotoxicity (27). When lymphocytes encounter antigen in the lymphoid tissue, they become activated. Then they leave the lymphoid organ as effector cells and enter the blood stream and in time migrate to the site where the initial antigen was encountered. Afferent lymphatics drain the villus lamina propria and PP into the mesenteric lymph nodes (MLN). The adhesion molecule L-selectin need to be expressed on the lymphocyte in order to enter peripheral tissues whereas α4β7 integrin need to be expressed on the lymphocyte in order to enter mucosal tissues, respectively, (25, 28-30). However, the entry of lymphocytes into the MLN requires both adhesion molecules to be expressed (31). Thus, MLN are a meeting point for peripheral and mucosal recirculation pathways.
Oral tolerance The feeding of soluble dietary proteins typically directs the immune response to a state of specific and active unresponsiveness, termed oral tolerance. Data on oral tolerance induction come mainly from rodent models, and the mechanisms behind oral tolerance remain undecided. There appears to be a very complex interaction of genetics, age, dose and timing of postnatal feeding, as well as antigenic structure and composition of the food protein, mucosal barrier mechanisms and local immune activation. It is an antigen-driven process and both microbes in the gut and food antigens are driving forces. Although data are lacking, it is believed that oral tolerance also operates in humans. Schematically, hyporesponsiveness to harmless antigens entering the GALT via the M-cells or through the intestinal surface epithelium may be mediated by 1) T cell anergy, which means that when the T cell is presented to antigens without co-stimulatory signals, the T cell becomes refractory to
Background
17
further stimulation by antigen 2) clonal deletion of antigen-specific T cells by apoptosis and 3) cytokine-mediated active suppression mediated via interleukin 10 (IL10) and transforming growth factor-β (TGF-β) produced by regulatory T cells. It has been considered that a high dose of antigen induces anergy and clonal deletion while multiple doses of low dose feeds are likely to induce cytokine-mediated active suppression. However, this dichotomy is challenged as it has been proposed that anergy and active regulation are not separate aspects of T cell function, (28-30, 32).
Influences of immunity on gut colonization As mentioned, microbial stimulation during infancy is suggested to be a prerequisite for the development of the mucosal immune system in the gut. This is based on work in animal models. It was demonstrated that stimulation with intestinal bacteria at the neonatal stage was necessary for induction of oral tolerance in germfree mice. However, if intestinal microbes were installed at an older age, this could not restore oral tolerance induction (20). In another mouse model, a complex intestinal microbiota induced oral tolerance, whereas monocolonization did not (22). For a variable period after birth, the intestinal barrier function, provided partly by secretory antibodies and immunoregulatory functions, is not fully developed. Starting immediately after birth, mucosal surfaces are colonized by a large variety of microorganisms and exposed to various protein antigens, the latter more pronounced in formula-fed than in breastfed infants. During weaning, the dietary antigenic exposure increases and the gut microbiota becomes more diverse, challenging the developing immune system in the gut. In a mouse model an increasing diversity of the gut microbiota was observed during early weaning, probably most likely due to a decline in maternal secretory immunoglobulin A (sIgA) supply by the milk. Maternal sIgA antibodies present in the milk are directed against the microorganisms in the mother’s environment and provide protection against these microorganisms also to the offspring. The main function of sIgA is to prevent bacteria and viruses already present on the surfaces of mucosal membranes from attaching to epithelial cells and enter the tissue. For several days, the levels of sIgA remained very low until endogenous secretion of sIgA had developed. The recovery of sIgA by the pups was then followed by a change in the composition of the gut microbiota, suggesting that the developed gut immune system might regulate the process of gut microbial diversification (33). It also suggests that during the period when maternal sIgA supply decreases and endogenous sIgA production is not fully developed, there is a period of increased vulnerability of intestinal mucosal surfaces to antigens from food and intestinal microbes. Data on this process in humans are scarce. However, it was demonstrated in a randomized clinical trial evaluating the effects of supplementation of Lactobacillus acidophilus LAVRI-A1 (LAVRI-A1) on sIgA levels in infants, that the strongest environmental predictor of the induction of sIgA was the introduction of complementary foods before the age of 6 months. In that study, fecal colonization by bifidobacteria was associated with increased levels of TGF-β (34). Maturation of the mucosal immune system in the gut and the induction of oral tolerance are influenced by gut microbial composition, nature and timing of exposure to food antigens, host factors and breastfeeding.
Background
17
further stimulation by antigen 2) clonal deletion of antigen-specific T cells by apoptosis and 3) cytokine-mediated active suppression mediated via interleukin 10 (IL10) and transforming growth factor-β (TGF-β) produced by regulatory T cells. It has been considered that a high dose of antigen induces anergy and clonal deletion while multiple doses of low dose feeds are likely to induce cytokine-mediated active suppression. However, this dichotomy is challenged as it has been proposed that anergy and active regulation are not separate aspects of T cell function, (28-30, 32).
Influences of immunity on gut colonization As mentioned, microbial stimulation during infancy is suggested to be a prerequisite for the development of the mucosal immune system in the gut. This is based on work in animal models. It was demonstrated that stimulation with intestinal bacteria at the neonatal stage was necessary for induction of oral tolerance in germfree mice. However, if intestinal microbes were installed at an older age, this could not restore oral tolerance induction (20). In another mouse model, a complex intestinal microbiota induced oral tolerance, whereas monocolonization did not (22). For a variable period after birth, the intestinal barrier function, provided partly by secretory antibodies and immunoregulatory functions, is not fully developed. Starting immediately after birth, mucosal surfaces are colonized by a large variety of microorganisms and exposed to various protein antigens, the latter more pronounced in formula-fed than in breastfed infants. During weaning, the dietary antigenic exposure increases and the gut microbiota becomes more diverse, challenging the developing immune system in the gut. In a mouse model an increasing diversity of the gut microbiota was observed during early weaning, probably most likely due to a decline in maternal secretory immunoglobulin A (sIgA) supply by the milk. Maternal sIgA antibodies present in the milk are directed against the microorganisms in the mother’s environment and provide protection against these microorganisms also to the offspring. The main function of sIgA is to prevent bacteria and viruses already present on the surfaces of mucosal membranes from attaching to epithelial cells and enter the tissue. For several days, the levels of sIgA remained very low until endogenous secretion of sIgA had developed. The recovery of sIgA by the pups was then followed by a change in the composition of the gut microbiota, suggesting that the developed gut immune system might regulate the process of gut microbial diversification (33). It also suggests that during the period when maternal sIgA supply decreases and endogenous sIgA production is not fully developed, there is a period of increased vulnerability of intestinal mucosal surfaces to antigens from food and intestinal microbes. Data on this process in humans are scarce. However, it was demonstrated in a randomized clinical trial evaluating the effects of supplementation of Lactobacillus acidophilus LAVRI-A1 (LAVRI-A1) on sIgA levels in infants, that the strongest environmental predictor of the induction of sIgA was the introduction of complementary foods before the age of 6 months. In that study, fecal colonization by bifidobacteria was associated with increased levels of TGF-β (34). Maturation of the mucosal immune system in the gut and the induction of oral tolerance are influenced by gut microbial composition, nature and timing of exposure to food antigens, host factors and breastfeeding.
Background
18
Allergic disease The prevalence of allergy and asthma has increased over the past 50 years and approximately 20% of the world population suffers from IgE-mediated allergic diseases. In Sweden, approximately one third of preschool children have eczema, asthma and/or allergic rhinoconjunctivits (35). Although there seems to be a strong hereditary component, the genetic factors and other disease mechanisms remain elusive. Individuals with a family history of atopy have an increased risk of developing IgE sensitization and develop typical symptoms of eczema, allergic asthma, allergic rhinitis and/or allergic conjunctivitis. However, this is a polygenetic disease and as of yet there are no reliable genetic and immunologic markers to identify an infant at risk which complicates primary prevention of IgE sensitization. Nonetheless, a positive family history is the most reliable predictor of allergy in infants (36).
Nomenclature The terminology in allergy and allergic-like reactions has been ambiguous. The nomenclature was revised by a Task Force of the European Academy of Allergology and Clinical Immunology (EAACI) in 2001, and then further revised by the Nomenclature Review Committee of the World Allergy Organization (WAO) in 2004 (37, 38). Thus, hypersensitivity is defined as “objectively reproducible symptoms or signs initiated by exposure to a defined stimulus at a dose tolerated by normal persons”. Allergy is defined as “a hypersensitivity reaction initiated by specific immunologic mechanisms”. Allergy can be either antibody-mediated or cell-mediated. Allergy is then further divided into an IgE-mediated or a non-IgE-mediated type. In the majority of patients with allergic symptoms from the mucosal membranes in the gastrointestinal tract and in the airways, the responsible antibody belongs to the IgE iso-type. In non-IgE-mediated allergy the mediators can be allergen-specific lymphocytes or IgG antibodies. Atopy is “a personal and/or familial tendency, usually in childhood or adolescence, to become sensitized and produce IgE-antibodies in response to ordinary exposures to allergens, usually proteins. As a consequence, these persons can develop typical symptoms of asthma, rhino-conjunctivitis, or eczema”. The term atopy should not be used until there is a documented IgE sensitization, i.e. by a confirmed rise in specific IgE antibodies or a positive skin prick test (SPT). Furthermore, an allergen is defined as “an antigen causing allergic disease”. The overall term for a local inflammation in the skin is dermatitis. Dermatitis is then divided into eczema, contact dermatitis and other forms of dermatitis. The term eczema replaces the former terms atopic dermatitis and atopic eczema/dermatitis syndrome. Eczema can be further defined as atopic or non-atopic, the former term referring to patients with an atopic constitution (Fig. 3). Another way to make a distinction between types of eczema is to use the terms IgE-associated and non-IgE-associated eczema. Below, IgE-associated eczema is used when sensitization is confirmed.
Background
18
Allergic disease The prevalence of allergy and asthma has increased over the past 50 years and approximately 20% of the world population suffers from IgE-mediated allergic diseases. In Sweden, approximately one third of preschool children have eczema, asthma and/or allergic rhinoconjunctivits (35). Although there seems to be a strong hereditary component, the genetic factors and other disease mechanisms remain elusive. Individuals with a family history of atopy have an increased risk of developing IgE sensitization and develop typical symptoms of eczema, allergic asthma, allergic rhinitis and/or allergic conjunctivitis. However, this is a polygenetic disease and as of yet there are no reliable genetic and immunologic markers to identify an infant at risk which complicates primary prevention of IgE sensitization. Nonetheless, a positive family history is the most reliable predictor of allergy in infants (36).
Nomenclature The terminology in allergy and allergic-like reactions has been ambiguous. The nomenclature was revised by a Task Force of the European Academy of Allergology and Clinical Immunology (EAACI) in 2001, and then further revised by the Nomenclature Review Committee of the World Allergy Organization (WAO) in 2004 (37, 38). Thus, hypersensitivity is defined as “objectively reproducible symptoms or signs initiated by exposure to a defined stimulus at a dose tolerated by normal persons”. Allergy is defined as “a hypersensitivity reaction initiated by specific immunologic mechanisms”. Allergy can be either antibody-mediated or cell-mediated. Allergy is then further divided into an IgE-mediated or a non-IgE-mediated type. In the majority of patients with allergic symptoms from the mucosal membranes in the gastrointestinal tract and in the airways, the responsible antibody belongs to the IgE iso-type. In non-IgE-mediated allergy the mediators can be allergen-specific lymphocytes or IgG antibodies. Atopy is “a personal and/or familial tendency, usually in childhood or adolescence, to become sensitized and produce IgE-antibodies in response to ordinary exposures to allergens, usually proteins. As a consequence, these persons can develop typical symptoms of asthma, rhino-conjunctivitis, or eczema”. The term atopy should not be used until there is a documented IgE sensitization, i.e. by a confirmed rise in specific IgE antibodies or a positive skin prick test (SPT). Furthermore, an allergen is defined as “an antigen causing allergic disease”. The overall term for a local inflammation in the skin is dermatitis. Dermatitis is then divided into eczema, contact dermatitis and other forms of dermatitis. The term eczema replaces the former terms atopic dermatitis and atopic eczema/dermatitis syndrome. Eczema can be further defined as atopic or non-atopic, the former term referring to patients with an atopic constitution (Fig. 3). Another way to make a distinction between types of eczema is to use the terms IgE-associated and non-IgE-associated eczema. Below, IgE-associated eczema is used when sensitization is confirmed.
Background
19
Figure 3. Nomenclature of skin symptoms according to WAO (38). Asthma is divided into allergic and non-allergic asthma. Allergic asthma is predominantly initiated by IgE antibodies, but it is recognized that there may be other immunological mechanisms initiating the inflammation in allergic asthma. The mechanisms initiating the inflammation in non-allergic asthma are not well defined.
Th1/Th2 concept, regulatory T cells and T cell activation Lymphocytes are the central cellular elements of adaptive immunity. Lymphocytes are divided into B and T lymphocytes (B and T cells). Both B and T cells have antigen receptors on their surface, so called B and T cell recptors, (BCR and TCR, respectively). B cells are fundamental for humoral immunity whereas T cells are responsible for cell-mediated immunity and immune regulation. Humoral immunity is mediated by antibodies that bind to epitopes. In cell-mediated immunity, cytotoxic T cells and cytokine-producing T cells are the effectors. Cytokines are secreted bioactive polypeptides that adjust the activity of the cell by which they are produced or another cell. Thus, cytokines produced by specific cell types upon activation can influence and regulate the resulting immune response. T cells are further divided in two major populations; CD4+ T helper (Th) and CD8+ cytotoxic T cells (CTL). Th cells are called T helper cells since they activate other immune cells e.g. B cells, CTL and macrophages, and they are essential for antibody production to proteins and glycoproteins. Differences in the patterns of cytokines secreted by the activated Th cells lead to different type of immune responses. The naïve Th cell (Th0 cell) produces interleukin 2 (IL2), IL4 and interferon-γ (IFN-γ). Depending on the complex interaction of the APC that presents antigen to the naïve Th cell, it can polarize into different directions depending on genetics, type and load of antigen, presence or absence of specific co-stimulatory molecules and other environmental factors e.g. the dominant cytokine environment (Fig. 4). Two types of polarized immune responses driven by the Th cells can be outlined and referred to as Th1 and Th2 responses, respectively (Fig. 4). These two types of
Dermatitis
Eczema Contact dermatitis Other dermatitis
Atopic eczema Non-atopic eczema Allergic Non-allergic
Background
19
Figure 3. Nomenclature of skin symptoms according to WAO (38). Asthma is divided into allergic and non-allergic asthma. Allergic asthma is predominantly initiated by IgE antibodies, but it is recognized that there may be other immunological mechanisms initiating the inflammation in allergic asthma. The mechanisms initiating the inflammation in non-allergic asthma are not well defined.
Th1/Th2 concept, regulatory T cells and T cell activation Lymphocytes are the central cellular elements of adaptive immunity. Lymphocytes are divided into B and T lymphocytes (B and T cells). Both B and T cells have antigen receptors on their surface, so called B and T cell recptors, (BCR and TCR, respectively). B cells are fundamental for humoral immunity whereas T cells are responsible for cell-mediated immunity and immune regulation. Humoral immunity is mediated by antibodies that bind to epitopes. In cell-mediated immunity, cytotoxic T cells and cytokine-producing T cells are the effectors. Cytokines are secreted bioactive polypeptides that adjust the activity of the cell by which they are produced or another cell. Thus, cytokines produced by specific cell types upon activation can influence and regulate the resulting immune response. T cells are further divided in two major populations; CD4+ T helper (Th) and CD8+ cytotoxic T cells (CTL). Th cells are called T helper cells since they activate other immune cells e.g. B cells, CTL and macrophages, and they are essential for antibody production to proteins and glycoproteins. Differences in the patterns of cytokines secreted by the activated Th cells lead to different type of immune responses. The naïve Th cell (Th0 cell) produces interleukin 2 (IL2), IL4 and interferon-γ (IFN-γ). Depending on the complex interaction of the APC that presents antigen to the naïve Th cell, it can polarize into different directions depending on genetics, type and load of antigen, presence or absence of specific co-stimulatory molecules and other environmental factors e.g. the dominant cytokine environment (Fig. 4). Two types of polarized immune responses driven by the Th cells can be outlined and referred to as Th1 and Th2 responses, respectively (Fig. 4). These two types of
Dermatitis
Eczema Contact dermatitis Other dermatitis
Atopic eczema Non-atopic eczema Allergic Non-allergic
Background
20
responses were first demonstrated in murine models, and later in humans, although the polarization in humans is not as clear-cut as in mice. Still, the Th1 and Th2 concept serves as a working model, albeit over-simplified. Early interleukin 4 (IL4) production supports Th2 polarization whereas IL12 and IFN-γ production in the absence of IL4 supports Th1 polarization. Cells of the innate immune system, e.g. dendritic cells (DC) and Natural Killer (NK) cells produce both the IL12 and IFN-γ that drive Th1 polarization. A Th1 response is characterized by increased levels of the pro-inflammatory cytokines interferon-gamma (IFN-γ), tumour necrosis factor beta (TNF-β) and IL2 without production of IL4, IL5, IL9 and IL13. The release of pro-inflammatory cytokines will have effects on the production of opsonising and complement-fixing antibodies by B cells, activation of macrophages and cell cytotoxicity. The Th1 cell helps the cytotoxic precursor T cell to develop into a CTL causing local inflammation. The Th1 response is typical for autoimmune diseases e.g. diabetes and celiac disease. The Th2 response, or humoral response, is characterized by production of IL4, IL5, IL9 and IL13, without the production of IFN-γ and TNF-β . A Th2 response activates B cells to develop into plasma cells, secrete immunoglobulins, favour eosinophil differentiation and activation but inhibit the function of phagocytic cells. This is the typical response in allergic reactions characterized by IgE reactivity. IL4 and IL13 induce B-cell switching to IgE-production while IL5 activates eosinophils (39-41). Until recently, the Th1 and Th2 subsets were considered to be the only CD4 effector responses. However, a third subset has been discovered and termed Th17 (Fig. 4). Th17 cells produce IL17, IL17F, IL22, IL6 and TNF-α and have been suggested to participate in both tissue inflammation and activation of neutrophils. The Th17 subset is distinct from and antagonized by cells from the Th1 and Th2 subsets (41, 42). T cells capable of suppressing the immune response, either by cell to cell contact and/or the production of down-regulatory cytokines, are referred to as regulatory T cells. Regulatory T cell types steer immune balance (Fig. 4). There are several families of regulatory T cells including CD4+ CD25+ T-regulatory (Treg) cells, inducing suppressive effects via cell contact and type 1 regulatory (Tr1) and Th3 cells exerting their effects via cytokines e.g. IL10 and TGF-β. Treg cells are thymus-derived and are characterized by their expression of CD25 (IL-2 receptor) and the transcription factor FoxP3. The production of Treg cells are induced by thymic expression of self-antigens and Treg cells are proposed to be of importance in the prevention of autoimmunity. The suppressor function of Treg cells appear to depend in part on TGF- β, but less on IL10. Th3 cells are gut derived and characterized by production of TGF-β (+ IL10), exerting their effects by mediating mucosal tolerance and antigen-specific IgA production. Tr1 cells are derived from the periphery and produce IL10 (+ TGF- β). However, their origin is unclear and as of yet it is not known whether they represent a distinctive developmental pathway or are derived from Th or Treg cells. There are data to suggest that production of IL10 by CD4+ cells may induce tolerant immune reponses (32, 43).
Background
20
responses were first demonstrated in murine models, and later in humans, although the polarization in humans is not as clear-cut as in mice. Still, the Th1 and Th2 concept serves as a working model, albeit over-simplified. Early interleukin 4 (IL4) production supports Th2 polarization whereas IL12 and IFN-γ production in the absence of IL4 supports Th1 polarization. Cells of the innate immune system, e.g. dendritic cells (DC) and Natural Killer (NK) cells produce both the IL12 and IFN-γ that drive Th1 polarization. A Th1 response is characterized by increased levels of the pro-inflammatory cytokines interferon-gamma (IFN-γ), tumour necrosis factor beta (TNF-β) and IL2 without production of IL4, IL5, IL9 and IL13. The release of pro-inflammatory cytokines will have effects on the production of opsonising and complement-fixing antibodies by B cells, activation of macrophages and cell cytotoxicity. The Th1 cell helps the cytotoxic precursor T cell to develop into a CTL causing local inflammation. The Th1 response is typical for autoimmune diseases e.g. diabetes and celiac disease. The Th2 response, or humoral response, is characterized by production of IL4, IL5, IL9 and IL13, without the production of IFN-γ and TNF-β . A Th2 response activates B cells to develop into plasma cells, secrete immunoglobulins, favour eosinophil differentiation and activation but inhibit the function of phagocytic cells. This is the typical response in allergic reactions characterized by IgE reactivity. IL4 and IL13 induce B-cell switching to IgE-production while IL5 activates eosinophils (39-41). Until recently, the Th1 and Th2 subsets were considered to be the only CD4 effector responses. However, a third subset has been discovered and termed Th17 (Fig. 4). Th17 cells produce IL17, IL17F, IL22, IL6 and TNF-α and have been suggested to participate in both tissue inflammation and activation of neutrophils. The Th17 subset is distinct from and antagonized by cells from the Th1 and Th2 subsets (41, 42). T cells capable of suppressing the immune response, either by cell to cell contact and/or the production of down-regulatory cytokines, are referred to as regulatory T cells. Regulatory T cell types steer immune balance (Fig. 4). There are several families of regulatory T cells including CD4+ CD25+ T-regulatory (Treg) cells, inducing suppressive effects via cell contact and type 1 regulatory (Tr1) and Th3 cells exerting their effects via cytokines e.g. IL10 and TGF-β. Treg cells are thymus-derived and are characterized by their expression of CD25 (IL-2 receptor) and the transcription factor FoxP3. The production of Treg cells are induced by thymic expression of self-antigens and Treg cells are proposed to be of importance in the prevention of autoimmunity. The suppressor function of Treg cells appear to depend in part on TGF- β, but less on IL10. Th3 cells are gut derived and characterized by production of TGF-β (+ IL10), exerting their effects by mediating mucosal tolerance and antigen-specific IgA production. Tr1 cells are derived from the periphery and produce IL10 (+ TGF- β). However, their origin is unclear and as of yet it is not known whether they represent a distinctive developmental pathway or are derived from Th or Treg cells. There are data to suggest that production of IL10 by CD4+ cells may induce tolerant immune reponses (32, 43).
Background
21
CD25+
CD4+ T cells
IL10TGF-β
Th1CD4+T cells IL2
IFN-γ
Th2CD4+ T cells
FoxP3+
CD4+ T cells
Treg
Th0CD4+ T cells
IL2
IFN-γ
IL4
Tr1, Th3
+
-
- -
-
Th17CD4+ T cells
IL17
-
IL4
IL5
Figure 4. The naïve Th0 cell can develop into Th1, Th2 or Th17 cells. Regulatory T cells (Treg, Tr1 and Th3 cells) are capable of suppressing the immune response. During pregnancy potentially dangerous T cell-mediated responses are down-regulated to protect the fetus. Recently, the prevailing notion of a strongly Th2-skewed type immune response during pregnancy has been challenged, and it has been suggested that a balanced Th1/Th2 immune response is necessary for a successful pregnancy (44). However, there are data to support deficient cell mediated immune defence mechanisms during the neonatal period leading to attenuated Th1-type immune responses. Several groups have demonstrated that neonatal IFN-γ responses are lower compared with adults (45, 46), and fetal IL13 levels were reported to be higher whereas IL4, IL10 and IFN-γ levels were lower compared with adult levels, supporting a Th-2 skew (47). Sensitization has been suggested to occur in utero, and house-dust mite allergen has been detected in amniotic fluid and umbilical cord blood (48, 49). The significance of these findings for future sensitization and allergy expression is uncertain (46). Recently it was suggested that allergen-specific IgE in cord blood results from transfer of maternal IgE to the fetus and not from fetal sensitization (50). It seems that the immune system shifts towards a balanced immune response during early childhood. In the allergic infant this does not happen, rather there is a further increase in Th2 type reactivity (46, 51).
Background
21
CD25+
CD4+ T cells
IL10TGF-β
Th1CD4+T cells IL2
IFN-γ
Th2CD4+ T cells
FoxP3+
CD4+ T cells
Treg
Th0CD4+ T cells
IL2
IFN-γ
IL4
Tr1, Th3
+
-
- -
-
Th17CD4+ T cells
IL17
-
IL4
IL5
Figure 4. The naïve Th0 cell can develop into Th1, Th2 or Th17 cells. Regulatory T cells (Treg, Tr1 and Th3 cells) are capable of suppressing the immune response. During pregnancy potentially dangerous T cell-mediated responses are down-regulated to protect the fetus. Recently, the prevailing notion of a strongly Th2-skewed type immune response during pregnancy has been challenged, and it has been suggested that a balanced Th1/Th2 immune response is necessary for a successful pregnancy (44). However, there are data to support deficient cell mediated immune defence mechanisms during the neonatal period leading to attenuated Th1-type immune responses. Several groups have demonstrated that neonatal IFN-γ responses are lower compared with adults (45, 46), and fetal IL13 levels were reported to be higher whereas IL4, IL10 and IFN-γ levels were lower compared with adult levels, supporting a Th-2 skew (47). Sensitization has been suggested to occur in utero, and house-dust mite allergen has been detected in amniotic fluid and umbilical cord blood (48, 49). The significance of these findings for future sensitization and allergy expression is uncertain (46). Recently it was suggested that allergen-specific IgE in cord blood results from transfer of maternal IgE to the fetus and not from fetal sensitization (50). It seems that the immune system shifts towards a balanced immune response during early childhood. In the allergic infant this does not happen, rather there is a further increase in Th2 type reactivity (46, 51).
Background
22
The central element in formation of both humoral and cell-mediated immune responses is the activation and clonal expansion of Th cells. Th cells recognize antigen only when it is combined with a major histocompability complex (MHC) class II molecule on professional APC, e.g. DC, B cells and macrophages. In short, the interaction of the TCR-CD3 complex with a processed antigenic peptide bound to a MHC class II molecule on the surface of an APC results in several biochemical events that induce the resting Th cell to proliferate and differentiate into a memory or effector cell. Co-stimulatory signals are required for full T cell activation. This signal is provided by interactions between CD28, a glycoprotein expressed on the T cell membrane, and a B7/CD80 molecule on the APC. In this thesis, T cells were activated polyclonally by anti-CD3 monoclonal antibody (mAb) plus anti-CD28 mAb and the subsequent cytokine response was assessed as a measure of T cell function. Differences in the patterns of cytokines secreted by the activated Th cells lead to different types of immune responses. We studied IL2 as a marker of general T cell activation, IL4 as a Th2 marker, IFN-γ as a Th1 marker and IL10 as a marker for regulatory T cell activity.
IgE-mediated hypersensitivity reaction The IgE-mediated hypersensitivity reaction is initiated by exposure to an allergen activating B cells to form IgE-secreting plasma cells. The secreted IgE molecules bind to IgE-specific Fc receptors, i.e. membrane glycoproteins with affinity for the Fc entity of the antibody molecule (the tail of the antibody), on the surface of tissue mast cells and blood basophils. The cells that become coated with IgE are sensitized. Subsequent exposure to the same allergen cross-links the membrane-bound IgE on sensitized cells which results in degranulation of the cells. The degranulation results in release of active mediators, e.g. histamine, cytokines and proteases that lead to smooth-muscle contraction, increased vascular permeability and vasodilation. Sensitization can be confirmed by assessing circulating specific IgE antibodies or by a skin prick test (SPT). When the allergen is introduced into the skin of a previously sensitized individual, IgE molecules on the surface of a mast cell cross-link and degranulation of the mast cell occurs. Pre-formed granulae containing histamine are released followed by progressive infiltration of the dermis by eosinophils and neutrophils resulting in a wheal and flare reaction of the skin that can be measured (52).
The atopic march The atopic march describes a pattern of clinical manifestations of allergy, beginning with eczema and food allergy, and progresses with respiratory allergies (Fig. 5), (53, 54), although this idea has been challenged (55). Food allergy most often occurs in the first 1 to 2 years of life with the process of sensitization, i.e. the immune system responds to specific food proteins with the development of allergen-specific IgE. Once sensitized, exposure to that food can cause adverse reactions. However, whereas food allergies in older children are mainly IgE-mediated, in young children non-IgE mediated mechanisms are recognized, although not fully understood. Most allergies to foods are outgrown in childhood. For instance, in industrialized countries allergy to cow’s milk protein (CMA) affects 2-3% of infants below 2 years of age
Background
22
The central element in formation of both humoral and cell-mediated immune responses is the activation and clonal expansion of Th cells. Th cells recognize antigen only when it is combined with a major histocompability complex (MHC) class II molecule on professional APC, e.g. DC, B cells and macrophages. In short, the interaction of the TCR-CD3 complex with a processed antigenic peptide bound to a MHC class II molecule on the surface of an APC results in several biochemical events that induce the resting Th cell to proliferate and differentiate into a memory or effector cell. Co-stimulatory signals are required for full T cell activation. This signal is provided by interactions between CD28, a glycoprotein expressed on the T cell membrane, and a B7/CD80 molecule on the APC. In this thesis, T cells were activated polyclonally by anti-CD3 monoclonal antibody (mAb) plus anti-CD28 mAb and the subsequent cytokine response was assessed as a measure of T cell function. Differences in the patterns of cytokines secreted by the activated Th cells lead to different types of immune responses. We studied IL2 as a marker of general T cell activation, IL4 as a Th2 marker, IFN-γ as a Th1 marker and IL10 as a marker for regulatory T cell activity.
IgE-mediated hypersensitivity reaction The IgE-mediated hypersensitivity reaction is initiated by exposure to an allergen activating B cells to form IgE-secreting plasma cells. The secreted IgE molecules bind to IgE-specific Fc receptors, i.e. membrane glycoproteins with affinity for the Fc entity of the antibody molecule (the tail of the antibody), on the surface of tissue mast cells and blood basophils. The cells that become coated with IgE are sensitized. Subsequent exposure to the same allergen cross-links the membrane-bound IgE on sensitized cells which results in degranulation of the cells. The degranulation results in release of active mediators, e.g. histamine, cytokines and proteases that lead to smooth-muscle contraction, increased vascular permeability and vasodilation. Sensitization can be confirmed by assessing circulating specific IgE antibodies or by a skin prick test (SPT). When the allergen is introduced into the skin of a previously sensitized individual, IgE molecules on the surface of a mast cell cross-link and degranulation of the mast cell occurs. Pre-formed granulae containing histamine are released followed by progressive infiltration of the dermis by eosinophils and neutrophils resulting in a wheal and flare reaction of the skin that can be measured (52).
The atopic march The atopic march describes a pattern of clinical manifestations of allergy, beginning with eczema and food allergy, and progresses with respiratory allergies (Fig. 5), (53, 54), although this idea has been challenged (55). Food allergy most often occurs in the first 1 to 2 years of life with the process of sensitization, i.e. the immune system responds to specific food proteins with the development of allergen-specific IgE. Once sensitized, exposure to that food can cause adverse reactions. However, whereas food allergies in older children are mainly IgE-mediated, in young children non-IgE mediated mechanisms are recognized, although not fully understood. Most allergies to foods are outgrown in childhood. For instance, in industrialized countries allergy to cow’s milk protein (CMA) affects 2-3% of infants below 2 years of age
Background
23
but is outgrown in about 50% of 1-year olds and in 90% of children before school age. The clinical symptoms of CMA are diverse, ranging from anaphylactic reactions to eczema, food-associated wheeze, infantile colic, gastro-eosophagal reflux, enterocolitis and constipation (56). The SPT and measurement of specific IgE antibodies are helpful in the diagnostic approach but the controlled oral food challenge is the gold standard (57). Eczema is common in young children. In a large prospective Swedish birth cohort study, the BAMSE study, the cumulative incidence of eczema from 0-4 years of age was 33%. Twenty-one % had non-IgE-mediated eczema, and 12% had IgE-mediated eczema. Twenty-seven % of the children without a positive family history (maternal or paternal) of allergies manifested eczema up to the age of four. The corresponding figures in children with a single or a double positive family history was 38% and 50%, respectively (35). Eczema is characterized by chronic erythematous skin lesions with a relapsing course, intense dryness of the skin and pruritus. In early infancy the eczematous lesions mainly affect the face (especially the cheeks and the chin), scalp, trunk and extensor surfaces of the extremities. The diaper area is usually spared. During childhood the eczema has a more chronic course with lichenifiation, papules and excoriations affecting the neck, wrists, ankles and flexural folds (54, 58). The skin barrier function is impaired in eczema, particularly in atopic eczema (59). In the BAMSE study, 9% of the boys and 4% of the girls had asthma at four years of age (60). The ISAAC phase I study noted worldwide variations in the prevalence of asthma symptoms with a 12-month prevalence of 8% in 6-7 year old Swedish children (61). In the OLIN-study, the prevalence of physician-diagnosed asthma in children in northern Sweden increased with age, from 6% in 7-8-year olds to 8% at age 11-12 years (62). In the ISAAC phase III study, the Swedish centre reported a 12-month prevalence of rhino-conjunctivitis of 7% in 13-14 year old children (63).
Figure 5. Schematic description of the atopic march. Adapted from JM Spergel et al (54) and printed with permission from the publisher.
Food allergy Eczema
Rhino- conjunctivitis
Asthma
Age
Background
23
but is outgrown in about 50% of 1-year olds and in 90% of children before school age. The clinical symptoms of CMA are diverse, ranging from anaphylactic reactions to eczema, food-associated wheeze, infantile colic, gastro-eosophagal reflux, enterocolitis and constipation (56). The SPT and measurement of specific IgE antibodies are helpful in the diagnostic approach but the controlled oral food challenge is the gold standard (57). Eczema is common in young children. In a large prospective Swedish birth cohort study, the BAMSE study, the cumulative incidence of eczema from 0-4 years of age was 33%. Twenty-one % had non-IgE-mediated eczema, and 12% had IgE-mediated eczema. Twenty-seven % of the children without a positive family history (maternal or paternal) of allergies manifested eczema up to the age of four. The corresponding figures in children with a single or a double positive family history was 38% and 50%, respectively (35). Eczema is characterized by chronic erythematous skin lesions with a relapsing course, intense dryness of the skin and pruritus. In early infancy the eczematous lesions mainly affect the face (especially the cheeks and the chin), scalp, trunk and extensor surfaces of the extremities. The diaper area is usually spared. During childhood the eczema has a more chronic course with lichenifiation, papules and excoriations affecting the neck, wrists, ankles and flexural folds (54, 58). The skin barrier function is impaired in eczema, particularly in atopic eczema (59). In the BAMSE study, 9% of the boys and 4% of the girls had asthma at four years of age (60). The ISAAC phase I study noted worldwide variations in the prevalence of asthma symptoms with a 12-month prevalence of 8% in 6-7 year old Swedish children (61). In the OLIN-study, the prevalence of physician-diagnosed asthma in children in northern Sweden increased with age, from 6% in 7-8-year olds to 8% at age 11-12 years (62). In the ISAAC phase III study, the Swedish centre reported a 12-month prevalence of rhino-conjunctivitis of 7% in 13-14 year old children (63).
Figure 5. Schematic description of the atopic march. Adapted from JM Spergel et al (54) and printed with permission from the publisher.
Food allergy Eczema
Rhino- conjunctivitis
Asthma
Age
Background
24
In the atopic child the atopic march progresses. A recent meta-analysis demonstrated that 1 in every 3 children with eczema develops asthma during later childhood, a figure that is lower than previously reported (64). However, early IgE sensitization and severe eczema are associated with an increased risk for the development of asthma (54).
Intestinal permeability in allergic individuals The rather high incidence of food allergies in infancy and early childhood might be explained by an incomplete mucosal barrier, increased gut permeability to large molecules and immature mucosal and systemic immune responses. Sensitization to food allergens can be regarded as a failure of normal tolerance induction or an abrogation of established tolerance, and is most common during infancy when mechanisms of tolerance are not fully developed. Increased gut permeability with increased absorption of macro-molecules have been reported in children and young adults with eczema and/or food allergy, (65) and in children with asthma (66). Thus, the entire mucosal immune system may be involved in allergic disease. However, whether this increased permeability is a primary inherited trait or reflects inflammation-driven gut mucosal barrier damage need further study (66).
Alterations of gut microbiota and disease
The hygiene hypothesis In 1989 Strachan demonstrated in an epidemiological study an inverse relationship between family size and the risk of allergic rhinitis. He hypothesized that in young children, infections transferred from older siblings might protect from allergic disease and that less microbial exposure in childhood might lie behind the increase in allergies in the Western world (67). Surprisingly, a strong association at the population level between the occurrence of diabetes type 1, a Th1-mediated disease, and asthma symptoms, as a proxy for a Th2-mediated disease, was demonstrated (68). Later, a modified version of the hygiene hypothesis proposed that general microbial encounter could stimulate the immune system towards a Th1 type immune response, and that the gut microbiota, which is established in early childhood, would have a major impact in driving these responses (23). Furthermore, it was proposed that microbial burden could increase the activity of regulatory T cells with down-regulating effects on both Th1- and Th2-mediated diseases (69). Thus, it was hypothesized that reduced microbial exposure early in life would lead to imprinting of aberrant immune response patterns.
Gut microbiota and allergy The decline in microbial exposure during early childhood is one of the most probable reasons for the increasing incidence of allergic disease in the Western world. As discussed, both epidemiological studies and gnotobiotic animal models have been supportive of such a hypothesis. In the late 1990s, Sepp et al demonstrated differences in gut microbial composition between Swedish and
Background
24
In the atopic child the atopic march progresses. A recent meta-analysis demonstrated that 1 in every 3 children with eczema develops asthma during later childhood, a figure that is lower than previously reported (64). However, early IgE sensitization and severe eczema are associated with an increased risk for the development of asthma (54).
Intestinal permeability in allergic individuals The rather high incidence of food allergies in infancy and early childhood might be explained by an incomplete mucosal barrier, increased gut permeability to large molecules and immature mucosal and systemic immune responses. Sensitization to food allergens can be regarded as a failure of normal tolerance induction or an abrogation of established tolerance, and is most common during infancy when mechanisms of tolerance are not fully developed. Increased gut permeability with increased absorption of macro-molecules have been reported in children and young adults with eczema and/or food allergy, (65) and in children with asthma (66). Thus, the entire mucosal immune system may be involved in allergic disease. However, whether this increased permeability is a primary inherited trait or reflects inflammation-driven gut mucosal barrier damage need further study (66).
Alterations of gut microbiota and disease
The hygiene hypothesis In 1989 Strachan demonstrated in an epidemiological study an inverse relationship between family size and the risk of allergic rhinitis. He hypothesized that in young children, infections transferred from older siblings might protect from allergic disease and that less microbial exposure in childhood might lie behind the increase in allergies in the Western world (67). Surprisingly, a strong association at the population level between the occurrence of diabetes type 1, a Th1-mediated disease, and asthma symptoms, as a proxy for a Th2-mediated disease, was demonstrated (68). Later, a modified version of the hygiene hypothesis proposed that general microbial encounter could stimulate the immune system towards a Th1 type immune response, and that the gut microbiota, which is established in early childhood, would have a major impact in driving these responses (23). Furthermore, it was proposed that microbial burden could increase the activity of regulatory T cells with down-regulating effects on both Th1- and Th2-mediated diseases (69). Thus, it was hypothesized that reduced microbial exposure early in life would lead to imprinting of aberrant immune response patterns.
Gut microbiota and allergy The decline in microbial exposure during early childhood is one of the most probable reasons for the increasing incidence of allergic disease in the Western world. As discussed, both epidemiological studies and gnotobiotic animal models have been supportive of such a hypothesis. In the late 1990s, Sepp et al demonstrated differences in gut microbial composition between Swedish and
Background
25
Estonian 1-year-old healthy children. Lactobacilli and eubacteria were more common in the gut microbiota of Estonian young children, whereas Swedish children had higher counts of clostridia, especially Clostridium difficile (C. difficile), compared with Estonian children (70). The microbiota of Estonian infants was in fact similar to that of Swedish infants in the 1960s. The same group then studied gut microbial composition in healthy and allergic infants in both these countries. They found that allergic infants were less often colonized by lactobacilli compared with non-allergic infants in both these countries. Furthermore, the counts of facultative aerobes were higher in allergic children, with higher counts of S. aureus in Swedish children and coliforms in Estonian children (71). In a following study, the same group prospectively followed gut microbial composition and allergy development in Swedish and Estonian infants. That study demonstrated lower colonization by bifidobacteria during the first year of life in infants who later developed allergy (72). Lower counts of bifidobacteria in young children with eczema compared with healthy controls were reported by another group (73). Still another group demonstrated a reduced ratio of bifidobacteria to clostridia in infants who later became sensitized (74). Whether or not certain bifidobacterial strains would be related to allergic outcomes was investigated in allergic and healthy breastfed infants. The frequency of colonization with Bifidobacterium bifidis (B. bifidis) was higher in the healthy infants whereas the allergic infants were more often colonized with B. adolescentis (75). Then again, two recent large prospective birth cohort studies using molecular biology techniques failed to demonstrate a protective effect on later allergy development by early colonization with bifidobacteria (2, 76). Other groups have studied the effects of caesarean delivery, which is associated with a delayed and altered establishment of gut microbiota compared with that of vaginal delivery (2, 6), on later manifestations of allergy. It has been observed that caesarean delivered infants are at increased risk of later development of asthma and allergic rhinitis (77, 78). Thus, there are indications of an association between altered gut microbial composition and allergy development, with differences in gut microbial composition even before disease symptoms occur. However, more studies are needed to clarify this issue.
Probiotics
Historical view and definition The concept of modulating gut microbial composition for therapeutic purposes is by no means a new idea. Modulation can be done by removing microorganisms by the use of antibiotics or by adding new organisms e.g. probiotics. In addition, providing different nutritional factors e.g. dietary fiber or resistant starches will also impact on the biota. The use of fermented milk goes back several hundred years to pre-biblical times. However, it was not until a century ago that the study of the health promoting effects of the consumption of soured milk was initiated by the pioneering work of The Nobel prize-winner Elie Metchnikoff. He proposed that soured milk could antagonize harmful bacteria in the lower gut and that regular ingestion of soured milk impacted upon the longevity of Bulgarians (79). At the same time period, Henri Tissier demonstrated that bifidobacteria were predominant in the gut microbiota of
Background
25
Estonian 1-year-old healthy children. Lactobacilli and eubacteria were more common in the gut microbiota of Estonian young children, whereas Swedish children had higher counts of clostridia, especially Clostridium difficile (C. difficile), compared with Estonian children (70). The microbiota of Estonian infants was in fact similar to that of Swedish infants in the 1960s. The same group then studied gut microbial composition in healthy and allergic infants in both these countries. They found that allergic infants were less often colonized by lactobacilli compared with non-allergic infants in both these countries. Furthermore, the counts of facultative aerobes were higher in allergic children, with higher counts of S. aureus in Swedish children and coliforms in Estonian children (71). In a following study, the same group prospectively followed gut microbial composition and allergy development in Swedish and Estonian infants. That study demonstrated lower colonization by bifidobacteria during the first year of life in infants who later developed allergy (72). Lower counts of bifidobacteria in young children with eczema compared with healthy controls were reported by another group (73). Still another group demonstrated a reduced ratio of bifidobacteria to clostridia in infants who later became sensitized (74). Whether or not certain bifidobacterial strains would be related to allergic outcomes was investigated in allergic and healthy breastfed infants. The frequency of colonization with Bifidobacterium bifidis (B. bifidis) was higher in the healthy infants whereas the allergic infants were more often colonized with B. adolescentis (75). Then again, two recent large prospective birth cohort studies using molecular biology techniques failed to demonstrate a protective effect on later allergy development by early colonization with bifidobacteria (2, 76). Other groups have studied the effects of caesarean delivery, which is associated with a delayed and altered establishment of gut microbiota compared with that of vaginal delivery (2, 6), on later manifestations of allergy. It has been observed that caesarean delivered infants are at increased risk of later development of asthma and allergic rhinitis (77, 78). Thus, there are indications of an association between altered gut microbial composition and allergy development, with differences in gut microbial composition even before disease symptoms occur. However, more studies are needed to clarify this issue.
Probiotics
Historical view and definition The concept of modulating gut microbial composition for therapeutic purposes is by no means a new idea. Modulation can be done by removing microorganisms by the use of antibiotics or by adding new organisms e.g. probiotics. In addition, providing different nutritional factors e.g. dietary fiber or resistant starches will also impact on the biota. The use of fermented milk goes back several hundred years to pre-biblical times. However, it was not until a century ago that the study of the health promoting effects of the consumption of soured milk was initiated by the pioneering work of The Nobel prize-winner Elie Metchnikoff. He proposed that soured milk could antagonize harmful bacteria in the lower gut and that regular ingestion of soured milk impacted upon the longevity of Bulgarians (79). At the same time period, Henri Tissier demonstrated that bifidobacteria were predominant in the gut microbiota of
Background
26
breastfed infants. He then proposed that administration of these bifid bacteria could restore gut microbial balance and resolve diarrheal disease. Thus, the concept of probiotics was born. The meaning of probiotics is “for life”. Fuller defined probiotics as “live microbial food supplements which beneficially affect the host animal by improving its microbial balance” (80). This definition was later revised by FAO/WHO as “live microorganisms which when ingested in adequate amounts confer a beneficial effect on the host” (81). The most commonly used species are lactobacilli and bifidobacteria, but other bacterial strains have been used as probiotics and also the yeast Saccharomyces boulardi. Prebiotics are “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health” (82). Such food ingredients are non-digestible oligosaccharides e.g. galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). Breastmilk is rich in oligosaccharides. Synbiotics refers to prebiotics in combination with probiotics (83).
Safety and guidelines Probiotics are a group of bacteria that are Generally Recognized As Safe (GRAS) (84). Bifidobacteria and lactobacilli are normal commensals of the mammalian microbiota and have been used in various types of foods for a long time. They rarely cause infections in humans. However, theoretically probiotics may cause systemic infections, deleterious metabolic activity, excessive immune stimulation and gene transfer (85). Epidemiological studies in Finland after the introduction of the probiotic L. rhamnosus GG (LGG) to the Finnish market, demonstrated no increase in lactobacilli-induced bacteremia after increased consumption of LGG (86). In a Swedish study the incidence of lactobacilli-induced bacteremia and the presence in blood cultures of three commercially available probiotic strains, including L. paracasei ssp. paracasei strain F19, were followed for five years. The incidence of bacteremia caused by lactobacilli constituted <1% of the total number of bacteremia cases with no increase during this five year-period. Lactobacilli-induced bacteremia was not caused by the three commercially available strains in any of the reported cases (87). Most of the rare cases of infection with lactobacilli occur in patients with severe underlying conditions. However, it is argued that the risk of infection of probiotic lactobacilli and bifidobacteria is similar to that of infection with commensal strains even in immuno-compromised individuals (85), whereas others suggest that probiotics should be used with caution in patients who are immuno-compromised, have cardiac valvular disease, short bowel, jejunostomy or a central venous catheter (88, 89). A joint FAO/WHO working group launched guidelines regarding evaluation of bacterial strains and defined data needed to be available to substantiate health claims as a probiotic strain (84).
Background
26
breastfed infants. He then proposed that administration of these bifid bacteria could restore gut microbial balance and resolve diarrheal disease. Thus, the concept of probiotics was born. The meaning of probiotics is “for life”. Fuller defined probiotics as “live microbial food supplements which beneficially affect the host animal by improving its microbial balance” (80). This definition was later revised by FAO/WHO as “live microorganisms which when ingested in adequate amounts confer a beneficial effect on the host” (81). The most commonly used species are lactobacilli and bifidobacteria, but other bacterial strains have been used as probiotics and also the yeast Saccharomyces boulardi. Prebiotics are “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health” (82). Such food ingredients are non-digestible oligosaccharides e.g. galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). Breastmilk is rich in oligosaccharides. Synbiotics refers to prebiotics in combination with probiotics (83).
Safety and guidelines Probiotics are a group of bacteria that are Generally Recognized As Safe (GRAS) (84). Bifidobacteria and lactobacilli are normal commensals of the mammalian microbiota and have been used in various types of foods for a long time. They rarely cause infections in humans. However, theoretically probiotics may cause systemic infections, deleterious metabolic activity, excessive immune stimulation and gene transfer (85). Epidemiological studies in Finland after the introduction of the probiotic L. rhamnosus GG (LGG) to the Finnish market, demonstrated no increase in lactobacilli-induced bacteremia after increased consumption of LGG (86). In a Swedish study the incidence of lactobacilli-induced bacteremia and the presence in blood cultures of three commercially available probiotic strains, including L. paracasei ssp. paracasei strain F19, were followed for five years. The incidence of bacteremia caused by lactobacilli constituted <1% of the total number of bacteremia cases with no increase during this five year-period. Lactobacilli-induced bacteremia was not caused by the three commercially available strains in any of the reported cases (87). Most of the rare cases of infection with lactobacilli occur in patients with severe underlying conditions. However, it is argued that the risk of infection of probiotic lactobacilli and bifidobacteria is similar to that of infection with commensal strains even in immuno-compromised individuals (85), whereas others suggest that probiotics should be used with caution in patients who are immuno-compromised, have cardiac valvular disease, short bowel, jejunostomy or a central venous catheter (88, 89). A joint FAO/WHO working group launched guidelines regarding evaluation of bacterial strains and defined data needed to be available to substantiate health claims as a probiotic strain (84).
Background
27
The guidelines are as follows; -Identification to the genus, species and strain level by phenotypic and genotypic methods -In vitro tests of resistance to gastric acidity and bile acids, adherence properties, antimicrobial activity, ability to reduce pathogen adhesion to surfaces and bile salt hydrolase activity -Determination of antibiotic resistance patterns -Assessment of certain metabolic activities e.g. D-lactate production and bile salt de-conjugation -Assessment of side-effects in human studies -Post-market surveillance of adverse effects in consumers
Proposed mechanisms of probiotics The common feature of probiotics is that they are non-pathogenic microorganisms. However, there is considerable diversity in demonstrated modes of action between single probiotic strains. Thus, there appear to be several mechanisms involved in mediating probiotic effects. Probiotics can have direct effects on the chyme, microbiota and effects related to changes in the microbial ecosystem. Also, they may exert effects on enterocytes and immunocompetent cells in the gut mucosa (90). Colonizing bacteria interact with the gastrointestinal mucosa and communicate with the underlying epithelial and mucosal lymphoid constituents, stimulating host defences in the gut. This communication has been termed bacterial-epithelial “cross-talk” (91). With the discovery of the Toll-like receptor (TLR) family, this communication is further understood. TLR are mainly located on APC and interact with molecular patterns on both pathogens and commensal bacteria, thus including probiotic bacteria. In humans, there are 11 known TLR that recognize molecular structures on micro-organisms, so called pathogen-associated molecular patterns (PAMP). TLR turn on the expression of antimicrobial genes and genes encoding inflammatory cytokines by several cell types, while activating DC, a professional APC, to initiate adaptive immune responses. DC have a unique capacity to activate resting naïve T cells and they act in-between innate and adaptive immunity to help shape a developing immune response. In mammals, The Toll pathway induces activation of the transcription factor Nuclear Factor кβ (NFкβ). Microbial substances e.g lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria can activate NFкβ in lymphocytes through this signalling pathway. NFкβ activates genes that contribute to adaptive immunity and secretion of pro-inflammatory cytokines. LPS signals via TLR4 in association with another receptor for LPS, called CD14. Another example is TLR2 which binds to lipoteic acid (LTA) in the cell wall of Gram-positive bacteria. TLR9 recognizes bacterial genomic DNA. NFкβ activate genes that contribute to adaptive immunity and secretion of pro-inflammatory cytokines (92).
Background
27
The guidelines are as follows; -Identification to the genus, species and strain level by phenotypic and genotypic methods -In vitro tests of resistance to gastric acidity and bile acids, adherence properties, antimicrobial activity, ability to reduce pathogen adhesion to surfaces and bile salt hydrolase activity -Determination of antibiotic resistance patterns -Assessment of certain metabolic activities e.g. D-lactate production and bile salt de-conjugation -Assessment of side-effects in human studies -Post-market surveillance of adverse effects in consumers
Proposed mechanisms of probiotics The common feature of probiotics is that they are non-pathogenic microorganisms. However, there is considerable diversity in demonstrated modes of action between single probiotic strains. Thus, there appear to be several mechanisms involved in mediating probiotic effects. Probiotics can have direct effects on the chyme, microbiota and effects related to changes in the microbial ecosystem. Also, they may exert effects on enterocytes and immunocompetent cells in the gut mucosa (90). Colonizing bacteria interact with the gastrointestinal mucosa and communicate with the underlying epithelial and mucosal lymphoid constituents, stimulating host defences in the gut. This communication has been termed bacterial-epithelial “cross-talk” (91). With the discovery of the Toll-like receptor (TLR) family, this communication is further understood. TLR are mainly located on APC and interact with molecular patterns on both pathogens and commensal bacteria, thus including probiotic bacteria. In humans, there are 11 known TLR that recognize molecular structures on micro-organisms, so called pathogen-associated molecular patterns (PAMP). TLR turn on the expression of antimicrobial genes and genes encoding inflammatory cytokines by several cell types, while activating DC, a professional APC, to initiate adaptive immune responses. DC have a unique capacity to activate resting naïve T cells and they act in-between innate and adaptive immunity to help shape a developing immune response. In mammals, The Toll pathway induces activation of the transcription factor Nuclear Factor кβ (NFкβ). Microbial substances e.g lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria can activate NFкβ in lymphocytes through this signalling pathway. NFкβ activates genes that contribute to adaptive immunity and secretion of pro-inflammatory cytokines. LPS signals via TLR4 in association with another receptor for LPS, called CD14. Another example is TLR2 which binds to lipoteic acid (LTA) in the cell wall of Gram-positive bacteria. TLR9 recognizes bacterial genomic DNA. NFкβ activate genes that contribute to adaptive immunity and secretion of pro-inflammatory cytokines (92).
Background
28
DC could be potential targets for probiotic bacteria. It has been suggested that immature DC in the lamina propria can extend the dendrite between the enterocytes into the gut lumen. Via TLR-signaling the DC becomes activated, leading to release of cytokines which steer the naïve Th cell to mature into balanced Th1, Th2 and T regulatory cell subsets (93) (Fig. 6).
Fig. 6. Probiotic bacteria may mediate their effects via TLR-signaling, leading to release of IL10 and IL12 that can steer the naïve Th cell to mature into balanced Th1, Th2 and T regulatory cell subsets.
Probiotics in the treatment of infectious disease Probiotics have been used in the treatment of acute infectious diarrhea based on the assumption that they operate against pathogens in the gut. Several clinical trials have evaluated the efficacy of probiotics in the treatment of infectious diarrhea. A meta-analysis of randomized controlled trials on probiotics in the treatment of acute diarrhea (defined as >3 loose or watery stools per 24 hours) in infants and children demonstrated that probiotics were effective, and especially in rotaviral gastroenteritis (94). A Cochrane review in 2004 identified 23 randomized controlled trials with a total of 1917 participants, evaluating probiotic treatment of infectious diarrhea. Probiotics reduced the risk of diarrhea at 3 days and the mean duration of diarrhea with about one day. The authors concluded that probiotics appear to be a useful addition to oral rehydration therapy in the treatment of acute infectious diarrhea in children and adults. However, there is still a lack of trials in developing countries where one would expect the effects to be greater (95).
Background
28
DC could be potential targets for probiotic bacteria. It has been suggested that immature DC in the lamina propria can extend the dendrite between the enterocytes into the gut lumen. Via TLR-signaling the DC becomes activated, leading to release of cytokines which steer the naïve Th cell to mature into balanced Th1, Th2 and T regulatory cell subsets (93) (Fig. 6).
Fig. 6. Probiotic bacteria may mediate their effects via TLR-signaling, leading to release of IL10 and IL12 that can steer the naïve Th cell to mature into balanced Th1, Th2 and T regulatory cell subsets.
Probiotics in the treatment of infectious disease Probiotics have been used in the treatment of acute infectious diarrhea based on the assumption that they operate against pathogens in the gut. Several clinical trials have evaluated the efficacy of probiotics in the treatment of infectious diarrhea. A meta-analysis of randomized controlled trials on probiotics in the treatment of acute diarrhea (defined as >3 loose or watery stools per 24 hours) in infants and children demonstrated that probiotics were effective, and especially in rotaviral gastroenteritis (94). A Cochrane review in 2004 identified 23 randomized controlled trials with a total of 1917 participants, evaluating probiotic treatment of infectious diarrhea. Probiotics reduced the risk of diarrhea at 3 days and the mean duration of diarrhea with about one day. The authors concluded that probiotics appear to be a useful addition to oral rehydration therapy in the treatment of acute infectious diarrhea in children and adults. However, there is still a lack of trials in developing countries where one would expect the effects to be greater (95).
Background
29
Probiotics in the treatment and prevention of allergic disease As discussed, both epidemiological and gnotobiotic animal models suggest that gut microbiota is a major stimulus of the maturation of the immune system. This has driven the idea of using probiotics, mainly bifidobacteria and lactobacilli, for treatment and prevention of allergic disease. Proposed mechanisms of probiotics are homeostasis of gut microbiota, stabilization of the intestinal barrier and control of inflammation (96). There are several studies that have evaluated the efficacy of probiotics in the treatment of eczema. The first study targeted infants with eczema and cow’s milk allergy (CMA). In that study, the Scoring Atopic Dermatitis (SCORAD) index was reduced by 50% when infants were fed a hydrolyzed whey-formula with LGG compared with the same formula without LGG (97). In another small study, addition of LGG or B. lactis led to faster recovery of the eczema compared with placebo (98). Then again, a study with a similar design to that of Isolauri et al (98), demonstrated no efficacy of LGG administration on eczema (99), and in two other studies there was no effect of LGG in infants with mild to moderate eczema compared with placebo (100, 101). However, in a larger study, LGG reduced the SCORAD index in infants with IgE-associated eczema whereas a MIX of four probiotic strains did not (102). In a study investigating the effects of probiotics on moderate to severe eczema, L. fermentum led to a significant reduction in SCORAD index compared with placebo (103). Moderate effects of probiotic administration have also been observed in a study in older children. This study had a crossover design and a combination of L. reuteri and L. rhamnosus improved the subjective symptoms in children with eczema but failed to reduce SCORAD index (104). Three randomized controlled trials have demonstrated preventive effects of perinatal administration of probiotics on eczema, including or confined to IgE-associated eczema (105-107), whereas 2 studies demonstrated no preventive effect (108, 109).
Lactobacillus F19
Isolation, colonization and safety Lactobacillus paracasei ssp. paracasei strain F19 (LF19) was originally isolated from the deep colonic mucus layer in patients without gastrointestinal disease. The strain was demonstrated to bind mucin and survived exposure to low pH and bile. Thus, the strain was expected to have a good possibility to survive transit through the gastrointestinal (GI) tract and establish in the GI mucin (110). The survival, ecology and safety of LF19 in human subjects were further investigated in a multicentre European research project, the PROBDEMO project. The probiotic was delivered in capsules and milk containing freeze-dried LF19 or in yoghurt. The doses ranged between 108-1010 CFU LF19. LF19 transiently colonized the colonic lumen and the mucosa (111). Monitoring of the fecal microbiota after the cessation of intake of LF19 demonstrated that some young children were colonized two weeks after the cessation, and two elderly subjects were still colonized by LF19 after 8 weeks. The trials also showed that strains indistinguishable from LF19 were identified by randomly amplified polymerase chain reaction (RAPD-PCR) in fecal samples of some subjects before administration of LF19. At the time of the trials,
Background
29
Probiotics in the treatment and prevention of allergic disease As discussed, both epidemiological and gnotobiotic animal models suggest that gut microbiota is a major stimulus of the maturation of the immune system. This has driven the idea of using probiotics, mainly bifidobacteria and lactobacilli, for treatment and prevention of allergic disease. Proposed mechanisms of probiotics are homeostasis of gut microbiota, stabilization of the intestinal barrier and control of inflammation (96). There are several studies that have evaluated the efficacy of probiotics in the treatment of eczema. The first study targeted infants with eczema and cow’s milk allergy (CMA). In that study, the Scoring Atopic Dermatitis (SCORAD) index was reduced by 50% when infants were fed a hydrolyzed whey-formula with LGG compared with the same formula without LGG (97). In another small study, addition of LGG or B. lactis led to faster recovery of the eczema compared with placebo (98). Then again, a study with a similar design to that of Isolauri et al (98), demonstrated no efficacy of LGG administration on eczema (99), and in two other studies there was no effect of LGG in infants with mild to moderate eczema compared with placebo (100, 101). However, in a larger study, LGG reduced the SCORAD index in infants with IgE-associated eczema whereas a MIX of four probiotic strains did not (102). In a study investigating the effects of probiotics on moderate to severe eczema, L. fermentum led to a significant reduction in SCORAD index compared with placebo (103). Moderate effects of probiotic administration have also been observed in a study in older children. This study had a crossover design and a combination of L. reuteri and L. rhamnosus improved the subjective symptoms in children with eczema but failed to reduce SCORAD index (104). Three randomized controlled trials have demonstrated preventive effects of perinatal administration of probiotics on eczema, including or confined to IgE-associated eczema (105-107), whereas 2 studies demonstrated no preventive effect (108, 109).
Lactobacillus F19
Isolation, colonization and safety Lactobacillus paracasei ssp. paracasei strain F19 (LF19) was originally isolated from the deep colonic mucus layer in patients without gastrointestinal disease. The strain was demonstrated to bind mucin and survived exposure to low pH and bile. Thus, the strain was expected to have a good possibility to survive transit through the gastrointestinal (GI) tract and establish in the GI mucin (110). The survival, ecology and safety of LF19 in human subjects were further investigated in a multicentre European research project, the PROBDEMO project. The probiotic was delivered in capsules and milk containing freeze-dried LF19 or in yoghurt. The doses ranged between 108-1010 CFU LF19. LF19 transiently colonized the colonic lumen and the mucosa (111). Monitoring of the fecal microbiota after the cessation of intake of LF19 demonstrated that some young children were colonized two weeks after the cessation, and two elderly subjects were still colonized by LF19 after 8 weeks. The trials also showed that strains indistinguishable from LF19 were identified by randomly amplified polymerase chain reaction (RAPD-PCR) in fecal samples of some subjects before administration of LF19. At the time of the trials,
Background
30
LF19 was not commercially produced and therefore the subjects could not have been exposed to this strain in foods (112, 113). In summary, these human feeding trials demonstrated that LF19 was well tolerated by young children, adults and elderly with no observed side effects. Furthermore, the trials demonstrated that LF19 or very closely related strains, is part of the indigenous microbiota in some persons in the Nordic countries (111). The antibiotic resistance profile of LF19 resembles that of the L. casei group, including Vancomycin resistance. Lactobacilli are intrinsically resistant to Vancomycin, and this trait is considered non-transmissible (89). There is no known plasmide-derived antibiotic resistance by LF19 (114). LF19 produces L-lactic acid, but no D-lactic acid and does not deconjugate bile salts (R Fondén, personal communication 2003).
Immunological effects in vitro and in animal models Stimulation of human peripheral blood mononuclear cells (PBMC) with different strains of live lactobacilli (including LF19) induced the production of TNF-α, IL6 and IL10 in vitro (115). Incubation of a suspension with LF19 on a monocyte cell line led to transcription of NFκB and caused production of IL1β, IL8 and IL10 (110). In a mouse model studying global gene expression in the distal ileum after ingestion of LF19, several components of B cell receptor signaling were upregulated in monocolonized mice (116).
Thus, LF19 fulfils the criteria of a probiotic, and was demonstrated to induce immune-stimulating effects in vitro.
Background
30
LF19 was not commercially produced and therefore the subjects could not have been exposed to this strain in foods (112, 113). In summary, these human feeding trials demonstrated that LF19 was well tolerated by young children, adults and elderly with no observed side effects. Furthermore, the trials demonstrated that LF19 or very closely related strains, is part of the indigenous microbiota in some persons in the Nordic countries (111). The antibiotic resistance profile of LF19 resembles that of the L. casei group, including Vancomycin resistance. Lactobacilli are intrinsically resistant to Vancomycin, and this trait is considered non-transmissible (89). There is no known plasmide-derived antibiotic resistance by LF19 (114). LF19 produces L-lactic acid, but no D-lactic acid and does not deconjugate bile salts (R Fondén, personal communication 2003).
Immunological effects in vitro and in animal models Stimulation of human peripheral blood mononuclear cells (PBMC) with different strains of live lactobacilli (including LF19) induced the production of TNF-α, IL6 and IL10 in vitro (115). Incubation of a suspension with LF19 on a monocyte cell line led to transcription of NFκB and caused production of IL1β, IL8 and IL10 (110). In a mouse model studying global gene expression in the distal ileum after ingestion of LF19, several components of B cell receptor signaling were upregulated in monocolonized mice (116).
Thus, LF19 fulfils the criteria of a probiotic, and was demonstrated to induce immune-stimulating effects in vitro.
Objectives
31
OBJECTIVES The general objective of this study was to investigate if feeding the probiotic LF19 during weaning could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, adaptive immunity, Th1/Th2 immune balance and allergy development. The specific objectives were to evaluate the following:
• The effects of feeding LF19 on fecal levels of lactobacilli and the pattern of SCFA as a proxy for gut microbial function
• The effects of feeding LF19 on infections and specific IgG antibody
responses to diphteria, tetanus and Haemophilus influenzae type b conjugate vaccines
• Age-dependent maturation of T cell function and the effects thereon of
feeding LF19
• The effects of feeding LF19 on the cumulative incidence of eczema at 13 months of age, immune balance and allergen-specific IgE levels
Objectives
31
OBJECTIVES The general objective of this study was to investigate if feeding the probiotic LF19 during weaning could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, adaptive immunity, Th1/Th2 immune balance and allergy development. The specific objectives were to evaluate the following:
• The effects of feeding LF19 on fecal levels of lactobacilli and the pattern of SCFA as a proxy for gut microbial function
• The effects of feeding LF19 on infections and specific IgG antibody
responses to diphteria, tetanus and Haemophilus influenzae type b conjugate vaccines
• Age-dependent maturation of T cell function and the effects thereon of
feeding LF19
• The effects of feeding LF19 on the cumulative incidence of eczema at 13 months of age, immune balance and allergen-specific IgE levels
Subjects and methods
32
SUBJECTS AND METHODS
Study design
3 4 5 6 7 8 9 10 11 12 13
LF19 feeding
Diary -----------------------------------------------------------------
Vaccination 1st 2nd 3rd
Blood sample 4th3rd2nd1st
Enrolment into study
Fecal sample ↑1st ↑2nd ↑3rd ↑4th
Age (mo)
Questionnaire x x x x x x x x x x
Figure 7. Flow chart for enrolment, vaccinations, feeding of the cereals, morbidity registration by diaries and questionnaires, blood and fecal samplings. The methods used are described in the respective Subjects and Methods section of each paper.
Subjects and methods
32
SUBJECTS AND METHODS
Study design
3 4 5 6 7 8 9 10 11 12 13
LF19 feeding
Diary -----------------------------------------------------------------
Vaccination 1st 2nd 3rd
Blood sample 4th3rd2nd1st
Enrolment into study
Fecal sample ↑1st ↑2nd ↑3rd ↑4th
Age (mo)
Questionnaire x x x x x x x x x x
Figure 7. Flow chart for enrolment, vaccinations, feeding of the cereals, morbidity registration by diaries and questionnaires, blood and fecal samplings. The methods used are described in the respective Subjects and Methods section of each paper.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
33
RESULTS
Characteristics of the participants At 4 months of age, we enrolled 180 infants. After excluding one infant due to exclusion criteria, (delivered by caesarean section), 89 infants were randomized to receive cereals with LF19 and 90 to receive cereals without the addition of LF19 (placebo). The drop-out rate was minimal. 94% (n=84) and 97% (n=90) of the infants completed the study in the probiotic- and placebo groups, respectively. The 8 dropouts were the result of withdrawal of consent by the parents. Thus, 171 infants completed the study. There were no statistically significant differences between the groups with respect to gender, gestational age, birth weight, older siblings, day care attendance, exposure to furred pets or smoking at home. There were no differences between the groups in atopic heredity; 66 and 61% of the infants in the probiotic- and placebo groups had at least one first grade relative with allergy and were classified as being at high-risk for allergy, respectively, (p=0.5). There were no differences between the groups with respect to the number and consistency of stools or in the frequency of spitting-up (unpublished data). CMA was diagnosed by an oral food challenge in 6 and 3 infants in the probiotic- and placebo groups, respectively (p=0.3). All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced into the infant diet, and all infants improved after elimination of cow’s milk protein. There were no differences between the two groups in growth (unpublished data). Thus, the cereals were well accepted including normal growth and with no demonstrated adverse effects.
Figure 8. Feeding cereals to one of the infants in the study.
Results
34
Effects of feeding probiotics during weaning on the functional status of gut microbiota (Paper 1) In the first paper, we aimed to determine if feeding probiotics during weaning would increase fecal lactobacilli counts and prolong the beneficial effects conferred by breastfeeding on gut microbial composition with subsequent effects on the SCFA pattern. Overall lactobacilli counts were higher during the intervention in the probiotic- compared with the placebo-group (p<0.001). There was an interaction of time and group (p=0.01, Huynh-Feldt). Follow-up analyses demonstrated that the difference in total lactobacilli counts between the groups tended to be statistically significant at 6½ months of age (p=0.09), and was statistically significant at 9 and 13 months of age (p<0.001), (data not shown). Thus, feeding probiotics increased lactobacilli counts compared with placebo, and the gap between the groups widened during the weaning period. Breastfeeding duration impacted on positive lactobacilli isolation rates. In the placebo group breastfeeding at the fecal sampling at 6½ months was associated with a higher frequency of lactobacilli in stool compared with completely weaned infants at that age, (p<0.005), whereas the frequency of lactobacilli in stool did not differ between breastfed and completely weaned infants in the probiotic group (Fig. 9, and data not shown).
Lactobacilli
+
-
-
Lacto-bacilli +
a) Breastfed infants b) Weaned infants
LF19 +
-
LF19 +
-
Probiotic
Placebo
Figure 9. In the placebo group, breastfeeding at the fecal sampling at 6½ months was associated with a higher frequency of lactobacilli (p<0.005), whereas there was no difference between breastfed or weaned infants in the frequency of LF19 in stool in the probiotic group at that age.
Results
34
Effects of feeding probiotics during weaning on the functional status of gut microbiota (Paper 1) In the first paper, we aimed to determine if feeding probiotics during weaning would increase fecal lactobacilli counts and prolong the beneficial effects conferred by breastfeeding on gut microbial composition with subsequent effects on the SCFA pattern. Overall lactobacilli counts were higher during the intervention in the probiotic- compared with the placebo-group (p<0.001). There was an interaction of time and group (p=0.01, Huynh-Feldt). Follow-up analyses demonstrated that the difference in total lactobacilli counts between the groups tended to be statistically significant at 6½ months of age (p=0.09), and was statistically significant at 9 and 13 months of age (p<0.001), (data not shown). Thus, feeding probiotics increased lactobacilli counts compared with placebo, and the gap between the groups widened during the weaning period. Breastfeeding duration impacted on positive lactobacilli isolation rates. In the placebo group breastfeeding at the fecal sampling at 6½ months was associated with a higher frequency of lactobacilli in stool compared with completely weaned infants at that age, (p<0.005), whereas the frequency of lactobacilli in stool did not differ between breastfed and completely weaned infants in the probiotic group (Fig. 9, and data not shown).
Lactobacilli
+
-
-
Lacto-bacilli +
a) Breastfed infants b) Weaned infants
LF19 +
-
LF19 +
-
Probiotic
Placebo
Figure 9. In the placebo group, breastfeeding at the fecal sampling at 6½ months was associated with a higher frequency of lactobacilli (p<0.005), whereas there was no difference between breastfed or weaned infants in the frequency of LF19 in stool in the probiotic group at that age.
Results
35
RAPD-PCR was used to verify the presence of LF19 to the strain level in the probiotic group. This analysis demonstrated the presence of LF19 in the fecal sample of one infant in the probiotic group before the intervention started and high rates of fecal recovery during the intervention in the probiotic group. In 64% of the 84 infants in the probiotic group, LF19 was isolated in all fecal samples during the intervention. In the fecal samples of two infants, LF19 was not detected at any time during the intervention despite reported intake. The fecal concentration of SCFA was assessed by gas liquid-chromatography. As expected, the concentrations of propionic and butyric acid increased during mid-late weaning, as did several of the branched-chain acids, (Fig 10, and data not shown). Overall, there were no differences in the concentration or proportion of the respective SCFA between the groups. There were interactions of time and group in the analyses of total amount of SCFA (p=0.02, Huynh-Feldt) and the concentration of acetic acid (p=0.009, Huynh-Feldt). Follow-up analyses could not verify any statistically significant differences between the groups at either sampling.
Acetic acid
0255075
100125150
ProbioticPlacebo
200400600
Propionic acid
0
5
10
15
20255075
4 6½ 13
Butyric acid
05
101520
255075
4 6½ 13
Con
cent
ratio
nsof
indi
cate
dSC
FA (m
mol
/kg)
Age (mo)
Figure 10. The concentration of acetic acid increased during early weaning whereas propionic and butyric acid increased later on during weaning in both groups. Statistical analyses were then adjusted for persistent colonization by LF19 during the intervention dichotomized as persistently colonized, (isolated in all three fecal samples during the intervention) or not colonized (isolated in < 3 fecal samples). In infants with persistent colonization by LF19 there was a lower concentration of iso-valeric- and iso-butyric acids compared with placebo and the correlation between these iso-acids was not as strong in the probiotic- as in the placebo group (p<0.05), (data not shown). There were no interactions. Follow-up analyses did not reach statistical significance at either sampling. In these infants the proportion of acetic acid was higher, while the proportions of several SCFA were lower (Table 1).
Results
35
RAPD-PCR was used to verify the presence of LF19 to the strain level in the probiotic group. This analysis demonstrated the presence of LF19 in the fecal sample of one infant in the probiotic group before the intervention started and high rates of fecal recovery during the intervention in the probiotic group. In 64% of the 84 infants in the probiotic group, LF19 was isolated in all fecal samples during the intervention. In the fecal samples of two infants, LF19 was not detected at any time during the intervention despite reported intake. The fecal concentration of SCFA was assessed by gas liquid-chromatography. As expected, the concentrations of propionic and butyric acid increased during mid-late weaning, as did several of the branched-chain acids, (Fig 10, and data not shown). Overall, there were no differences in the concentration or proportion of the respective SCFA between the groups. There were interactions of time and group in the analyses of total amount of SCFA (p=0.02, Huynh-Feldt) and the concentration of acetic acid (p=0.009, Huynh-Feldt). Follow-up analyses could not verify any statistically significant differences between the groups at either sampling.
Acetic acid
0255075
100125150
ProbioticPlacebo
200400600
Propionic acid
0
5
10
15
20255075
4 6½ 13
Butyric acid
05
101520
255075
4 6½ 13
Con
cent
ratio
nsof
indi
cate
dSC
FA (m
mol
/kg)
Age (mo)
Figure 10. The concentration of acetic acid increased during early weaning whereas propionic and butyric acid increased later on during weaning in both groups. Statistical analyses were then adjusted for persistent colonization by LF19 during the intervention dichotomized as persistently colonized, (isolated in all three fecal samples during the intervention) or not colonized (isolated in < 3 fecal samples). In infants with persistent colonization by LF19 there was a lower concentration of iso-valeric- and iso-butyric acids compared with placebo and the correlation between these iso-acids was not as strong in the probiotic- as in the placebo group (p<0.05), (data not shown). There were no interactions. Follow-up analyses did not reach statistical significance at either sampling. In these infants the proportion of acetic acid was higher, while the proportions of several SCFA were lower (Table 1).
Results
36
Table 1. The proportion of acetic acid was higher, while the proportion of several short-chain fatty acids (SCFA) was lower in infants persistently colonized by LF19 compared with the placebo group Short-chain fatty acid In comparison with the
placebo group p-value
acetic acid ↑ 0.002 propionic acid ↓ 0.003 iso-butyric ↓ 0.001 butyric - 0.7 iso-valeric ↓ 0.06 valeric ↓ 0.007* iso-caproic - 0.9 caproic - 0.2 * The difference disappeared when adjusting for breastfeeding at entry. There was an interaction of group and breastfeeding (p=0.03, Huynh-Feldt). In summary, feeding probiotics during weaning maintained high fecal lactobacilli counts and induced functional differences in the gut microbiota compared with placebo, with possible effects on gut barrier function.
Results
36
Table 1. The proportion of acetic acid was higher, while the proportion of several short-chain fatty acids (SCFA) was lower in infants persistently colonized by LF19 compared with the placebo group Short-chain fatty acid In comparison with the
placebo group p-value
acetic acid ↑ 0.002 propionic acid ↓ 0.003 iso-butyric ↓ 0.001 butyric - 0.7 iso-valeric ↓ 0.06 valeric ↓ 0.007* iso-caproic - 0.9 caproic - 0.2 * The difference disappeared when adjusting for breastfeeding at entry. There was an interaction of group and breastfeeding (p=0.03, Huynh-Feldt). In summary, feeding probiotics during weaning maintained high fecal lactobacilli counts and induced functional differences in the gut microbiota compared with placebo, with possible effects on gut barrier function.
Results
37
Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines (Paper II) The preventive effects of feeding LF19 on infections were evaluated by prospectively recorded data on infectious symptoms and antibiotic use. Furthermore, we monitored the specific IgG antibody responses to two protein antigens, i.e. diphtheria- and tetanus toxoid, and one polysaccharide antigen Haemophilus influenzae type b capsular polysaccharide (HibPS), to study the potential effects of LF19 on T cell-dependent humoral immunity. The number of days with fever, respiratory illness or diarrhoea did not differ between the groups. Adjusting for seasonal variation, siblings, day-care attendance or smoking at home did not affect the outcome. Twenty-one % (n=84) and 32% (n=87) of the infants in the probiotic- and placebo groups were at least once prescribed antibiotics during the intervention, (p=0.1), (unpublished data). The diagnoses that resulted in antibiotic prescription were otitis media, pneumonia, tonsillitis, scarlatina and urinary tract infection. There was a trend towards less episodes of antibiotic prescriptions in the probiotic group compared with the placebo group; with a mean of 0.3 (0.5 SD) versus 0.5 (0.8 SD) episodes, respectively (p=0.05), (unpublished data). Infants in the probiotic group had slightly fewer days with antibiotics compared with placebo (p=0.044). As expected, the antibody response to all three antigens was markedly higher after the third compared with the second dose. Overall, there was no effect of LF19 on the specific antibody responses to any of the antigens. However, LF19 enhanced anti-diphteria toxin concentrations during the course of vaccination when adjusting for breastfeeding duration and colonization with LF19 (p=0.024). There was an interaction of the intervention and persistent colonization with LF19 on anti-tetanus toxoid concentrations during the course of vaccination (p=0.035). However, follow-up analyses did not reach statistical significance at any time point. In contrast, the anti-HibPS concentrations were higher after the first and second dose of Hib vaccine in infants breastfed < 6 months compared to those breastfed ≥6 months, (p<0.05), with no effect by LF19. Thus, the effects by LF19 on vaccine responses might have been mediated by induced changes in the gut microbiota with subsequent immune-stimulating effects. However, the effects by LF19 were modulated by breastfeeding duration and persistent colonization by LF19, (Fig. 11). After the third dose there was no influence of probiotic intake or breastfeeding duration on IgG specific antibody concentrations to any of the antigens. All infants reached specific IgG antibody concentrations to diphtheria and tetanus toxoids above 1.0 IU, considered an indication of long term protection of diphtheria and tetanus. All infants reached anti-HibPS concentrations above the protective level 0.15 μg/ml when immunization was completed, and all infants but one in the probiotic- and two
Results
37
Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines (Paper II) The preventive effects of feeding LF19 on infections were evaluated by prospectively recorded data on infectious symptoms and antibiotic use. Furthermore, we monitored the specific IgG antibody responses to two protein antigens, i.e. diphtheria- and tetanus toxoid, and one polysaccharide antigen Haemophilus influenzae type b capsular polysaccharide (HibPS), to study the potential effects of LF19 on T cell-dependent humoral immunity. The number of days with fever, respiratory illness or diarrhoea did not differ between the groups. Adjusting for seasonal variation, siblings, day-care attendance or smoking at home did not affect the outcome. Twenty-one % (n=84) and 32% (n=87) of the infants in the probiotic- and placebo groups were at least once prescribed antibiotics during the intervention, (p=0.1), (unpublished data). The diagnoses that resulted in antibiotic prescription were otitis media, pneumonia, tonsillitis, scarlatina and urinary tract infection. There was a trend towards less episodes of antibiotic prescriptions in the probiotic group compared with the placebo group; with a mean of 0.3 (0.5 SD) versus 0.5 (0.8 SD) episodes, respectively (p=0.05), (unpublished data). Infants in the probiotic group had slightly fewer days with antibiotics compared with placebo (p=0.044). As expected, the antibody response to all three antigens was markedly higher after the third compared with the second dose. Overall, there was no effect of LF19 on the specific antibody responses to any of the antigens. However, LF19 enhanced anti-diphteria toxin concentrations during the course of vaccination when adjusting for breastfeeding duration and colonization with LF19 (p=0.024). There was an interaction of the intervention and persistent colonization with LF19 on anti-tetanus toxoid concentrations during the course of vaccination (p=0.035). However, follow-up analyses did not reach statistical significance at any time point. In contrast, the anti-HibPS concentrations were higher after the first and second dose of Hib vaccine in infants breastfed < 6 months compared to those breastfed ≥6 months, (p<0.05), with no effect by LF19. Thus, the effects by LF19 on vaccine responses might have been mediated by induced changes in the gut microbiota with subsequent immune-stimulating effects. However, the effects by LF19 were modulated by breastfeeding duration and persistent colonization by LF19, (Fig. 11). After the third dose there was no influence of probiotic intake or breastfeeding duration on IgG specific antibody concentrations to any of the antigens. All infants reached specific IgG antibody concentrations to diphtheria and tetanus toxoids above 1.0 IU, considered an indication of long term protection of diphtheria and tetanus. All infants reached anti-HibPS concentrations above the protective level 0.15 μg/ml when immunization was completed, and all infants but one in the probiotic- and two
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
LF19 LF19
Gutmicrobiota
Gutmicrobiota
Breastfeedingduration
Breastfeedingduration
Vaccineresponse
Vaccineresponse
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
LF19 LF19
Gutmicrobiota
Gutmicrobiota
Breastfeedingduration
Breastfeedingduration
Vaccineresponse
Vaccineresponse
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
LF19 LF19
Gutmicrobiota
Gutmicrobiota
Breastfeedingduration
Breastfeedingduration
Vaccineresponse
Vaccineresponse
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
Results
38
in the placebo group reached concentrations above 1.0 g/ml, considered the threshold level for long-term protection, respectively.
In summary, infants fed LF19 did not have fewer infections. Days with antibiotic prescriptions were slightly reduced in the probiotic group. However, persistent colonization by LF19 increased the ability to raise immune responses to the protein antigens during the course of vaccination with more marked effects in infants breastfed < 6 months. In contrast, there was a strong influence on anti-HibPS concentrations by breastfeeding duration, with no effect of LF19.
Vaccine response
Breastfeeding duration
Gut microbiota
LF19
Figure 11. The effects by LF19 on specific IgG antibody response were modulated by breastfeeding duration and persistent colonization by LF19 during the intervention.
Results
39
Maturation of T cell function in the human infant and effects thereon of feeding probiotics during weaning (Paper III) We investigated age-dependent maturation of T cell function and if feeding LF19 during weaning would impact thereon. We assessed IL2, IL10, IL4 and IFN-γ mRNA expression levels in PBMC subjected to polyclonal or specific T cell activation. T cell function was assessed as the ability of PBMC to respond to the polyclonal T cell activators anti-CD3 mAb plus anti-CD28 mAb in vitro, using IL2 as a marker of general T cell activation, IL4 as a Th2 marker, IFN-γ as a Th1 marker and IL10 as a marker for regulatory T cell activity. Analysis was performed at 5½ and 13 months of age, i.e. after 1½ and 9 months of probiotic intake. T cell function in adaptive immunity was monitored as IL2, IL4, IL10 and IFN-γ expression after in vitro challenge of PBMC with the T cell-dependent antigen tetanus toxoid (TT), which is also a component of the pentavalent combination vaccines (diphtheria- and tetanus toxoid, acellular pertussis, polio and Hib-conjugate vaccines) which was administered to the infants at 3, 5½ and 12 months of age. After polyclonal T cell activation, infants in both groups expressed the highest levels of IL2 compared with the other cytokines, both at 5½ and 13 months of age, followed by IFN-γ, whereas the expression of IL4 and IL10 was low (Table 2 and data not shown). The IL2 expression was increased at 13 months compared with 5½ months of age in the placebo group (p=0.005), whereas the levels were comparable in the probiotic group (data not shown). Infants in both groups increased their capacity to express both IFN-γ and IL4 mRNA from 5½ to 13 months of age, (p<0.005) while IL10 mRNA levels remained low at both samplings. Even though the expression of IL2, IFN-γ and IL4 mRNA increased from 5½ to 13 months of age in infants in the placebo group, median levels were still lower compared with adult levels. In adults, there was about a five-fold higher response for IL2 and IL4 mRNA, and a 30-fold higher response for IFN-γ mRNA. IL10 levels were similar in infants and adults (Table 2). Table 2. Median cytokine mRNA expression levels following polyclonal T cell activation in infants in the placebo group and adults IL2 IFN-γ IL4 IL10 N At 5½ mo 139 (76-259)1 7 (2-24) 0.4 (0.1-1) 1 (0.3-2) 77 At 13 mo 175 (94-290) 26 (12-104) 2 (0.9-4) 0.8 (0.3-3) 83 Adults 1014 (425-1756) 846 (452-1213) 11 (5-30) 3 (0.1-7) 6 p=0.0012 p<0.001 p=0.001 p=0.2 1 Median (25-75th percentile) Cytokine mRNA levels following 6 hours of polyclonal T cell activation with anti-CD3 mAb plus antiCD28 mAb in vitro. Cytokine mRNA expression level after activation was calculated as mRNA copies/18S rRNA unit (U) in cells incubated with stimulants minus mRNA copies/18S rRNA U in cells incubated with medium alone. Results are displayed as mRNA copies per 18SrRNA U. One 18S RNA U was defined as the signal obtained by 10 pg of a pool of total RNA extracted from PBMC stimulated with anti-CD3 mAb, corresponding to approximately 100 lymphocytes. 2Adult values were compared with values at 13 months of age by using the Mann Whitney U test.
Results
39
Maturation of T cell function in the human infant and effects thereon of feeding probiotics during weaning (Paper III) We investigated age-dependent maturation of T cell function and if feeding LF19 during weaning would impact thereon. We assessed IL2, IL10, IL4 and IFN-γ mRNA expression levels in PBMC subjected to polyclonal or specific T cell activation. T cell function was assessed as the ability of PBMC to respond to the polyclonal T cell activators anti-CD3 mAb plus anti-CD28 mAb in vitro, using IL2 as a marker of general T cell activation, IL4 as a Th2 marker, IFN-γ as a Th1 marker and IL10 as a marker for regulatory T cell activity. Analysis was performed at 5½ and 13 months of age, i.e. after 1½ and 9 months of probiotic intake. T cell function in adaptive immunity was monitored as IL2, IL4, IL10 and IFN-γ expression after in vitro challenge of PBMC with the T cell-dependent antigen tetanus toxoid (TT), which is also a component of the pentavalent combination vaccines (diphtheria- and tetanus toxoid, acellular pertussis, polio and Hib-conjugate vaccines) which was administered to the infants at 3, 5½ and 12 months of age. After polyclonal T cell activation, infants in both groups expressed the highest levels of IL2 compared with the other cytokines, both at 5½ and 13 months of age, followed by IFN-γ, whereas the expression of IL4 and IL10 was low (Table 2 and data not shown). The IL2 expression was increased at 13 months compared with 5½ months of age in the placebo group (p=0.005), whereas the levels were comparable in the probiotic group (data not shown). Infants in both groups increased their capacity to express both IFN-γ and IL4 mRNA from 5½ to 13 months of age, (p<0.005) while IL10 mRNA levels remained low at both samplings. Even though the expression of IL2, IFN-γ and IL4 mRNA increased from 5½ to 13 months of age in infants in the placebo group, median levels were still lower compared with adult levels. In adults, there was about a five-fold higher response for IL2 and IL4 mRNA, and a 30-fold higher response for IFN-γ mRNA. IL10 levels were similar in infants and adults (Table 2). Table 2. Median cytokine mRNA expression levels following polyclonal T cell activation in infants in the placebo group and adults IL2 IFN-γ IL4 IL10 N At 5½ mo 139 (76-259)1 7 (2-24) 0.4 (0.1-1) 1 (0.3-2) 77 At 13 mo 175 (94-290) 26 (12-104) 2 (0.9-4) 0.8 (0.3-3) 83 Adults 1014 (425-1756) 846 (452-1213) 11 (5-30) 3 (0.1-7) 6 p=0.0012 p<0.001 p=0.001 p=0.2 1 Median (25-75th percentile) Cytokine mRNA levels following 6 hours of polyclonal T cell activation with anti-CD3 mAb plus antiCD28 mAb in vitro. Cytokine mRNA expression level after activation was calculated as mRNA copies/18S rRNA unit (U) in cells incubated with stimulants minus mRNA copies/18S rRNA U in cells incubated with medium alone. Results are displayed as mRNA copies per 18SrRNA U. One 18S RNA U was defined as the signal obtained by 10 pg of a pool of total RNA extracted from PBMC stimulated with anti-CD3 mAb, corresponding to approximately 100 lymphocytes. 2Adult values were compared with values at 13 months of age by using the Mann Whitney U test.
Results
40
As expected, the response to in vitro challenge with TT was lower than the response following polyclonal activation. Infants in both groups increased their capacity to express IL2, IFN-γ and IL4 mRNA (p<0.005) from 5½ to 13 months of age. In contrast, the IL10 mRNA levels remained low, with no differences between the two samplings (data not shown). At 13 months of age, after 9 months of probiotic intake, we observed differences in the response to polyclonal activation between the groups. IL2 expression was higher in the placebo group, median (25-75th percentile) 175 (94-290) mRNA copies/18S rRNA U compared with 117 (57-242) in the probiotic group, (p=0.02). At that age, the median IFN-γ expression was higher in the probiotic group, 40 (12-85) mRNA copies/18S rRNA U, versus 26 (12-104) mRNA copies/18S rRNA U, although the difference was not statistically significant. There were no differences between the groups in reponse to in vitro challenge with TT. Since feeding LF19 to this study population increased the capacity to raise specific IgG immune responses to diphtheria toxoid during the course of vaccination, with the same trend for TT, we analyzed correlations between TT-specific IgG serum concentrations and response to in vitro challenge with TT. At 13 months of age, i.e. 4 weeks after the third dose, TT specific antibody concentrations correlated to IL2 and IFN-γ expression after TT challenge in vitro, (rs= 0,31 and 0.26, p<0.05, respectively) but not to IL4- and IL10 mRNA levels in the placebo group. In the probiotic group, the TT specific antibody concentrations correlated to TT-challenge induced IL2, IL4 and IFN-γ mRNA levels (rs= 0.30, 0.31 and 0.33, p<0.05, respectively) but not to IL10. In summary, infants in both groups demonstrated specific as well as global maturation of T cell immunity with increased capacity to express both Th1 and Th2 cytokines during the second half of infancy, even though the cytokine expression was still lower than that of adults. Furthermore, infants in the placebo group had higher IL2 expression levels after polyclonal T cell activation at 13 months of age compared with infants fed probiotics, with a trend towards higher IFN-γ expression levels in infants in the probiotic group at that age.
Results
40
As expected, the response to in vitro challenge with TT was lower than the response following polyclonal activation. Infants in both groups increased their capacity to express IL2, IFN-γ and IL4 mRNA (p<0.005) from 5½ to 13 months of age. In contrast, the IL10 mRNA levels remained low, with no differences between the two samplings (data not shown). At 13 months of age, after 9 months of probiotic intake, we observed differences in the response to polyclonal activation between the groups. IL2 expression was higher in the placebo group, median (25-75th percentile) 175 (94-290) mRNA copies/18S rRNA U compared with 117 (57-242) in the probiotic group, (p=0.02). At that age, the median IFN-γ expression was higher in the probiotic group, 40 (12-85) mRNA copies/18S rRNA U, versus 26 (12-104) mRNA copies/18S rRNA U, although the difference was not statistically significant. There were no differences between the groups in reponse to in vitro challenge with TT. Since feeding LF19 to this study population increased the capacity to raise specific IgG immune responses to diphtheria toxoid during the course of vaccination, with the same trend for TT, we analyzed correlations between TT-specific IgG serum concentrations and response to in vitro challenge with TT. At 13 months of age, i.e. 4 weeks after the third dose, TT specific antibody concentrations correlated to IL2 and IFN-γ expression after TT challenge in vitro, (rs= 0,31 and 0.26, p<0.05, respectively) but not to IL4- and IL10 mRNA levels in the placebo group. In the probiotic group, the TT specific antibody concentrations correlated to TT-challenge induced IL2, IL4 and IFN-γ mRNA levels (rs= 0.30, 0.31 and 0.33, p<0.05, respectively) but not to IL10. In summary, infants in both groups demonstrated specific as well as global maturation of T cell immunity with increased capacity to express both Th1 and Th2 cytokines during the second half of infancy, even though the cytokine expression was still lower than that of adults. Furthermore, infants in the placebo group had higher IL2 expression levels after polyclonal T cell activation at 13 months of age compared with infants fed probiotics, with a trend towards higher IFN-γ expression levels in infants in the probiotic group at that age.
Results
41
Probiotics during weaning reduce the incidence of eczema (Paper IV) In paper IV we investigated the preventive effects of feeding probiotics during weaning on the cumulative incidence of eczema. The cumulative incidence of respiratory allergies was also recorded. We used the IFN-γ/IL4 mRNA ratio expression levels in PBMC subjected to polyclonal T cell activation with anti-CD3 mAb plus anti-CD28 mAb, as a proxy for Th1/Th2 immune balance. Total and specific IgE levels were analyzed for effects of LF19 as well. Eczema was defined on the basis of questionnaires and diaries as an itchy rash for at least two weeks with typical distribution and dry skin and/or doctor’s diagnosis of eczema. The cumulative incidence of eczema at 13 months of age was 9/84 [(11%) (4-17%, 95% CI)] and 19/87 [(22%) (13-31%, 95% CI)] in the probiotic- and placebo groups, respectively (p=0.049), (Fig. 12). The number needed to treat was 9 (6.5-11.5, 95% CI).
0
10
20
30
40
HR
HR
HRHR
p=0.049
p=0.038
ns
ns
AsthmaAsthmaEczemaEczema
Figure 12. Cumulative incidence of eczma and asthma at 13 months of age in the probiotic (open bars) and placebo (hatched bars) groups. Whiskers depict the 95% CI. The Chi-2 test was used for comparisons between the groups. HR=infants at high-risk of allergy. In the high-risk infants, i.e. infants with at least one first grade relative with allergy, the cumulative incidence of eczema was 6/55 [(11%) (2-19%, 95% CI)] in the probiotic group, compared with 14/53 [(26%) (14-39% 95% CI)] in the placebo group (p=0.038). Asthma was diagnosed by a physician in 2/84, (2%) and 5/87, (6%) of the infants in the probiotic- and placebo groups, respectively (p=0.4). The corresponding figures in high-risk infants were 2/55, 4% and 5/53, 9 %, respectively, (p=0.3). All infants diagnosed with asthma had a family history of allergy, (Fig. 12). One infant in the probiotic group was diagnosed with allergic rhino-conjunctivitis by a physician.
Results
41
Probiotics during weaning reduce the incidence of eczema (Paper IV) In paper IV we investigated the preventive effects of feeding probiotics during weaning on the cumulative incidence of eczema. The cumulative incidence of respiratory allergies was also recorded. We used the IFN-γ/IL4 mRNA ratio expression levels in PBMC subjected to polyclonal T cell activation with anti-CD3 mAb plus anti-CD28 mAb, as a proxy for Th1/Th2 immune balance. Total and specific IgE levels were analyzed for effects of LF19 as well. Eczema was defined on the basis of questionnaires and diaries as an itchy rash for at least two weeks with typical distribution and dry skin and/or doctor’s diagnosis of eczema. The cumulative incidence of eczema at 13 months of age was 9/84 [(11%) (4-17%, 95% CI)] and 19/87 [(22%) (13-31%, 95% CI)] in the probiotic- and placebo groups, respectively (p=0.049), (Fig. 12). The number needed to treat was 9 (6.5-11.5, 95% CI).
0
10
20
30
40
HR
HR
HRHR
p=0.049
p=0.038
ns
ns
AsthmaAsthmaEczemaEczema
Figure 12. Cumulative incidence of eczma and asthma at 13 months of age in the probiotic (open bars) and placebo (hatched bars) groups. Whiskers depict the 95% CI. The Chi-2 test was used for comparisons between the groups. HR=infants at high-risk of allergy. In the high-risk infants, i.e. infants with at least one first grade relative with allergy, the cumulative incidence of eczema was 6/55 [(11%) (2-19%, 95% CI)] in the probiotic group, compared with 14/53 [(26%) (14-39% 95% CI)] in the placebo group (p=0.038). Asthma was diagnosed by a physician in 2/84, (2%) and 5/87, (6%) of the infants in the probiotic- and placebo groups, respectively (p=0.4). The corresponding figures in high-risk infants were 2/55, 4% and 5/53, 9 %, respectively, (p=0.3). All infants diagnosed with asthma had a family history of allergy, (Fig. 12). One infant in the probiotic group was diagnosed with allergic rhino-conjunctivitis by a physician.
Results
42
At 13 months of age the IFN- /IL4 mRNA ratio was higher in the probiotic group median (25-75th percentile) 26 (12-71) compared with 16 (6-48) in the placebo group (p=0.040). Also in high-risk infants, this ratio was higher at 13 months in the LF19-group although this difference did not reach statistical significance, (data not shown). At 13 months of age, total IgE and specific IgE levels to cow’s milk, egg white, cat and dog were analyzed. Even though there was an effect of LF19 on the Th1/Th2 immune balance, there was no effect of LF19 on the frequency of sensitization. In summary, feeding LF19 during weaning reduced the cumulative incidence of eczema in infancy, and we suggest that the mechanism could be mediated, at least partly, by a higher Th1/Th2 ratio in infants receiving LF19.
Results
42
At 13 months of age the IFN- /IL4 mRNA ratio was higher in the probiotic group median (25-75th percentile) 26 (12-71) compared with 16 (6-48) in the placebo group (p=0.040). Also in high-risk infants, this ratio was higher at 13 months in the LF19-group although this difference did not reach statistical significance, (data not shown). At 13 months of age, total IgE and specific IgE levels to cow’s milk, egg white, cat and dog were analyzed. Even though there was an effect of LF19 on the Th1/Th2 immune balance, there was no effect of LF19 on the frequency of sensitization. In summary, feeding LF19 during weaning reduced the cumulative incidence of eczema in infancy, and we suggest that the mechanism could be mediated, at least partly, by a higher Th1/Th2 ratio in infants receiving LF19.
General discussion
43
GENERAL DISCUSSION The early gut microbial composition with critical periods for its establishment being the immediate postnatal period and weaning, are likely to impact on the developing immune system, both locally in the gut and systemically. In the present study we attempted to maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, adaptive immunity, Th1/Th2 immune balance and allergic disease.
Probiotic effects on homeostasis of gut microbiota
Dose, compliance and timing During weaning the direct immunological effects conferred by breastfeeding, e.g. supply of sIgA, and the stimulating effects of breast milk on colonization with bifidobacteria and lactobacilli cease. The rationale for introducing LF19 during the period of complementary feeding was in an attempt to maintain the presence of lactobacilli during a dynamic period of increased antigen exposure both from a more diverse microbiota and from food stuffs, with possible effects on gut microbial function and possibly on imprinting of immune responses. We hypothesized that this period could provide an opportunity for stimulation of gut microbial function and the maturing immune system by feeding the probiotic LF19. LF19 was chosen since it fulfilled the criteria of a probiotic and doses between 108-1010 CFU/day had been administered without side effects in human feeding trials (111, 113). At the start of the study, little was known about the minimum dose needed to ensure colonization by probiotic bacteria, including LF19. The present study targeted infants from 4 months of age, and the youngest children previously studied were 1 year-olds, which led us to choose the lower daily dose of 108 CFU for safety concerns. Later, a study designed at investigating the dose-response relationship on colonization of the intestine when feeding infants a formula with LGG at a dose of 108, 109 or 1010 CFU/day, reported successful temporary colonization at all these dosages (117). Different probiotic strains differ in the capacity to temporarily colonize the intestine why these findings cannot be extrapolated to effects of LF19. However, administration of gelatine capsules containing LF19 at a dose of 1010 CFU/day to healthy, fully weaned one-year old children demonstrated a fecal recovery of LF19 at a median log10 6 CFU/g faeces after 3 weeks intake (113). This figure is equivalent with the figures at 6½ and 9 months of age in the probiotic group in the present study, using the lower daily dose of 108 CFU. However, at 13 months of age, LF19 at a dose of 108 CFU/day resulted in a fecal recovery of LF19 one log10 lower compared with the dose of 1010 CFU/day to children of a similar age in the study by Sullivan et al (113). The lower counts of LF19 at 13 months of age coincides with a decreasing frequency of colonization by LF19, possibly reflecting the establishment of a more diversified microbiota or a developing resistance to colonization by the host. No dose-response relationship was investigated in this study. Thus, if a higher dose of LF19 would have exerted stronger effects on gut microbial function and immunological markers is not certain but cannot be ruled out.
General discussion
43
GENERAL DISCUSSION The early gut microbial composition with critical periods for its establishment being the immediate postnatal period and weaning, are likely to impact on the developing immune system, both locally in the gut and systemically. In the present study we attempted to maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, adaptive immunity, Th1/Th2 immune balance and allergic disease.
Probiotic effects on homeostasis of gut microbiota
Dose, compliance and timing During weaning the direct immunological effects conferred by breastfeeding, e.g. supply of sIgA, and the stimulating effects of breast milk on colonization with bifidobacteria and lactobacilli cease. The rationale for introducing LF19 during the period of complementary feeding was in an attempt to maintain the presence of lactobacilli during a dynamic period of increased antigen exposure both from a more diverse microbiota and from food stuffs, with possible effects on gut microbial function and possibly on imprinting of immune responses. We hypothesized that this period could provide an opportunity for stimulation of gut microbial function and the maturing immune system by feeding the probiotic LF19. LF19 was chosen since it fulfilled the criteria of a probiotic and doses between 108-1010 CFU/day had been administered without side effects in human feeding trials (111, 113). At the start of the study, little was known about the minimum dose needed to ensure colonization by probiotic bacteria, including LF19. The present study targeted infants from 4 months of age, and the youngest children previously studied were 1 year-olds, which led us to choose the lower daily dose of 108 CFU for safety concerns. Later, a study designed at investigating the dose-response relationship on colonization of the intestine when feeding infants a formula with LGG at a dose of 108, 109 or 1010 CFU/day, reported successful temporary colonization at all these dosages (117). Different probiotic strains differ in the capacity to temporarily colonize the intestine why these findings cannot be extrapolated to effects of LF19. However, administration of gelatine capsules containing LF19 at a dose of 1010 CFU/day to healthy, fully weaned one-year old children demonstrated a fecal recovery of LF19 at a median log10 6 CFU/g faeces after 3 weeks intake (113). This figure is equivalent with the figures at 6½ and 9 months of age in the probiotic group in the present study, using the lower daily dose of 108 CFU. However, at 13 months of age, LF19 at a dose of 108 CFU/day resulted in a fecal recovery of LF19 one log10 lower compared with the dose of 1010 CFU/day to children of a similar age in the study by Sullivan et al (113). The lower counts of LF19 at 13 months of age coincides with a decreasing frequency of colonization by LF19, possibly reflecting the establishment of a more diversified microbiota or a developing resistance to colonization by the host. No dose-response relationship was investigated in this study. Thus, if a higher dose of LF19 would have exerted stronger effects on gut microbial function and immunological markers is not certain but cannot be ruled out.
General discussion
44
Compliance was high. The recommended intake was at least one serving daily, and mean consumption was 0.7 servings daily, with no differences between the groups. However, there was a variation in intake of cereals between the infants during the 9 month intervention period. Obviously, this variation was affected by differences in daily intake, but also by intervals with low consumption of cereals by some infants due to loss of appetite, infections etc. Also, 9 infants, 6 infants in the probiotic- and 3 infants in the placebo group, respectively, were diagnosed with CMA and had to stop consumption of the cereals that contained cow’s milk protein. This lead to a variation between infants in the dose of ingested LF19. Mean daily cereal consumption in the probiotic group correlated moderately to isolation of LF19 at all samplings, and we decided to adjust the statistical analyses for persistent colonization with LF19 during the intervention as a measure to at least partly adjust for the variation in ingested dose of LF19. We recognize that host factors affecting colonization (12, 118) and the possibility of false negative cultures, are aspects that in addition to intake of cereals (dose and regularity), might have affected persistent colonization by LF19 during the intervention.
Colonization with lactobacilli The intervention was successful in terms of maintaining high fecal lactobacilli counts in the probiotic group during the intervention. We observed an expanding gap between the two groups both in counts of cultivable lactobacilli and frequency of fecal samples with cultivable lactobacilli. At 13 months of age, LF19 was isolated in 71% of the infants in the probiotic group. Lactobacilli were isolated in stool in 87% of the infants in the probiotic group compared with 46 % in the placebo group at that age. In comparison, lactobacilli were isolated in stool in 17% at 12 months of age in the AllergyFlora study (119). The latter study demonstrated low frequency of lactobacilli in stool during the first year of life, reaching a nadir at 12 months of age, with an upsurge again at 18 months of age presumably due to increased consumption of fermented foods. In agreement with the findings from the AllergyFlora study, breastfeeding at the fecal sampling at 6½ months in the placebo group in our study was associated with a higher frequency of fecal recovery of cultivable lactobacilli, suggesting a stimulating effect by breastfeeding. Breastfeeding beyond 6 months was also associated with higher frequency of colonization by lactobacilli and bifidobacteria in another study (34). It has been suggested that breast milk might even provide these bacteria (10, 11). However, adding the probiotic LF19 in weaning food maintained the presence of lactobacilli in stool in both breastfed and weaned infants. Recent data concerning changes of the predominant gut microbiota during the period of introduction of complementary foods in humans are scarce. Amarri et al investigated the effects of complementary feeding on gut microbial composition from 4-9 months of age in healthy infants. Exclusive breastfeeding was recommended until the age of 4 months and complementary feeding was started at the parents’ own discretion. The frequency of infants receiving only breast milk was high, 73%, at the end of 7 months, declining to 36% at the end of 9 months. They found dominance of bifidobacteria in these exclusively breastfed infants before the start of complementary feeding, and breastfed infants maintained high counts of bifidobacteria. Counts of enterobacteria and enterococci increased with age.
General discussion
44
Compliance was high. The recommended intake was at least one serving daily, and mean consumption was 0.7 servings daily, with no differences between the groups. However, there was a variation in intake of cereals between the infants during the 9 month intervention period. Obviously, this variation was affected by differences in daily intake, but also by intervals with low consumption of cereals by some infants due to loss of appetite, infections etc. Also, 9 infants, 6 infants in the probiotic- and 3 infants in the placebo group, respectively, were diagnosed with CMA and had to stop consumption of the cereals that contained cow’s milk protein. This lead to a variation between infants in the dose of ingested LF19. Mean daily cereal consumption in the probiotic group correlated moderately to isolation of LF19 at all samplings, and we decided to adjust the statistical analyses for persistent colonization with LF19 during the intervention as a measure to at least partly adjust for the variation in ingested dose of LF19. We recognize that host factors affecting colonization (12, 118) and the possibility of false negative cultures, are aspects that in addition to intake of cereals (dose and regularity), might have affected persistent colonization by LF19 during the intervention.
Colonization with lactobacilli The intervention was successful in terms of maintaining high fecal lactobacilli counts in the probiotic group during the intervention. We observed an expanding gap between the two groups both in counts of cultivable lactobacilli and frequency of fecal samples with cultivable lactobacilli. At 13 months of age, LF19 was isolated in 71% of the infants in the probiotic group. Lactobacilli were isolated in stool in 87% of the infants in the probiotic group compared with 46 % in the placebo group at that age. In comparison, lactobacilli were isolated in stool in 17% at 12 months of age in the AllergyFlora study (119). The latter study demonstrated low frequency of lactobacilli in stool during the first year of life, reaching a nadir at 12 months of age, with an upsurge again at 18 months of age presumably due to increased consumption of fermented foods. In agreement with the findings from the AllergyFlora study, breastfeeding at the fecal sampling at 6½ months in the placebo group in our study was associated with a higher frequency of fecal recovery of cultivable lactobacilli, suggesting a stimulating effect by breastfeeding. Breastfeeding beyond 6 months was also associated with higher frequency of colonization by lactobacilli and bifidobacteria in another study (34). It has been suggested that breast milk might even provide these bacteria (10, 11). However, adding the probiotic LF19 in weaning food maintained the presence of lactobacilli in stool in both breastfed and weaned infants. Recent data concerning changes of the predominant gut microbiota during the period of introduction of complementary foods in humans are scarce. Amarri et al investigated the effects of complementary feeding on gut microbial composition from 4-9 months of age in healthy infants. Exclusive breastfeeding was recommended until the age of 4 months and complementary feeding was started at the parents’ own discretion. The frequency of infants receiving only breast milk was high, 73%, at the end of 7 months, declining to 36% at the end of 9 months. They found dominance of bifidobacteria in these exclusively breastfed infants before the start of complementary feeding, and breastfed infants maintained high counts of bifidobacteria. Counts of enterobacteria and enterococci increased with age.
General discussion
45
Lactobacilli increased and then decreased during the study period (120), which is consistent with other studies reporting transient colonization by lactobacilli from birth until weaning (1, 2). Even though lactobacilli are considered transient colonizers, it has been proposed that administration of probiotics in early infancy might lead to permanent colonization of the intestine. However, 6 months after the cessation of perinatal feeding of LGG, there was no observed permanent establishment of the strain in infant feces (121). Consistent with these findings, feeding a formula with LGG and B. longum did not induce permanent colonization by either of the strains (122). In the present study, we did not monitor the presence of LF19 in feces after the cessation of intake. Clinical trials have demonstrated effects of probiotic feeding on gut microbial composition. Feeding formula supplemented with B. lactis to preterm infants increased the counts of bifidobacteria and reduced the counts of enterobacteria and clostridia compared with placebo (123). Sensitized infants weaned to an extensively hydrolyzed formula with B. lactis had lower counts of E. coli and Bacteroides compared with infants weaned to the same formula without any addition (124). Feeding LGG or placebo to mothers before delivery and then to infants until 6 months of age demonstrated less clostridia in the placebo group at 6 months of age, but less lactobacilli/enterococci and clostridia at 2 years of age in the probiotic compared with the placebo group (125). Feeding LAVRI-A1 to infants for 6 months induced higher rates of colonization by lactobacilli, with trends towards a lower frequency of colonization by coliforms and a higher frequency of colonization by bifidobacteria (108). In contrast, feeding a formula with LGG and B. longum to infants for 6 months postnatally did not affect general gut microbial composition (122). Although not unequivocally, there are data to support effects by probiotic supplementation during infancy not only by increased counts and frequency of the ingested probiotic bacterium, but also on general gut microbial composition. In the present study, persistent colonization by LF19 induced changes in the fecal SCFA pattern, i.e. functional differences of the gut microbiota. In agreement with our findings, very young anthroposophic children demonstrated changes in the pattern of several SCFA, with a higher proportion of acetic and lower proportions of propionic-, iso-butyric-, iso-valeric- and valeric acids in stool compared with children with a traditional lifestyle (126). The changes we observed in the probiotic group resemble the changes in young anthroposophic children. People with an anthroposophic lifestyle have a high and regular intake of organically produced and fermented foods, and a restrictive use of antibiotics and vaccinations. In the present study, there were no differences between the groups in lifestyle factors e.g. older siblings, exposure to furred pets, breastfeeding duration or immunizations. All infants participating in the present study were immunized according to the national immunization schedule. Infants in the probiotic group had slightly less antibiotic use compared with infants in the placebo group, which could have affected gut microbial composition and hence, gut microbial function. However, the differences in fecal SCFA pattern remained after adjusting for antibiotic use. The vast majority of infants were prescribed penicillin V, which is not expected to influence the gut microbial composition as much as broad spectrum antibiotics. Thus, the changes observed in the fecal pattern of SCFA in the probiotic group are most likely to be induced by probiotic feeding. Actually, anthroposophic school-children had less allergic
General discussion
45
Lactobacilli increased and then decreased during the study period (120), which is consistent with other studies reporting transient colonization by lactobacilli from birth until weaning (1, 2). Even though lactobacilli are considered transient colonizers, it has been proposed that administration of probiotics in early infancy might lead to permanent colonization of the intestine. However, 6 months after the cessation of perinatal feeding of LGG, there was no observed permanent establishment of the strain in infant feces (121). Consistent with these findings, feeding a formula with LGG and B. longum did not induce permanent colonization by either of the strains (122). In the present study, we did not monitor the presence of LF19 in feces after the cessation of intake. Clinical trials have demonstrated effects of probiotic feeding on gut microbial composition. Feeding formula supplemented with B. lactis to preterm infants increased the counts of bifidobacteria and reduced the counts of enterobacteria and clostridia compared with placebo (123). Sensitized infants weaned to an extensively hydrolyzed formula with B. lactis had lower counts of E. coli and Bacteroides compared with infants weaned to the same formula without any addition (124). Feeding LGG or placebo to mothers before delivery and then to infants until 6 months of age demonstrated less clostridia in the placebo group at 6 months of age, but less lactobacilli/enterococci and clostridia at 2 years of age in the probiotic compared with the placebo group (125). Feeding LAVRI-A1 to infants for 6 months induced higher rates of colonization by lactobacilli, with trends towards a lower frequency of colonization by coliforms and a higher frequency of colonization by bifidobacteria (108). In contrast, feeding a formula with LGG and B. longum to infants for 6 months postnatally did not affect general gut microbial composition (122). Although not unequivocally, there are data to support effects by probiotic supplementation during infancy not only by increased counts and frequency of the ingested probiotic bacterium, but also on general gut microbial composition. In the present study, persistent colonization by LF19 induced changes in the fecal SCFA pattern, i.e. functional differences of the gut microbiota. In agreement with our findings, very young anthroposophic children demonstrated changes in the pattern of several SCFA, with a higher proportion of acetic and lower proportions of propionic-, iso-butyric-, iso-valeric- and valeric acids in stool compared with children with a traditional lifestyle (126). The changes we observed in the probiotic group resemble the changes in young anthroposophic children. People with an anthroposophic lifestyle have a high and regular intake of organically produced and fermented foods, and a restrictive use of antibiotics and vaccinations. In the present study, there were no differences between the groups in lifestyle factors e.g. older siblings, exposure to furred pets, breastfeeding duration or immunizations. All infants participating in the present study were immunized according to the national immunization schedule. Infants in the probiotic group had slightly less antibiotic use compared with infants in the placebo group, which could have affected gut microbial composition and hence, gut microbial function. However, the differences in fecal SCFA pattern remained after adjusting for antibiotic use. The vast majority of infants were prescribed penicillin V, which is not expected to influence the gut microbial composition as much as broad spectrum antibiotics. Thus, the changes observed in the fecal pattern of SCFA in the probiotic group are most likely to be induced by probiotic feeding. Actually, anthroposophic school-children had less allergic
General discussion
46
manifestations and fewer of these children were sensitized compared with children with a traditional lifestyle (127). Whether the induced changes in gut microbial function in the probiotic group may impact on later allergies and sensitization need to be followed. The main end product from glucose fermentation by lactobacilli is lactic acid, and we speculate that the increased proportion of acetic acid in the probiotic group is due to an increase in bifidobacteria counts that amongst other species may metabolize carbohydrates to acetic acid (12). Ingestion of other probiotic strains led to an increase in bifidobacteria counts (108). Thus, we hypothesize that feeding LF19 may have created an intestinal milieu favouring the maintenance of bifidobacteria. It could be expected that supplementation with probiotic bacteria e.g. specific strains of lactobacilli would compete for space and nutrients with other lactobacilli in their ecological niche. However, feeding LF19 to elderly led to a transient increase in lactobacilli other than LF19 (113). Again, feeding a probiotic e.g. LF19 might have created an intestinal milieu favoring the thrift of other lactobacilli. The production of SCFA is a feature of groups of bacteria. However, iso-caproic acid is suggested to specifically indicate the presence of C. difficile (128). C. difficile has previously been associated with allergy development. Even though LF19 was previously demonstrated to decrease the numbers of clostridia in a Simulator of the Human Intestinal Microbial Ecosystem (SHIME) in vitro model (129), we observed no effects of LF19 on concentrations or proportions of iso-caproic acid. However, infants with detectable levels of iso-caproic acid at all samplings that could be included for statistical comparison were few. Thus, the power to detect differences between the groups was low. Counts of clostridia have been demonstrated to be higher in children with specific IgE to food or inhalant allergens (130). In the present study, there was a trend towards a higher prevalence of iso-caproic acid in stool in infants who manifested eczema during the intervention, suggesting a relationship between early presence of C. difficile and development of eczema. This finding is in agreement with data from a prospective birth cohort study, where allergic infants had an altered SCFA pattern with higher concentrations of iso-caproic acid compared with non-allergic infants (131). Then again, two large prospective birth cohort studies came to different conclusions about the association between clostridia and allergy development. Colonization by C. difficile at 1 month of age was associated with allergy development at 2 years of age in one of them (76), whereas there was no association in the other (2). It has been proposed that microbial diversity is important in the maturation of regulatory immune mechanisms. Infants in developing countries with a low prevalence of allergy have a faster strain-turnover compared with infants in industrialized countries (5). Anthroposophic children have a higher diversity of their gut microbiota (132), and in the AllergyFlora study there was reduced diversity in the fecal microbiota at 1 week of age in infants who later developed IgE-associated eczema (133). Therefore, a more thorough understanding of the induced changes by prebiotics and probiotics on gut microbial composition are needed.
General discussion
46
manifestations and fewer of these children were sensitized compared with children with a traditional lifestyle (127). Whether the induced changes in gut microbial function in the probiotic group may impact on later allergies and sensitization need to be followed. The main end product from glucose fermentation by lactobacilli is lactic acid, and we speculate that the increased proportion of acetic acid in the probiotic group is due to an increase in bifidobacteria counts that amongst other species may metabolize carbohydrates to acetic acid (12). Ingestion of other probiotic strains led to an increase in bifidobacteria counts (108). Thus, we hypothesize that feeding LF19 may have created an intestinal milieu favouring the maintenance of bifidobacteria. It could be expected that supplementation with probiotic bacteria e.g. specific strains of lactobacilli would compete for space and nutrients with other lactobacilli in their ecological niche. However, feeding LF19 to elderly led to a transient increase in lactobacilli other than LF19 (113). Again, feeding a probiotic e.g. LF19 might have created an intestinal milieu favoring the thrift of other lactobacilli. The production of SCFA is a feature of groups of bacteria. However, iso-caproic acid is suggested to specifically indicate the presence of C. difficile (128). C. difficile has previously been associated with allergy development. Even though LF19 was previously demonstrated to decrease the numbers of clostridia in a Simulator of the Human Intestinal Microbial Ecosystem (SHIME) in vitro model (129), we observed no effects of LF19 on concentrations or proportions of iso-caproic acid. However, infants with detectable levels of iso-caproic acid at all samplings that could be included for statistical comparison were few. Thus, the power to detect differences between the groups was low. Counts of clostridia have been demonstrated to be higher in children with specific IgE to food or inhalant allergens (130). In the present study, there was a trend towards a higher prevalence of iso-caproic acid in stool in infants who manifested eczema during the intervention, suggesting a relationship between early presence of C. difficile and development of eczema. This finding is in agreement with data from a prospective birth cohort study, where allergic infants had an altered SCFA pattern with higher concentrations of iso-caproic acid compared with non-allergic infants (131). Then again, two large prospective birth cohort studies came to different conclusions about the association between clostridia and allergy development. Colonization by C. difficile at 1 month of age was associated with allergy development at 2 years of age in one of them (76), whereas there was no association in the other (2). It has been proposed that microbial diversity is important in the maturation of regulatory immune mechanisms. Infants in developing countries with a low prevalence of allergy have a faster strain-turnover compared with infants in industrialized countries (5). Anthroposophic children have a higher diversity of their gut microbiota (132), and in the AllergyFlora study there was reduced diversity in the fecal microbiota at 1 week of age in infants who later developed IgE-associated eczema (133). Therefore, a more thorough understanding of the induced changes by prebiotics and probiotics on gut microbial composition are needed.
General discussion
47
Probiotic effects on adaptive immunity
Effects on infections There was no effect of LF19 on number of infections, possibly explained by the long breastfeeding duration and comparatively low number of infections during the first year of life in these otherwise healthy Swedish infants. In a randomized trial in Israel, L. reuteri and B. lactis reduced number of days and episodes with diarrhoea in infants of similar age as in the present trial (134). In that study, L. reuteri also reduced absence from day care and courses of antibiotics. Neither probiotic affected respiratory illnesses. However, all infants were weaned from breastfeeding when entering the trial and it was carried out in a day care setting, in contrast to the present trial where the majority of infants were breastfed and cared for at home. In contrast to the results from the study in Israel, Abrahamsson et al observed no preventive effect on infections by administration of L. reuteri to healthy Swedish infants compared with placebo (107). Then again, in a Finnish large-scale randomized trial in children attending day care, intake of LGG reduced the incidence of respiratory infections and courses of antibiotics compared with placebo, although adjusting for age removed the differences (135). Feeding LF19 slightly reduced days with antibiotic prescriptions, which might suggest a preventive effect by LF19 on bacterial infections. In contrast to the preventive effects by probiotics on antibiotic prescriptions observed in our study and the studies in Finland and in Israel, Abrahamsson et al quite unexpectedly observed a slight increase in courses of antibiotics in the L. reuteri-supplemented group (107). Most infants in that study were prescribed antibiotics because of otitis media. Breastfeeding is demonstrated to protect against otitis media (136). Accordingly, the high breastfeeding rate in both groups in that study might have protected infants in both groups. In fact, there was a trend towards longer breastfeeding duration in the placebo group compared with the probiotic group (107). It is of note that the difference in days with antibiotic treatment between the two groups in the present study is rather small, and that in the Finnish study adjusting for age removed the difference in antibiotic courses between the groups. Thus, the most marked effect on the prevention of bacterial infection by probiotics (L. reuteri) was observed in non-breastfed infants in Israel. Even though meta-analyses demonstrate a moderate effect of probiotics by reducing the duration of viral gastroenteritis (94, 95), there are studies in infants and young children that show no preventive effect by probiotic feeding on viral gastroenteritis. In one of them, LGG did not prevent nosocomial rotavirus infection, whereas breastfeeding was protective (137). In a study investigating the effects of LGG in undernourished Peruvian children, the incidence of diarrhoea was reduced in the probiotic- compared with the placebo group, but only in non-breastfed infants and young children. The authors suggest that even though probiotics stimulate some of the effects of breastfeeding, the impact of breastfeeding on the gut microbiota is superior to that of probiotics (138). In summary, probiotics might prevent infections to some degree but the effect appears to be more pronounced in areas with heavy infectious exposure and in non-breastfed infants and young children. Our data support this view. However, all of the abovementioned studies are comparatively small, except one. There is still a need for randomized controlled large-scale trials both in industrialized and developing countries.
General discussion
47
Probiotic effects on adaptive immunity
Effects on infections There was no effect of LF19 on number of infections, possibly explained by the long breastfeeding duration and comparatively low number of infections during the first year of life in these otherwise healthy Swedish infants. In a randomized trial in Israel, L. reuteri and B. lactis reduced number of days and episodes with diarrhoea in infants of similar age as in the present trial (134). In that study, L. reuteri also reduced absence from day care and courses of antibiotics. Neither probiotic affected respiratory illnesses. However, all infants were weaned from breastfeeding when entering the trial and it was carried out in a day care setting, in contrast to the present trial where the majority of infants were breastfed and cared for at home. In contrast to the results from the study in Israel, Abrahamsson et al observed no preventive effect on infections by administration of L. reuteri to healthy Swedish infants compared with placebo (107). Then again, in a Finnish large-scale randomized trial in children attending day care, intake of LGG reduced the incidence of respiratory infections and courses of antibiotics compared with placebo, although adjusting for age removed the differences (135). Feeding LF19 slightly reduced days with antibiotic prescriptions, which might suggest a preventive effect by LF19 on bacterial infections. In contrast to the preventive effects by probiotics on antibiotic prescriptions observed in our study and the studies in Finland and in Israel, Abrahamsson et al quite unexpectedly observed a slight increase in courses of antibiotics in the L. reuteri-supplemented group (107). Most infants in that study were prescribed antibiotics because of otitis media. Breastfeeding is demonstrated to protect against otitis media (136). Accordingly, the high breastfeeding rate in both groups in that study might have protected infants in both groups. In fact, there was a trend towards longer breastfeeding duration in the placebo group compared with the probiotic group (107). It is of note that the difference in days with antibiotic treatment between the two groups in the present study is rather small, and that in the Finnish study adjusting for age removed the difference in antibiotic courses between the groups. Thus, the most marked effect on the prevention of bacterial infection by probiotics (L. reuteri) was observed in non-breastfed infants in Israel. Even though meta-analyses demonstrate a moderate effect of probiotics by reducing the duration of viral gastroenteritis (94, 95), there are studies in infants and young children that show no preventive effect by probiotic feeding on viral gastroenteritis. In one of them, LGG did not prevent nosocomial rotavirus infection, whereas breastfeeding was protective (137). In a study investigating the effects of LGG in undernourished Peruvian children, the incidence of diarrhoea was reduced in the probiotic- compared with the placebo group, but only in non-breastfed infants and young children. The authors suggest that even though probiotics stimulate some of the effects of breastfeeding, the impact of breastfeeding on the gut microbiota is superior to that of probiotics (138). In summary, probiotics might prevent infections to some degree but the effect appears to be more pronounced in areas with heavy infectious exposure and in non-breastfed infants and young children. Our data support this view. However, all of the abovementioned studies are comparatively small, except one. There is still a need for randomized controlled large-scale trials both in industrialized and developing countries.
General discussion
48
Effects on specific antibody responses to common vaccines The effects of probiotics on antigen specific responses to oral vaccines have been studied. Ingestion of LGG increased the IgA secretion following oral rota virus vaccination in infants (139), and LGG tended to enhance the specific IgA secretion in healthy adults following oral Salmonella typhii vaccine (140). Specific IgG antibody responses depend on the functional interplay of antigen presenting cells, antigen specific T helper cells, intercellular cytokine communication and the differentiation of antigen specific B-cells into antibody-producing plasma cells. Thus, measuring the specific IgG antibody response is a useful proxy for the evaluation of adaptive immune responses. We demonstrated that feeding LF19 increased the specific IgG antibody response to diphtheria toxin during the course of vaccination with more marked effects in infants breastfed less than 6 months, with the same trend in response to TT. We observed correlations between the TT specific IgG antibody concentrations and IL4 and IFN-γ expression after TT challenge in vitro in the probiotic group, whereas there was no correlation between TT specific antibody concentrations and IL4 and a slightly weaker correlation to IFN-γ in the placebo group. Functional deficiencies of T cells during infancy have been demonstrated with a diminished capacity to produce several cytokines, in particular IFN-γ (141), resulting in decreased CTL function and ability to offer adequate B cell help for efficacious antibody production (142). Also, it was demonstrated that neonatal monocytes had a markedly lesser response to ligands of several TLR, including bacterial lipopeptides and LPS, with a lower release of TNF-α (143). There are data to support that DC immaturity in infancy limit the capacity to express vaccine specific T cell memory since DC supplementation enhanced TT-specific reactivity in 12-months old infants (144). Probiotic bacteria have been demonstrated to affect APC function. Probiotics increased DC function in the adult human gut (145), and enhanced the maturation of monocytes in infant animals (146). Therefore, we speculate that LF19 might have driven APC maturity, with subsequent effects on Th cells reflected as increased capacity to raise immune responses to the protein antigens during the course of vaccination and stronger correlations between B and T cell TT-specific responses when primary immunization was complete. In comparison, one study has investigated the effects of probiotic supplementation to infants on TT-specific T cell responses. They found lower IL10 responses to TT in the probiotic group, but no differences between the groups in IL4 or IFN-γ responses (147). There was no effect by LF19 on the anti-HibPS concentrations or in the proportion of infants reaching protective anti-HibPS concentrations. This is in contrast to the findings by Kukkonen et al, who demonstrated that perinatal administration of a mix of four probiotic strains and prebiotic GOS increased the frequency of infants reaching protective anti-HibPS concentrations after primary immunization compared with placebo. In this quite small study, they reported no differences between the groups in specific IgG concentrations to HibPS or diphtheria- and tetanus toxoids (148). The inconsistency in results might be due to the use of different probiotic strains with dissimilar immune-stimulating capacity, as well as differences in nature and dose of antigen, infant age and level of maternal antibodies (149).
General discussion
48
Effects on specific antibody responses to common vaccines The effects of probiotics on antigen specific responses to oral vaccines have been studied. Ingestion of LGG increased the IgA secretion following oral rota virus vaccination in infants (139), and LGG tended to enhance the specific IgA secretion in healthy adults following oral Salmonella typhii vaccine (140). Specific IgG antibody responses depend on the functional interplay of antigen presenting cells, antigen specific T helper cells, intercellular cytokine communication and the differentiation of antigen specific B-cells into antibody-producing plasma cells. Thus, measuring the specific IgG antibody response is a useful proxy for the evaluation of adaptive immune responses. We demonstrated that feeding LF19 increased the specific IgG antibody response to diphtheria toxin during the course of vaccination with more marked effects in infants breastfed less than 6 months, with the same trend in response to TT. We observed correlations between the TT specific IgG antibody concentrations and IL4 and IFN-γ expression after TT challenge in vitro in the probiotic group, whereas there was no correlation between TT specific antibody concentrations and IL4 and a slightly weaker correlation to IFN-γ in the placebo group. Functional deficiencies of T cells during infancy have been demonstrated with a diminished capacity to produce several cytokines, in particular IFN-γ (141), resulting in decreased CTL function and ability to offer adequate B cell help for efficacious antibody production (142). Also, it was demonstrated that neonatal monocytes had a markedly lesser response to ligands of several TLR, including bacterial lipopeptides and LPS, with a lower release of TNF-α (143). There are data to support that DC immaturity in infancy limit the capacity to express vaccine specific T cell memory since DC supplementation enhanced TT-specific reactivity in 12-months old infants (144). Probiotic bacteria have been demonstrated to affect APC function. Probiotics increased DC function in the adult human gut (145), and enhanced the maturation of monocytes in infant animals (146). Therefore, we speculate that LF19 might have driven APC maturity, with subsequent effects on Th cells reflected as increased capacity to raise immune responses to the protein antigens during the course of vaccination and stronger correlations between B and T cell TT-specific responses when primary immunization was complete. In comparison, one study has investigated the effects of probiotic supplementation to infants on TT-specific T cell responses. They found lower IL10 responses to TT in the probiotic group, but no differences between the groups in IL4 or IFN-γ responses (147). There was no effect by LF19 on the anti-HibPS concentrations or in the proportion of infants reaching protective anti-HibPS concentrations. This is in contrast to the findings by Kukkonen et al, who demonstrated that perinatal administration of a mix of four probiotic strains and prebiotic GOS increased the frequency of infants reaching protective anti-HibPS concentrations after primary immunization compared with placebo. In this quite small study, they reported no differences between the groups in specific IgG concentrations to HibPS or diphtheria- and tetanus toxoids (148). The inconsistency in results might be due to the use of different probiotic strains with dissimilar immune-stimulating capacity, as well as differences in nature and dose of antigen, infant age and level of maternal antibodies (149).
General discussion
49
Probiotic effects in the prevention of allergy
Prevention of eczema and respiratory allergies We observed a reduction in the cumulative incidence of eczema at 13 months of age in the probiotic group. In comparison, 3 randomized placebo-controlled trials have demonstrated preventive effects of perinatal administration of probiotics on eczema, including or confined to IgE-associated eczema (105-107). In the first study LGG was administered to mothers before delivery and then to mothers or infants until 6 months of age (106). The cumulative incidence of eczema was reduced by 50% in the probiotic group and the preventive effect of LGG on eczema extended to 4 and 7 years of age (150, 151). However, there was no effect of LGG on sensitization, and the mechanism was suggested to be IgE-independent. In another study, administration of L. reuteri to mothers before delivery and to infants until 12 months of age prevented IgE-associated eczema, but not eczema (107). Then again, in a study with a larger sample size, administration of four probiotic strains to mothers before delivery, and the same probiotic mix together with GOS to their infants until 6 months of age prevented both eczema and IgE-associated eczema (105). Conversely, intake of LAVRI-A1 from birth until 6 months of age did not reduce the risk of developing eczema (108). Recently, a study with a similar design to that of Kalliomäki et al demonstrated no preventive effect of LGG on eczema or sensitization (109) (Table 3). None of the above-mentioned prevention studies have demonstrated a preventive effect on respiratory allergies. As of yet, there are only a few published studies on probiotics in the prevention of allergy, and two meta-analyses came to different conclusions regarding recommendations of probiotics in the treatment and prevention of allergy (152, 153).
General discussion
49
Probiotic effects in the prevention of allergy
Prevention of eczema and respiratory allergies We observed a reduction in the cumulative incidence of eczema at 13 months of age in the probiotic group. In comparison, 3 randomized placebo-controlled trials have demonstrated preventive effects of perinatal administration of probiotics on eczema, including or confined to IgE-associated eczema (105-107). In the first study LGG was administered to mothers before delivery and then to mothers or infants until 6 months of age (106). The cumulative incidence of eczema was reduced by 50% in the probiotic group and the preventive effect of LGG on eczema extended to 4 and 7 years of age (150, 151). However, there was no effect of LGG on sensitization, and the mechanism was suggested to be IgE-independent. In another study, administration of L. reuteri to mothers before delivery and to infants until 12 months of age prevented IgE-associated eczema, but not eczema (107). Then again, in a study with a larger sample size, administration of four probiotic strains to mothers before delivery, and the same probiotic mix together with GOS to their infants until 6 months of age prevented both eczema and IgE-associated eczema (105). Conversely, intake of LAVRI-A1 from birth until 6 months of age did not reduce the risk of developing eczema (108). Recently, a study with a similar design to that of Kalliomäki et al demonstrated no preventive effect of LGG on eczema or sensitization (109) (Table 3). None of the above-mentioned prevention studies have demonstrated a preventive effect on respiratory allergies. As of yet, there are only a few published studies on probiotics in the prevention of allergy, and two meta-analyses came to different conclusions regarding recommendations of probiotics in the treatment and prevention of allergy (152, 153).
General discussion
50
Table 3. Effects of probiotic supplementation in the prevention of eczema in randomized placebo-controlled trials Population Intervention Outcome First author,
year
≥ 1 Allergic 1st grade relative
L. rhamnosus GG 1 x 1010
CFU/day (77 mothers) and placebo (82 mothers) 2-4 weeks before delivery and then to their infants (or to the mother if breastfeeding) until 6 mo of age.
Eczema at 2 years of age 23% in probiotic vs 46% in placebo group*.
Kalliomäki et al, 2001
≥ 1 Allergic 1st grade relative
L. rhamnosus GG 5 x 109 CFU twice daily (54 mothers) and placebo (51 mothers) 4-6 weeks before delivery and then to their infants until 6 mo of age.
Eczema at 2 years of age 28% in probiotic vs 27% in placebo group, (ns).
Kopp et al, 2008
≥ 1 Allergic 1st grade relative
L. reuteri 1 x 108 CFU/day (117 mothers) and placebo (115 mothers) 4 weeks before delivery and then to their infants until 12 mo of age.
Eczema at 2 years of age 36% in probiotic vs 34% in placebo group, (ns). IgE-associated eczema at 2 years of age 8% in probiotic vs 20% in placebo group*.
Abrahamsson et al, 2007
≥ 1 Allergic Parent
Probiotic MIX (610 mothers) and placebo (613 mothers) 2-4 weeks before delivery and MIX+GOS or placebo to infants until 6 mo of age.
Eczema at 2 years of age 26% in probiotic vs 32% in placebo group*. IgE-associated eczema at 2 years of age 12% in probiotic vs 18% in placebo group*.
Kukkonen et al, 2007
Allergic mother
L. acidophilus (LAVRI-A1) 3x 108 CFU/day (n=115) and placebo (n=111) from birth until 6 months of age.
Eczema at 1 year of age 26% in probiotic vs 23% in placebo group, (ns).
Taylor et al, 2007
Healthy infants
L. paracasei ssp. paracasei strain F19 1 x 108 CFU/day (n=89) and placebo (n=90) from 4 to 13 months of age.
Eczema at 13 months of age 11% in probiotic vs 22% in placebo group*.
This study
*The difference was statistically significant.
General discussion
50
Table 3. Effects of probiotic supplementation in the prevention of eczema in randomized placebo-controlled trials Population Intervention Outcome First author,
year
≥ 1 Allergic 1st grade relative
L. rhamnosus GG 1 x 1010
CFU/day (77 mothers) and placebo (82 mothers) 2-4 weeks before delivery and then to their infants (or to the mother if breastfeeding) until 6 mo of age.
Eczema at 2 years of age 23% in probiotic vs 46% in placebo group*.
Kalliomäki et al, 2001
≥ 1 Allergic 1st grade relative
L. rhamnosus GG 5 x 109 CFU twice daily (54 mothers) and placebo (51 mothers) 4-6 weeks before delivery and then to their infants until 6 mo of age.
Eczema at 2 years of age 28% in probiotic vs 27% in placebo group, (ns).
Kopp et al, 2008
≥ 1 Allergic 1st grade relative
L. reuteri 1 x 108 CFU/day (117 mothers) and placebo (115 mothers) 4 weeks before delivery and then to their infants until 12 mo of age.
Eczema at 2 years of age 36% in probiotic vs 34% in placebo group, (ns). IgE-associated eczema at 2 years of age 8% in probiotic vs 20% in placebo group*.
Abrahamsson et al, 2007
≥ 1 Allergic Parent
Probiotic MIX (610 mothers) and placebo (613 mothers) 2-4 weeks before delivery and MIX+GOS or placebo to infants until 6 mo of age.
Eczema at 2 years of age 26% in probiotic vs 32% in placebo group*. IgE-associated eczema at 2 years of age 12% in probiotic vs 18% in placebo group*.
Kukkonen et al, 2007
Allergic mother
L. acidophilus (LAVRI-A1) 3x 108 CFU/day (n=115) and placebo (n=111) from birth until 6 months of age.
Eczema at 1 year of age 26% in probiotic vs 23% in placebo group, (ns).
Taylor et al, 2007
Healthy infants
L. paracasei ssp. paracasei strain F19 1 x 108 CFU/day (n=89) and placebo (n=90) from 4 to 13 months of age.
Eczema at 13 months of age 11% in probiotic vs 22% in placebo group*.
This study
*The difference was statistically significant.
General discussion
51
Sensitization and cow’s milk allergy Immune programming starts early in life and several approaches to prevent allergy development and IgE sensitization have been applied over the years. Dietary measures in the prevention of allergy have included elimination of dietary antigens during pregnancy and breastfeeding with no evidence of long-term prevention of allergy (154). Studies exploring the effects of breastfeeding in the prevention of allergy have come to different conclusions, and an apparent shortcoming is the lack of randomized studies due to ethical reasons (155, 156). One explanation of the contrasting results of breastfeeding in the prevention of allergy might be that breast milk composition varies between mothers (157). Today, the view regarding allergy prevention has shifted from allergen avoidance towards tolerance induction (158). Some probiotic bacterial strains have been observed to induce the production of Th1 or regulatory cytokines in vitro. In a mouse model, administration of L. casei strain Shirota led to increased production of Th1 cytokines and decreased production of Th2 cytokines, with subsequently lower IgE levels (159). In vitro studies in humans have identified IL10 - inducing lactic acid bacteria, which down-regulate Th2 cytokines (160). We observed no effect by feeding LF19 on sensitization. Notably, few infants in both groups were sensitized why the statistical power to detect differences was low. However, none of the studies demonstrating preventive effects of probiotics on eczema and IgE-associated eczema observed effects on sensitization (105-107), except in subgroup analyses, (105, 107). At this age, total and specific IgE-antibody levels overlap between atopic and non-atopic children (161, 162), which is why follow-up at later ages might reveal effects of probiotics on sensitization per se. Data from one follow-up study of feeding LGG in allergy-prevention are available. At the ages 4 and 7, sustaining effects of intake of LGG on the incidence of eczema were reported but there was no effect on sensitization or respiratory allergies. If anything, there was a trend towards higher rates of sensitization and respiratory allergies in the probiotic compared with the placebo group at these ages (150, 151). One of the prevention studies actually reported higher rates of sensitization at 12 months of age in infants receiving LAVRI-A1 compared with placebo (108), again stressing the need for follow-up of the infants at later ages. In the present study, the incidence of cow’s milk allergy was somewhat higher than expected, 5% versus reported 2-3%. An IgE-association (positive SPT at the time of diagnosis) was demonstrated in 5 out of 9 infants. Of these, 4 infants were fed probiotics and 1 infant placebo. All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced to the infant diet, and all infants improved after elimination of cow’s milk protein. That administration of a few doses of LF19 would have elicited such reactions to cow’s milk protein seems unlikely. In a small study investigating the effects of probiotics in the treatment of CMA and eczema, elimination of cow’s milk protein in addition to intake of LGG alleviated eczema severity and intestinal inflammation (97). In a larger study, administration of LGG to very young children with IgE-associated eczema and suspected CMA alleviated eczema severity. In that study a mix of probiotic strains had no effect on CMA in contrast to the beneficial effects by LGG, but the probiotic mix caused no harmful side-effect either (102).
General discussion
51
Sensitization and cow’s milk allergy Immune programming starts early in life and several approaches to prevent allergy development and IgE sensitization have been applied over the years. Dietary measures in the prevention of allergy have included elimination of dietary antigens during pregnancy and breastfeeding with no evidence of long-term prevention of allergy (154). Studies exploring the effects of breastfeeding in the prevention of allergy have come to different conclusions, and an apparent shortcoming is the lack of randomized studies due to ethical reasons (155, 156). One explanation of the contrasting results of breastfeeding in the prevention of allergy might be that breast milk composition varies between mothers (157). Today, the view regarding allergy prevention has shifted from allergen avoidance towards tolerance induction (158). Some probiotic bacterial strains have been observed to induce the production of Th1 or regulatory cytokines in vitro. In a mouse model, administration of L. casei strain Shirota led to increased production of Th1 cytokines and decreased production of Th2 cytokines, with subsequently lower IgE levels (159). In vitro studies in humans have identified IL10 - inducing lactic acid bacteria, which down-regulate Th2 cytokines (160). We observed no effect by feeding LF19 on sensitization. Notably, few infants in both groups were sensitized why the statistical power to detect differences was low. However, none of the studies demonstrating preventive effects of probiotics on eczema and IgE-associated eczema observed effects on sensitization (105-107), except in subgroup analyses, (105, 107). At this age, total and specific IgE-antibody levels overlap between atopic and non-atopic children (161, 162), which is why follow-up at later ages might reveal effects of probiotics on sensitization per se. Data from one follow-up study of feeding LGG in allergy-prevention are available. At the ages 4 and 7, sustaining effects of intake of LGG on the incidence of eczema were reported but there was no effect on sensitization or respiratory allergies. If anything, there was a trend towards higher rates of sensitization and respiratory allergies in the probiotic compared with the placebo group at these ages (150, 151). One of the prevention studies actually reported higher rates of sensitization at 12 months of age in infants receiving LAVRI-A1 compared with placebo (108), again stressing the need for follow-up of the infants at later ages. In the present study, the incidence of cow’s milk allergy was somewhat higher than expected, 5% versus reported 2-3%. An IgE-association (positive SPT at the time of diagnosis) was demonstrated in 5 out of 9 infants. Of these, 4 infants were fed probiotics and 1 infant placebo. All infants with CMA reacted with symptoms from the skin or gastrointestinal tract shortly after introduction of the cereal, which was the first milk protein-based weaning food introduced to the infant diet, and all infants improved after elimination of cow’s milk protein. That administration of a few doses of LF19 would have elicited such reactions to cow’s milk protein seems unlikely. In a small study investigating the effects of probiotics in the treatment of CMA and eczema, elimination of cow’s milk protein in addition to intake of LGG alleviated eczema severity and intestinal inflammation (97). In a larger study, administration of LGG to very young children with IgE-associated eczema and suspected CMA alleviated eczema severity. In that study a mix of probiotic strains had no effect on CMA in contrast to the beneficial effects by LGG, but the probiotic mix caused no harmful side-effect either (102).
General discussion
52
Gut barrier function We observed a stronger correlation between iso-butyric and iso-valeric acids in the placebo- compared with the probiotic group at 13 months of age. Furthermore, the concentration of iso-butyric and iso-valeric acids in infants with persistent colonization by LF19 was lower compared with placebo. Branched-chain SCFA, i.e. iso-forms, are metabolic products resulting from partial digestion of proteins and lipids. A follow-up of a birth cohort study demonstrated that the correlation between iso-butyric and iso-valeric acids was stronger in sensitized than non-sensitized four-year-olds, possibly due to higher intestinal epithelial desquamation in sensitized children (E Norin, personal communication). In children with celiac disease, the concentrations of iso-butyric and iso-valeric acids in stool were higher compared with healthy children, suggesting an altered gut microbial composition in the small intestine, a rapid small bowel passage and/or less metabolic activity due to the small bowel enteropathy, leading to an excess of partially digested nutrients reaching the colon (163). One of the proposed mechanisms of probiotics is stabilization of the intestinal barrier. Feeding LGG to suckling rats increased gut mucosal integrity (164). Allergic individuals have increased intestinal permeability (65, 66). In a small study elimination of cow’s milk protein in addition to intake of LGG alleviated both eczema severity and intestinal inflammation, reflected by decreased fecal α1-antitrypsin and increased TGF-β levels (97). The combination of L. reuteri and L. rhamnosus relieved gastrointestinal symptoms and reversed the increased permeability in children with eczema (165). Thus, we propose that the lower concentration of iso-butyric and iso-valeric acids, and the lower correlation between these iso-acids in infants with persistent colonization by LF19 during the intervention compared with placebo, might reflect a more intact mucosal barrier and a more complete digestion. Further, we suggest that the preventive effect by LF19 on eczema is at least partly due to an increased intestinal integrity. If so, the induced changes in SCFA pattern may impact on later sensitization and need to be followed.
Immune-stimulating effects
Maternal and fetal immune responses It has been suggested that probiotic administration to mothers before delivery might be a prerequisite to obtain allergy-preventive and immune-stimulating effects (107, 166, 167). This was suggested since three studies with preventive effects on eczema, including or confined to IgE-associated eczema, started the probiotic intervention before delivery (105-107), as opposed to within 48 hours after delivery in one study that failed to demonstrate a preventive effect (108). Indeed, intake of LGG by pregnant mothers was demonstrated to affect the composition of bifidobacterial species in their infants, but no other effects were reported in this rather small study (168). In another study, LGG was demonstrated to increase the production of IL10 and IFN-γ in vitro, but not in vivo in PBMC in the supplemented mothers or in cord blood mononuclear cells (CBMC) of their infants (169). Prebiotic (GOS/FOS) intake
General discussion
52
Gut barrier function We observed a stronger correlation between iso-butyric and iso-valeric acids in the placebo- compared with the probiotic group at 13 months of age. Furthermore, the concentration of iso-butyric and iso-valeric acids in infants with persistent colonization by LF19 was lower compared with placebo. Branched-chain SCFA, i.e. iso-forms, are metabolic products resulting from partial digestion of proteins and lipids. A follow-up of a birth cohort study demonstrated that the correlation between iso-butyric and iso-valeric acids was stronger in sensitized than non-sensitized four-year-olds, possibly due to higher intestinal epithelial desquamation in sensitized children (E Norin, personal communication). In children with celiac disease, the concentrations of iso-butyric and iso-valeric acids in stool were higher compared with healthy children, suggesting an altered gut microbial composition in the small intestine, a rapid small bowel passage and/or less metabolic activity due to the small bowel enteropathy, leading to an excess of partially digested nutrients reaching the colon (163). One of the proposed mechanisms of probiotics is stabilization of the intestinal barrier. Feeding LGG to suckling rats increased gut mucosal integrity (164). Allergic individuals have increased intestinal permeability (65, 66). In a small study elimination of cow’s milk protein in addition to intake of LGG alleviated both eczema severity and intestinal inflammation, reflected by decreased fecal α1-antitrypsin and increased TGF-β levels (97). The combination of L. reuteri and L. rhamnosus relieved gastrointestinal symptoms and reversed the increased permeability in children with eczema (165). Thus, we propose that the lower concentration of iso-butyric and iso-valeric acids, and the lower correlation between these iso-acids in infants with persistent colonization by LF19 during the intervention compared with placebo, might reflect a more intact mucosal barrier and a more complete digestion. Further, we suggest that the preventive effect by LF19 on eczema is at least partly due to an increased intestinal integrity. If so, the induced changes in SCFA pattern may impact on later sensitization and need to be followed.
Immune-stimulating effects
Maternal and fetal immune responses It has been suggested that probiotic administration to mothers before delivery might be a prerequisite to obtain allergy-preventive and immune-stimulating effects (107, 166, 167). This was suggested since three studies with preventive effects on eczema, including or confined to IgE-associated eczema, started the probiotic intervention before delivery (105-107), as opposed to within 48 hours after delivery in one study that failed to demonstrate a preventive effect (108). Indeed, intake of LGG by pregnant mothers was demonstrated to affect the composition of bifidobacterial species in their infants, but no other effects were reported in this rather small study (168). In another study, LGG was demonstrated to increase the production of IL10 and IFN-γ in vitro, but not in vivo in PBMC in the supplemented mothers or in cord blood mononuclear cells (CBMC) of their infants (169). Prebiotic (GOS/FOS) intake
General discussion
53
by pregnant mothers increased the proportions of bifidobacteria in the maternal gut but had no direct effect on bacterial transfer between mother and infant. There was no indication of impact of prebiotic intake by mothers on fetal immune parameters, as assessed by phenotyping of lymphocyte subsets and cytokine pattern in CBMC (170). The effects of feeding a MIX of probiotic strains to mothers prior to delivery and then to their infants in combination with prebiotics until 6 months of age on fetal immune responses was investigated by Marschan et al. Consistent with the findings by Kopp et al, intake of probiotics by mothers during pregnancy did not affect CBMC immune responses (169, 171). The immune system of the infant is influenced by maternal immunity, both during gestation and during the breastfeeding period. IgE-levels and cytokine responses were demonstrated to correlate between infants and their mothers (172). Notably, in the study by Abrahamsson et al, skin prick test reactivity was less frequent in infants of allergic mothers in the probiotic group (107). The effects of probiotic ingestion by pregnant mothers on the composition of breast milk have also been studied. LGG to mothers during breastfeeding increased TGF-β2 levels in mature milk, although with no association to allergic outcome or sensitization of their infants (173). Intake of L. reuteri by mothers prior to delivery lowered TGF-β2 levels in colostrum, and this decrease was associated with less IgE-sensitization in early childhood (167). This could at least partly explain the preventive effects of L. reuteri in the prevention of IgE-associated eczema (107). It is of note however, that breast milk TGF-β2 levels in mature milk was not associated with future sensitization. In that context, prenatal administration to mothers might be crucial for the modulation of colostral milk composition, with possible effects on future sensitization. However, data are only available from a limited number of studies and more studies are needed to assess effects by prenatal probiotic intake on maternal and fetal immune responses.
Infant immune responses Opposite to the lack of demonstrated effects on fetal immune responses by prenatal probiotic supplementation, the present study demonstrates that probiotics administered during weaning may modulate T cell function. As of yet, little is known about the optimal timing of probiotic supplementation to human infants. However, the timing of the initiation of probiotic feeding was studied in a mouse model. The effects of feeding L. johnsooni NCC533 (La1) to mice during early, mid and late weaning on sIgA production by pups were studied. Feeding La1 during mid weaning increased sIgA production by pups, whereas feeding La1 during early weaning showed negative effects on sIgA production. Feeding La1 during the late phase had no effects on sIgA production. The authors suggest that during early weaning, the pups’ intestines were still protected by maternal sIgA, whereas during late weaning the endogenous production by sIgA was already induced, protecting the pup intestine from contact with La1. Thus, the mid weaning period emerged as a crucial period for contact between the probiotic and the gut mucosa with subsequent immune- stimulating effects. The authors then investigated administration of La1 during mid weaning in a mouse model of eczema, with preventive effects on the development of eczematous lesions in conjunction with increased fecal sIgA levels (174).
General discussion
53
by pregnant mothers increased the proportions of bifidobacteria in the maternal gut but had no direct effect on bacterial transfer between mother and infant. There was no indication of impact of prebiotic intake by mothers on fetal immune parameters, as assessed by phenotyping of lymphocyte subsets and cytokine pattern in CBMC (170). The effects of feeding a MIX of probiotic strains to mothers prior to delivery and then to their infants in combination with prebiotics until 6 months of age on fetal immune responses was investigated by Marschan et al. Consistent with the findings by Kopp et al, intake of probiotics by mothers during pregnancy did not affect CBMC immune responses (169, 171). The immune system of the infant is influenced by maternal immunity, both during gestation and during the breastfeeding period. IgE-levels and cytokine responses were demonstrated to correlate between infants and their mothers (172). Notably, in the study by Abrahamsson et al, skin prick test reactivity was less frequent in infants of allergic mothers in the probiotic group (107). The effects of probiotic ingestion by pregnant mothers on the composition of breast milk have also been studied. LGG to mothers during breastfeeding increased TGF-β2 levels in mature milk, although with no association to allergic outcome or sensitization of their infants (173). Intake of L. reuteri by mothers prior to delivery lowered TGF-β2 levels in colostrum, and this decrease was associated with less IgE-sensitization in early childhood (167). This could at least partly explain the preventive effects of L. reuteri in the prevention of IgE-associated eczema (107). It is of note however, that breast milk TGF-β2 levels in mature milk was not associated with future sensitization. In that context, prenatal administration to mothers might be crucial for the modulation of colostral milk composition, with possible effects on future sensitization. However, data are only available from a limited number of studies and more studies are needed to assess effects by prenatal probiotic intake on maternal and fetal immune responses.
Infant immune responses Opposite to the lack of demonstrated effects on fetal immune responses by prenatal probiotic supplementation, the present study demonstrates that probiotics administered during weaning may modulate T cell function. As of yet, little is known about the optimal timing of probiotic supplementation to human infants. However, the timing of the initiation of probiotic feeding was studied in a mouse model. The effects of feeding L. johnsooni NCC533 (La1) to mice during early, mid and late weaning on sIgA production by pups were studied. Feeding La1 during mid weaning increased sIgA production by pups, whereas feeding La1 during early weaning showed negative effects on sIgA production. Feeding La1 during the late phase had no effects on sIgA production. The authors suggest that during early weaning, the pups’ intestines were still protected by maternal sIgA, whereas during late weaning the endogenous production by sIgA was already induced, protecting the pup intestine from contact with La1. Thus, the mid weaning period emerged as a crucial period for contact between the probiotic and the gut mucosa with subsequent immune- stimulating effects. The authors then investigated administration of La1 during mid weaning in a mouse model of eczema, with preventive effects on the development of eczematous lesions in conjunction with increased fecal sIgA levels (174).
General discussion
54
In the present study, T cell function following polyclonal activation increased in both groups during the second half of infancy, although levels were still lower compared with adult levels. Our data support development of both the Th1 and Th2 arm of immune response during this period. The effects of LF19 on T cell function were modest but measurable. At 13 months of age the IL2 expression was lower in the probiotic group compared with the placebo group, whereas the IFN-γ expression tended to be higher in the probiotic group. Naïve Th cells produce IL2 upon activation, whereas the Th1 cell response is tilted towards IFN-γ production (175). We speculate that the feeding of LF19 has driven the T cell response towards a Th1 type immune response following polyclonal activation whereas infants in the placebo group express a Th0 type response. IL4 and IFN-γ are counter-regulators (176), and when analyzing the IFN-γ/IL4 mRNA ratio as a proxy for propensity of type of immune response, this ratio was higher in the probiotic group after 9 months of supplementation with LF19. Even though the response of PBMC to polyclonal activation could reflect immune system maturation induced in the mucosa due to mucosal and systemic recirculation pathways, this response might be lesser than if we had studied the response in mucosal biopsies. However, that was not an option in these otherwise healthy infants due to ethical reasons. We infer that the increased Th1/Th2 ratio at least partly explains the reduced incidence of eczema in the probiotic group. If so, this effect is in agreement with the observed effects of probiotics in the treatment of eczema. Supplementation with LGG to infants and very young children with CMA and IgE-associated eczema enhanced their IFN-γ responses to polyclonal T cell activation compared with placebo, whereas a mix of four probiotic strains, including LGG, did not (177). Supplementation with Lactobacillus fermentum PCCTM to infants with IgE-associated eczema reduced the disease severity and augmented polyclonal IFN-γ responses by T cells compared with placebo (178). In further support of immune-stimulating effects by probiotics on infant immune responses, feeding a mix of probiotic strains to mothers before delivery and then synbiotics to their infants for 6 months did not affect fetal immune parameters, but impacted upon infant immune parameters (179). A profile consistent to that of low-grade inflammation after 6 months of probiotic intake in infants supplemented with probiotics was observed. The plasma levels of IL10, CRP, total IgA and IgE were increased in the probiotic group compared with the placebo group. The authors also observed that augmented plasma CRP levels at 6 months of age were associated with a decreased risk of eczema and allergic disease at 2 years of age. The rise in total IgE in the probiotic group might seem paradoxical, but the authors demonstrated that this rise was not correlated to allergen-specific IgE levels. The authors propose that probiotics may induce chronic low-grade inflammation during ingestion, resembling the immunological response to helminth infection with induced increases in IL10 and total IgE. Despite the induction of a Th2 response in helminth infection, helminth infection protects against allergy expression, possibly explained by induction of regulatory mechanisms in the intestine (180). Marschan et al further propose that induction of inflammation is the link between probiotic immune-stimulating effects and tolerance induction (179).
General discussion
54
In the present study, T cell function following polyclonal activation increased in both groups during the second half of infancy, although levels were still lower compared with adult levels. Our data support development of both the Th1 and Th2 arm of immune response during this period. The effects of LF19 on T cell function were modest but measurable. At 13 months of age the IL2 expression was lower in the probiotic group compared with the placebo group, whereas the IFN-γ expression tended to be higher in the probiotic group. Naïve Th cells produce IL2 upon activation, whereas the Th1 cell response is tilted towards IFN-γ production (175). We speculate that the feeding of LF19 has driven the T cell response towards a Th1 type immune response following polyclonal activation whereas infants in the placebo group express a Th0 type response. IL4 and IFN-γ are counter-regulators (176), and when analyzing the IFN-γ/IL4 mRNA ratio as a proxy for propensity of type of immune response, this ratio was higher in the probiotic group after 9 months of supplementation with LF19. Even though the response of PBMC to polyclonal activation could reflect immune system maturation induced in the mucosa due to mucosal and systemic recirculation pathways, this response might be lesser than if we had studied the response in mucosal biopsies. However, that was not an option in these otherwise healthy infants due to ethical reasons. We infer that the increased Th1/Th2 ratio at least partly explains the reduced incidence of eczema in the probiotic group. If so, this effect is in agreement with the observed effects of probiotics in the treatment of eczema. Supplementation with LGG to infants and very young children with CMA and IgE-associated eczema enhanced their IFN-γ responses to polyclonal T cell activation compared with placebo, whereas a mix of four probiotic strains, including LGG, did not (177). Supplementation with Lactobacillus fermentum PCCTM to infants with IgE-associated eczema reduced the disease severity and augmented polyclonal IFN-γ responses by T cells compared with placebo (178). In further support of immune-stimulating effects by probiotics on infant immune responses, feeding a mix of probiotic strains to mothers before delivery and then synbiotics to their infants for 6 months did not affect fetal immune parameters, but impacted upon infant immune parameters (179). A profile consistent to that of low-grade inflammation after 6 months of probiotic intake in infants supplemented with probiotics was observed. The plasma levels of IL10, CRP, total IgA and IgE were increased in the probiotic group compared with the placebo group. The authors also observed that augmented plasma CRP levels at 6 months of age were associated with a decreased risk of eczema and allergic disease at 2 years of age. The rise in total IgE in the probiotic group might seem paradoxical, but the authors demonstrated that this rise was not correlated to allergen-specific IgE levels. The authors propose that probiotics may induce chronic low-grade inflammation during ingestion, resembling the immunological response to helminth infection with induced increases in IL10 and total IgE. Despite the induction of a Th2 response in helminth infection, helminth infection protects against allergy expression, possibly explained by induction of regulatory mechanisms in the intestine (180). Marschan et al further propose that induction of inflammation is the link between probiotic immune-stimulating effects and tolerance induction (179).
General discussion
55
It is possible that the higher IFN-γ/IL4 ratio in the probiotic group could have been induced by regulatory cell populations. Probiotic bacteria have been demonstrated to induce regulatory T cell populations. In an animal model, probiotics induced Tregs in the gut (181), and as mentioned in vitro studies in humans have identified IL10 - inducing lactic acid-producing bacteria, which down-regulate Th2 cytokines (160). Tr1 cells are induced in the presence of IL10 and exert their suppressive function by the production of IL10 (182). Previously, LF19 induced IL10 production of human PBMC in vitro as well as IL10 production in a monocyte cell line (110, 115). We observed no effects of LF19 on IL10 mRNA levels following polyclonal activation of PBMC. However, in vitro and in vivo immune responses may differ. Despite that, we suspect that 6 hours of polyclonal activation was not optimal for this particular cytokine since levels were low even in adults following polyclonal activation. Another key immuno-regulatory cytokine in oral tolerance induction is TGF-β . It would have been of interest to study the effects of LF19 on the expression of TGF-β, but during the preceding time-curve experiments, TGF-β levels did not change following polyclonal activation of PBMC, why this was not an option in our setting. In one study the effects of LAVRI-A1 on the frequency of T reg cells and the expression of the transcription factor Foxp3 in infants were studied. An up-regulated Foxp3 mRNA expression in PBMC following activation with allergens in infants with eczema compared with infants without eczema was noted. However there was no effect of feeding LAVRI-A1 to infants for 6 months on the frequency of CD4+CD25+ T cells or Foxp3 mRNA expression (183). Furthermore, specific ligands to assess TLR 2 and 4 functions were used, and even though there were no statistically significant differences between the groups, there were trends for higher TNF- α and IFN-γ responses following TLR2 activation in the probiotic group (184). Also, as previously discussed, in that study there was no effect of LAVRI-A1 on eczema incidence, and infants at 12 months of age were sensitized to a higher extent in the probiotic group than infants in the placebo group (184). Probiotic strains should be judged on their individual merits. The inconsistency in the results of the immune-stimulating effects of probiotics in primary prevention of allergy might be explained by the use of different probiotic strains but also due to differences in host and environmental factors (166), (Table 4). Table 4. Probiotic, host and environmental factors that may impact upon clinical outcome measures in the infant Probiotic Mother Infant Strain Dose Viability Timing Duration
Genetic background Environment Allergic status Breast milk composition
Genetic background Environment Mode of delivery Feeding mode
General discussion
55
It is possible that the higher IFN-γ/IL4 ratio in the probiotic group could have been induced by regulatory cell populations. Probiotic bacteria have been demonstrated to induce regulatory T cell populations. In an animal model, probiotics induced Tregs in the gut (181), and as mentioned in vitro studies in humans have identified IL10 - inducing lactic acid-producing bacteria, which down-regulate Th2 cytokines (160). Tr1 cells are induced in the presence of IL10 and exert their suppressive function by the production of IL10 (182). Previously, LF19 induced IL10 production of human PBMC in vitro as well as IL10 production in a monocyte cell line (110, 115). We observed no effects of LF19 on IL10 mRNA levels following polyclonal activation of PBMC. However, in vitro and in vivo immune responses may differ. Despite that, we suspect that 6 hours of polyclonal activation was not optimal for this particular cytokine since levels were low even in adults following polyclonal activation. Another key immuno-regulatory cytokine in oral tolerance induction is TGF-β . It would have been of interest to study the effects of LF19 on the expression of TGF-β, but during the preceding time-curve experiments, TGF-β levels did not change following polyclonal activation of PBMC, why this was not an option in our setting. In one study the effects of LAVRI-A1 on the frequency of T reg cells and the expression of the transcription factor Foxp3 in infants were studied. An up-regulated Foxp3 mRNA expression in PBMC following activation with allergens in infants with eczema compared with infants without eczema was noted. However there was no effect of feeding LAVRI-A1 to infants for 6 months on the frequency of CD4+CD25+ T cells or Foxp3 mRNA expression (183). Furthermore, specific ligands to assess TLR 2 and 4 functions were used, and even though there were no statistically significant differences between the groups, there were trends for higher TNF- α and IFN-γ responses following TLR2 activation in the probiotic group (184). Also, as previously discussed, in that study there was no effect of LAVRI-A1 on eczema incidence, and infants at 12 months of age were sensitized to a higher extent in the probiotic group than infants in the placebo group (184). Probiotic strains should be judged on their individual merits. The inconsistency in the results of the immune-stimulating effects of probiotics in primary prevention of allergy might be explained by the use of different probiotic strains but also due to differences in host and environmental factors (166), (Table 4). Table 4. Probiotic, host and environmental factors that may impact upon clinical outcome measures in the infant Probiotic Mother Infant Strain Dose Viability Timing Duration
Genetic background Environment Allergic status Breast milk composition
Genetic background Environment Mode of delivery Feeding mode
General discussion
56
Strengths and weaknesses of the study This study investigates the effects of feeding LF19 on clinical outcomes as well as development of gut microbial composition and immune system maturation. A strength of the study design is that we followed infants prospectively during the weaning period. A strength in the results is the very low drop-out rate and the high number of collected blood and fecal samples available for analysis. The diagnosis of eczema was based on questionnaires and diaries. We used a previously validated definition of eczema demonstrated to have high sensitivity (92%) and specificity (100%) compared to clinical diagnosis by a dermatologist (185). A weakness of the study is that we have no data on eczema severity. Alternatively, a clincial examination and classification of eczema severity using the SCORAD index could have been applied. On the other hand, assessing the cumulative incidence as done here instead of point prevalence could actually be a strength. If the children are examined only once, eczema might be missed due to the typically relapsing course of the disease and that some children might have outgrown the eczema by that age. Ultimately, repeated clinical examinations with assessment of eczema severity and collection of data from diaries and questionnaires could have been used. In the diagnosis of CMA infants were put on a cow’s milk protein elimination diet and the subsequent food challenge was open. In general, infants rarely report subjective symptoms, but the challenge being open and not blinded might have led to an over-diagnosis (57). Another weakness is the comparatively low statistical power for the clinical outcome variables. A larger study sample would have allowed us to assess the effects of LF19 on overall infections, respiratory allergies, IgE sensitization and to undertake subgroup-analyses distinguishing between IgE and non-IgE mediated eczema. Also, trends of probiotic effects on T cell function might have been more evident with a larger study sample. Regarding ethical considerations, the study protocol might have been demanding for some families. In the majority of cases, the research nurses delivered the cereals and visited the families in their homes for the collection of diaries, completion of questionnaires, anthropometric measures and venous punctures. The venous puncture that could be potentially stressful for the infant was done in a familial environment, thus reducing the tension of both infants and their parents. Anaesthetic cream was applied before the venous puncture. Even though we took the abovementioned measures, it cannot be completely ruled out that that the procedures were stressful for some of the infants and their parents. However, some parents enjoyed the support and close contact with the experienced study nurse, and the possibility of getting an appointment with the study doctors in case of illness of the infant.
General discussion
56
Strengths and weaknesses of the study This study investigates the effects of feeding LF19 on clinical outcomes as well as development of gut microbial composition and immune system maturation. A strength of the study design is that we followed infants prospectively during the weaning period. A strength in the results is the very low drop-out rate and the high number of collected blood and fecal samples available for analysis. The diagnosis of eczema was based on questionnaires and diaries. We used a previously validated definition of eczema demonstrated to have high sensitivity (92%) and specificity (100%) compared to clinical diagnosis by a dermatologist (185). A weakness of the study is that we have no data on eczema severity. Alternatively, a clincial examination and classification of eczema severity using the SCORAD index could have been applied. On the other hand, assessing the cumulative incidence as done here instead of point prevalence could actually be a strength. If the children are examined only once, eczema might be missed due to the typically relapsing course of the disease and that some children might have outgrown the eczema by that age. Ultimately, repeated clinical examinations with assessment of eczema severity and collection of data from diaries and questionnaires could have been used. In the diagnosis of CMA infants were put on a cow’s milk protein elimination diet and the subsequent food challenge was open. In general, infants rarely report subjective symptoms, but the challenge being open and not blinded might have led to an over-diagnosis (57). Another weakness is the comparatively low statistical power for the clinical outcome variables. A larger study sample would have allowed us to assess the effects of LF19 on overall infections, respiratory allergies, IgE sensitization and to undertake subgroup-analyses distinguishing between IgE and non-IgE mediated eczema. Also, trends of probiotic effects on T cell function might have been more evident with a larger study sample. Regarding ethical considerations, the study protocol might have been demanding for some families. In the majority of cases, the research nurses delivered the cereals and visited the families in their homes for the collection of diaries, completion of questionnaires, anthropometric measures and venous punctures. The venous puncture that could be potentially stressful for the infant was done in a familial environment, thus reducing the tension of both infants and their parents. Anaesthetic cream was applied before the venous puncture. Even though we took the abovementioned measures, it cannot be completely ruled out that that the procedures were stressful for some of the infants and their parents. However, some parents enjoyed the support and close contact with the experienced study nurse, and the possibility of getting an appointment with the study doctors in case of illness of the infant.
General discussion
57
Future aspects We have gained some knowledge and even more questions have arisen. Investigations regarding aspects of the ecosystem in gut and the intricate interplay with the immune system are faced by their enormous complexity. However, in the near future we will try to add some more pieces to the puzzle. First, a more in-depth analysis of gut microbial composition and the effects by LF19 thereon in this study population is underway. We will further investigate the impact by LF19 on regulatory T cell function in this material, as well as markers of inflammation. In addition, it is of outmost necessity to follow these children and investigate if the preventive effects on eczema extend to school-age with effects on immune programming and respiratory allergies.
General discussion
57
Future aspects We have gained some knowledge and even more questions have arisen. Investigations regarding aspects of the ecosystem in gut and the intricate interplay with the immune system are faced by their enormous complexity. However, in the near future we will try to add some more pieces to the puzzle. First, a more in-depth analysis of gut microbial composition and the effects by LF19 thereon in this study population is underway. We will further investigate the impact by LF19 on regulatory T cell function in this material, as well as markers of inflammation. In addition, it is of outmost necessity to follow these children and investigate if the preventive effects on eczema extend to school-age with effects on immune programming and respiratory allergies.
Conclusions
58
CONCLUSIONS The early gut microbial composition is likely to impact on the developing immune system, both locally in the gut and systemically. During weaning, there is a major increase in exposure to food antigens and a diversifying gut microbiota, challenging the developing immune system. In the present study we attempted to maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition with potential immune-programming effects, by adding the probiotic bacterium LF19 to weaning cereals. The intervention was successful and maintained high fecal lactobacilli counts during weaning in the probiotic group. Feeding LF19 was safe with no observed adverse effects. Infants in both groups demonstrated specific as well as global immune system maturation with increased capacity to express both Th1 and Th2 cytokines. However, we observed no preventive effects on infections, but a slight reduction in days with antibiotic treatment. Furthermore, feeding LF19 reduced the risk of eczema in conjunction with an enhanced Th1/Th2 ratio following polyclonal T cell activation. We also observed functional differences in the gut microbiota induced by LF19, with possible effects on gut barrier function. Thus, we suggest that the reduced risk of eczema in the probiotic group partly depends on effects of LF19 on T cell function and partly by increased gut mucosal integrity. Finally, it is hypothesized that the gut microbiota and the developing immune system can be modulated by administration of probiotic bacteria to infants. The findings of the present thesis support this hypothesis, and demonstrate that probiotic feeding initiated during weaning may impact upon gut microbial function, T cell function in adaptive immunity and reduce the risk of eczema.
Conclusions
58
CONCLUSIONS The early gut microbial composition is likely to impact on the developing immune system, both locally in the gut and systemically. During weaning, there is a major increase in exposure to food antigens and a diversifying gut microbiota, challenging the developing immune system. In the present study we attempted to maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition with potential immune-programming effects, by adding the probiotic bacterium LF19 to weaning cereals. The intervention was successful and maintained high fecal lactobacilli counts during weaning in the probiotic group. Feeding LF19 was safe with no observed adverse effects. Infants in both groups demonstrated specific as well as global immune system maturation with increased capacity to express both Th1 and Th2 cytokines. However, we observed no preventive effects on infections, but a slight reduction in days with antibiotic treatment. Furthermore, feeding LF19 reduced the risk of eczema in conjunction with an enhanced Th1/Th2 ratio following polyclonal T cell activation. We also observed functional differences in the gut microbiota induced by LF19, with possible effects on gut barrier function. Thus, we suggest that the reduced risk of eczema in the probiotic group partly depends on effects of LF19 on T cell function and partly by increased gut mucosal integrity. Finally, it is hypothesized that the gut microbiota and the developing immune system can be modulated by administration of probiotic bacteria to infants. The findings of the present thesis support this hypothesis, and demonstrate that probiotic feeding initiated during weaning may impact upon gut microbial function, T cell function in adaptive immunity and reduce the risk of eczema.
Populärvetenskaplig sammanfattning
59
POPULÄRVETENSKAPLIG SAMMANFATTNING Bakgrund: Tarmfloran och immunförsvaret är i nära samverkan. En tarmflora rik på laktobaciller och bifidobakterier är typisk för ammade men inte för ersättningsuppfödda barn. Probiotika, främst mjölksyra-bildande laktobaciller och bifidobakterier, är ofarliga hälsobefrämjande bakterier som tillförs via livsmedel eller i annan form. Probiotika har föreslagits ha effekt vid olika sjukdomstillstånd genom att stabilisera tarmfloran och tarmens barriärfunktion och/eller kontrollera inflammation. Med få undantag saknas kontrollerade kliniska prövningar som styrker påstådd verkan av probiotika. Den mest påtagliga effekten ses vid virusorsakad magsjuka, där studier har visat att intag av vissa laktobacillstammar kan förkorta diarréperioden med ett knappt dygn. Det finns skillnader i tarmflorans sammansättning mellan friska barn och barn med allergi. Immunförsvaret kan delas in i T-hjälparcell typ 1- och 2-svar (Th1- och Th2-svar) vilka normalt ska vara i balans. T-hjälparcellerna styr immunceller via utsöndring av sk cytokiner. Th1-svar är viktigt för den cellförmedlade immuniteten och ett förstärkt svar kan sättas i samband med autoimmuna sjukdomar. Th2-svar är å andra sidan viktigt för den antikroppsförmedlade immuniteten och ett för kraftigt Th2-svar är typiskt vid allergiska sjukdomar. Normalt finns en balans mellan Th1- och Th2-svar som förhindrar för kraftfulla reaktioner. Hos det nyfödda barnet dominerar dock Th2-svar. Några studier har visat att behandling med vissa laktobacill-stammar kan minska eksemets svårighetsgrad och återställa Th1/Th2-balansen hos barn med eksem. I ett fåtal studier har man undersökt om probiotika-tillförsel tidigt i livet kan förebygga insjuknande i allergier. Resultaten från dessa studier är inte entydiga. Syfte: Syftet med vår studie var att undersöka om daglig tillförsel av den probiotiska bakterien Lactobacillus F19 under avvänjningen kunde bidra till att bibehålla den gynnsamma tarmflora amning ger upphov till och reglera immunförsvarsbalansen med minskad allergirisk som följd. Metod: Friska spädbarn lottades till intag av barngröt med (89 barn) eller utan probiotika (sk placebo, 90 barn) från 4 till 13 månaders ålder. Föräldrarna förde dagbok över grötintag, amningstillfällen och sjukdomssymtom. En forskningssjuksköterska intervjuade dem varje månad avseende sjukdomar, läkemedelsbehandling och allergiutveckling. Tillväxten kontrollerades. I avföringsprov analyserades förekomsten av Lactobacillus F19 och mönstret av korta fettsyror som markör för tarmflorans funktion. I blodprov mättes antikroppar mot difteri, stelkramp och Haemophilus influenzae typ b. Fördelningen av vita blodkroppar och cytokinsvar på isolerat mRNA från vita blodkroppar som markörer för Th1- och Th2-svar studerades. IgE-antikroppar mättes. Huvudresultat: Det fanns inga biverkningar av att tillföra Lactobacillus F19 i barngröt, utan snarare gynnsamma effekter. Färre barn hade eller hade haft eksem vid 13 månaders ålder i probiotika- jämfört med placebogruppen och de hade också mer laktobaciller i avföringen under studietiden. De barn som regelbundet åt gröt med probiotika hade ett förändrat mönster av korta fettsyror i avföringen jämfört
Populärvetenskaplig sammanfattning
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POPULÄRVETENSKAPLIG SAMMANFATTNING Bakgrund: Tarmfloran och immunförsvaret är i nära samverkan. En tarmflora rik på laktobaciller och bifidobakterier är typisk för ammade men inte för ersättningsuppfödda barn. Probiotika, främst mjölksyra-bildande laktobaciller och bifidobakterier, är ofarliga hälsobefrämjande bakterier som tillförs via livsmedel eller i annan form. Probiotika har föreslagits ha effekt vid olika sjukdomstillstånd genom att stabilisera tarmfloran och tarmens barriärfunktion och/eller kontrollera inflammation. Med få undantag saknas kontrollerade kliniska prövningar som styrker påstådd verkan av probiotika. Den mest påtagliga effekten ses vid virusorsakad magsjuka, där studier har visat att intag av vissa laktobacillstammar kan förkorta diarréperioden med ett knappt dygn. Det finns skillnader i tarmflorans sammansättning mellan friska barn och barn med allergi. Immunförsvaret kan delas in i T-hjälparcell typ 1- och 2-svar (Th1- och Th2-svar) vilka normalt ska vara i balans. T-hjälparcellerna styr immunceller via utsöndring av sk cytokiner. Th1-svar är viktigt för den cellförmedlade immuniteten och ett förstärkt svar kan sättas i samband med autoimmuna sjukdomar. Th2-svar är å andra sidan viktigt för den antikroppsförmedlade immuniteten och ett för kraftigt Th2-svar är typiskt vid allergiska sjukdomar. Normalt finns en balans mellan Th1- och Th2-svar som förhindrar för kraftfulla reaktioner. Hos det nyfödda barnet dominerar dock Th2-svar. Några studier har visat att behandling med vissa laktobacill-stammar kan minska eksemets svårighetsgrad och återställa Th1/Th2-balansen hos barn med eksem. I ett fåtal studier har man undersökt om probiotika-tillförsel tidigt i livet kan förebygga insjuknande i allergier. Resultaten från dessa studier är inte entydiga. Syfte: Syftet med vår studie var att undersöka om daglig tillförsel av den probiotiska bakterien Lactobacillus F19 under avvänjningen kunde bidra till att bibehålla den gynnsamma tarmflora amning ger upphov till och reglera immunförsvarsbalansen med minskad allergirisk som följd. Metod: Friska spädbarn lottades till intag av barngröt med (89 barn) eller utan probiotika (sk placebo, 90 barn) från 4 till 13 månaders ålder. Föräldrarna förde dagbok över grötintag, amningstillfällen och sjukdomssymtom. En forskningssjuksköterska intervjuade dem varje månad avseende sjukdomar, läkemedelsbehandling och allergiutveckling. Tillväxten kontrollerades. I avföringsprov analyserades förekomsten av Lactobacillus F19 och mönstret av korta fettsyror som markör för tarmflorans funktion. I blodprov mättes antikroppar mot difteri, stelkramp och Haemophilus influenzae typ b. Fördelningen av vita blodkroppar och cytokinsvar på isolerat mRNA från vita blodkroppar som markörer för Th1- och Th2-svar studerades. IgE-antikroppar mättes. Huvudresultat: Det fanns inga biverkningar av att tillföra Lactobacillus F19 i barngröt, utan snarare gynnsamma effekter. Färre barn hade eller hade haft eksem vid 13 månaders ålder i probiotika- jämfört med placebogruppen och de hade också mer laktobaciller i avföringen under studietiden. De barn som regelbundet åt gröt med probiotika hade ett förändrat mönster av korta fettsyror i avföringen jämfört
Populärvetenskaplig sammanfattning
60
med de barn som ätit placebogröten. Vidare såg vi att de barn som fått probiotika hade en högre Th1/Th2-kvot och därmed bättre balans. Tolkningen av dessa resultat skulle kunna vara att den lägre förekomsten av eksem är förmedlad dels via en förändrad funktion av tarmfloran med effekter på tarmens barriärfunktion, dels via ett förändrat immunsvar. Däremot fanns det inga skillnader mellan grupperna i nivåer eller förekomst av IgE-antikroppar, dvs antikroppar som är drivande i den allergiska processen. Vi såg också att de barn som fick probiotika och hade ammats kortare än 6 månader, svarade med starkare antikroppssvar mot difteri under den tid som vaccinationerna pågick. Däremot var det ingen skillnad mellan grupperna när barnen hade fått alla sprutor, dvs barnen i bägge grupperna hade ett gott antikroppsskydd. Till skillnad från vissa andra studier kunde vi inte påvisa någon förebyggande effekt av probiotika på infektioner. Infektioner i denna åldersgrupp är framför allt virusinfektioner. En förklaring till avsaknaden av effekt kan vara att barnen i denna studie ammades längre än i de studier där probiotikaintag förhindrade infektioner. Det är klarlagt i flera studier att amning i sig skyddar mot infektioner. Andra förklaringar kan vara att vi använt en annan probiotisk bakterie och/eller att förekomsten av infektioner hos dessa barn som ännu inte börjat förskolan är så pass låg att det är svårt att kunna påvisa statistiska skillnader. Däremot hade barnen i probiotikagruppen något färre dagar med antibiotikabehandling än placebogruppen som skulle kunna tyda på att LF19 skyddar mot bakteriella infektioner och sekundärinfektioner. Slutsats: Det är säkert att tillföra den probiotiska bakterien Lactobacillus F19 i barngröt under avvänjningen. Tillförsel av Lactobacillus F19 minskar förekomsten av eksem, och våra resultat tyder på att förändringar i tarmflorans funktion samt en gynnsam immunförsvarsbalans skulle kunna förklara denna effekt. Däremot påvisade vi inte några effekter av probiotikatillförsel på nivåer eller förekomst av IgE-antikroppar eller insjuknande i infektioner. Det är viktigt att i en förnyad undersökning av barnen studera om de gynnsamma effekterna av tillförsel av Lactobacillus F19 kvarstår i skolåldern och om de även innefattar astma och hösnuva.
Populärvetenskaplig sammanfattning
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med de barn som ätit placebogröten. Vidare såg vi att de barn som fått probiotika hade en högre Th1/Th2-kvot och därmed bättre balans. Tolkningen av dessa resultat skulle kunna vara att den lägre förekomsten av eksem är förmedlad dels via en förändrad funktion av tarmfloran med effekter på tarmens barriärfunktion, dels via ett förändrat immunsvar. Däremot fanns det inga skillnader mellan grupperna i nivåer eller förekomst av IgE-antikroppar, dvs antikroppar som är drivande i den allergiska processen. Vi såg också att de barn som fick probiotika och hade ammats kortare än 6 månader, svarade med starkare antikroppssvar mot difteri under den tid som vaccinationerna pågick. Däremot var det ingen skillnad mellan grupperna när barnen hade fått alla sprutor, dvs barnen i bägge grupperna hade ett gott antikroppsskydd. Till skillnad från vissa andra studier kunde vi inte påvisa någon förebyggande effekt av probiotika på infektioner. Infektioner i denna åldersgrupp är framför allt virusinfektioner. En förklaring till avsaknaden av effekt kan vara att barnen i denna studie ammades längre än i de studier där probiotikaintag förhindrade infektioner. Det är klarlagt i flera studier att amning i sig skyddar mot infektioner. Andra förklaringar kan vara att vi använt en annan probiotisk bakterie och/eller att förekomsten av infektioner hos dessa barn som ännu inte börjat förskolan är så pass låg att det är svårt att kunna påvisa statistiska skillnader. Däremot hade barnen i probiotikagruppen något färre dagar med antibiotikabehandling än placebogruppen som skulle kunna tyda på att LF19 skyddar mot bakteriella infektioner och sekundärinfektioner. Slutsats: Det är säkert att tillföra den probiotiska bakterien Lactobacillus F19 i barngröt under avvänjningen. Tillförsel av Lactobacillus F19 minskar förekomsten av eksem, och våra resultat tyder på att förändringar i tarmflorans funktion samt en gynnsam immunförsvarsbalans skulle kunna förklara denna effekt. Däremot påvisade vi inte några effekter av probiotikatillförsel på nivåer eller förekomst av IgE-antikroppar eller insjuknande i infektioner. Det är viktigt att i en förnyad undersökning av barnen studera om de gynnsamma effekterna av tillförsel av Lactobacillus F19 kvarstår i skolåldern och om de även innefattar astma och hösnuva.
Acknowledgements
61
ACKNOWLEDGEMENTS First of all, I would like to recognize all the infants and their families who participated in this study, making this project possible at all. Thank you! This thesis and the project behind it is a joint effort of a dynamic team of dedicated persons to whom I wish to express my deepest gratitude. Olle Hernell- my main supervisor for inviting me to explore this exciting area of research and for your visions, commitment, great scientific skills and support whenever needed. Marie-Louise Hammarström- my supervisor for your vast scientific experience, innovative ideas, fruitful discussions and support. Leif Gothefors- my supervisor for your contagious enthusiasm and for being a mentor both in the clinical and scientific fields. Sten Hammarström-for valuable advice and discussions during this project. Hans Stenlund-always ready to give expert advice in the area of Statistics and always with patience and great pedagogic skills. Gisela Dahlquist- for sharing advice along the way. Margareta Henriksson and RuthGerd Larsson- for invaluable contact with the infants and their families, for keeping the infants in the study and for successfully obtaining blood samples from invisibly thin blood vessels. Margareta Bäckman for helping out with the practicalities when the other research nurses needed a hand and for friendship and good cooperation in other projects. Yvonne Andersson- for your invaluable contribution to this project in numerous aspects, including your skill and thorough experience in the lab, advice and friendship over the years. Marianne Sjöstedt for the dedicated and skilful work with the qRT-PCR analyses. Anne Israelsson for designing the primers and probes for the qRT-PCR analyses. Helén Fält, Elisabeth Granström, Carina Lagerqvist, and Lotta Westman for your commitment, skilful assistance with laboratory analyses and useful comments. Helena Brännström, Helena Harding, Karin Moström, Anna Nordström and Ulla Norman for excellent assistance with administrative matters. Marta Granström for sharing your vast experience about infant vaccine responses, their methodology and for stimulating discussions. Ingrid Yones for skilful assistance with laboratory analyses.
Acknowledgements
61
ACKNOWLEDGEMENTS First of all, I would like to recognize all the infants and their families who participated in this study, making this project possible at all. Thank you! This thesis and the project behind it is a joint effort of a dynamic team of dedicated persons to whom I wish to express my deepest gratitude. Olle Hernell- my main supervisor for inviting me to explore this exciting area of research and for your visions, commitment, great scientific skills and support whenever needed. Marie-Louise Hammarström- my supervisor for your vast scientific experience, innovative ideas, fruitful discussions and support. Leif Gothefors- my supervisor for your contagious enthusiasm and for being a mentor both in the clinical and scientific fields. Sten Hammarström-for valuable advice and discussions during this project. Hans Stenlund-always ready to give expert advice in the area of Statistics and always with patience and great pedagogic skills. Gisela Dahlquist- for sharing advice along the way. Margareta Henriksson and RuthGerd Larsson- for invaluable contact with the infants and their families, for keeping the infants in the study and for successfully obtaining blood samples from invisibly thin blood vessels. Margareta Bäckman for helping out with the practicalities when the other research nurses needed a hand and for friendship and good cooperation in other projects. Yvonne Andersson- for your invaluable contribution to this project in numerous aspects, including your skill and thorough experience in the lab, advice and friendship over the years. Marianne Sjöstedt for the dedicated and skilful work with the qRT-PCR analyses. Anne Israelsson for designing the primers and probes for the qRT-PCR analyses. Helén Fält, Elisabeth Granström, Carina Lagerqvist, and Lotta Westman for your commitment, skilful assistance with laboratory analyses and useful comments. Helena Brännström, Helena Harding, Karin Moström, Anna Nordström and Ulla Norman for excellent assistance with administrative matters. Marta Granström for sharing your vast experience about infant vaccine responses, their methodology and for stimulating discussions. Ingrid Yones for skilful assistance with laboratory analyses.
Acknowledgements
62
Helena Käyhty for welcoming me in Helsinki and sharing your expertise in the field of serologic evaluations of vaccines. Leena Saarinen for excellent assistance with laboratory analyses and Anu Nurkka for useful disucssions. Elisabeth Norin for sharing your knowledge in the field of microbiology and helpful discussions. Anna-Karin Persson for skilful assistance with laboratory analyses. Catharina Tennefors and Lars-Börje Sjöberg at Semper AB for valuable contributions in the planning and performance of this project, including development of the study product and for support over the years. Ragne Fondén and Ulla Svensson at Arla Foods AB for support during this project and for interesting discussions about LF19. Janet Håkansson and Astrid Walles-Granberg for stimulating discussions and excellent assistance with laboratory analyses. Åsa Sullivan and Ann-Chatrin Palmgren for excellent assistance with laboratory analyses. Aamir Mukhdoomi-for meticulous work with data entry. Anna Möllsten, Luis Cobian and Michael Haney for useful advice on layout and editing of the frame text. Former and present colleagues at the Department of Pediatrics for sharing thoughts and advice over the years, especially, Anneli Ivarsson, Berit Kriström, Torbjörn Lind, Sussie Lindquist, Christian Möller, Solveig Petersen, Auste Pundziute-Lyckå, Olof Sandström, Sven-Arne Silfverdal and Inger Öhlund. Magnus Domellöf, Göte Forsberg, Leif Gothefors, Ulrika Norén Nyström, Annika Rydberg, Svante Sjöstedt and Anna Winberg, who in addition to good advice also shared friendship, songs and laughter. My close friends, in particular Ulrika NN for being excellent company when travelling this path and Mia Malby for reminding me to enjoy the travel. My parents Benita and Stig, for love and support, and my brother Ulf and his family for being there and putting up a supportive attitude during the last hectic weeks of thesis writing. My companion in life Björn, for standing resolutely by my side for better and for worse and my most precious sons David and Lukas for reminding me every day “what it is all about”.
Acknowledgements
62
Helena Käyhty for welcoming me in Helsinki and sharing your expertise in the field of serologic evaluations of vaccines. Leena Saarinen for excellent assistance with laboratory analyses and Anu Nurkka for useful disucssions. Elisabeth Norin for sharing your knowledge in the field of microbiology and helpful discussions. Anna-Karin Persson for skilful assistance with laboratory analyses. Catharina Tennefors and Lars-Börje Sjöberg at Semper AB for valuable contributions in the planning and performance of this project, including development of the study product and for support over the years. Ragne Fondén and Ulla Svensson at Arla Foods AB for support during this project and for interesting discussions about LF19. Janet Håkansson and Astrid Walles-Granberg for stimulating discussions and excellent assistance with laboratory analyses. Åsa Sullivan and Ann-Chatrin Palmgren for excellent assistance with laboratory analyses. Aamir Mukhdoomi-for meticulous work with data entry. Anna Möllsten, Luis Cobian and Michael Haney for useful advice on layout and editing of the frame text. Former and present colleagues at the Department of Pediatrics for sharing thoughts and advice over the years, especially, Anneli Ivarsson, Berit Kriström, Torbjörn Lind, Sussie Lindquist, Christian Möller, Solveig Petersen, Auste Pundziute-Lyckå, Olof Sandström, Sven-Arne Silfverdal and Inger Öhlund. Magnus Domellöf, Göte Forsberg, Leif Gothefors, Ulrika Norén Nyström, Annika Rydberg, Svante Sjöstedt and Anna Winberg, who in addition to good advice also shared friendship, songs and laughter. My close friends, in particular Ulrika NN for being excellent company when travelling this path and Mia Malby for reminding me to enjoy the travel. My parents Benita and Stig, for love and support, and my brother Ulf and his family for being there and putting up a supportive attitude during the last hectic weeks of thesis writing. My companion in life Björn, for standing resolutely by my side for better and for worse and my most precious sons David and Lukas for reminding me every day “what it is all about”.
References
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144. Upham JW, Rate A, Rowe J, Kusel M, Sly PD, Holt PG. Dendritic cell immaturity during infancy restricts the capacity to express vaccine-specific T-cell memory. Infect Immun. 2006 Feb;74(2):1106-12. 145. Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P, et al. Modulation of human dendritic cell phenotype and function by probiotic bacteria. Gut. 2004 Nov;53(11):1602-9. 146. Benyacoub J, Czarnecki-Maulden GL, Cavadini C, Sauthier T, Anderson RE, Schiffrin EJ, et al. Supplementation of food with Enterococcus faecium (SF68) stimulates immune functions in young dogs. J Nutr. 2003 Apr;133(4):1158-62. 147. Taylor AL, Hale J, Wiltschut J, Lehmann H, Dunstan JA, Prescott SL. Effects of probiotic supplementation for the first 6 months of life on allergen- and vaccine-specific immune responses. Clin Exp Allergy. 2006 Oct;36(10):1227-35. 148. Kukkonen K, Nieminen T, Poussa T, Savilahti E, Kuitunen M. Effect of probiotics on vaccine antibody responses in infancy--a randomized placebo-controlled double-blind trial. Pediatr Allergy Immunol. 2006 Sep;17(6):416-21. 149. Siegrist CA, Cordova M, Brandt C, Barrios C, Berney M, Tougne C, et al. Determinants of infant responses to vaccines in presence of maternal antibodies. Vaccine. 1998 Aug-Sep;16(14-15):1409-14. 150. Kalliomäki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet. 2003 May 31;361(9372):1869-71. 151. Kalliomäki M, Salminen S, Poussa T, Isolauri E. Probiotics during the first 7 years of life: a cumulative risk reduction of eczema in a randomized, placebo-controlled trial. J Allergy Clin Immunol. 2007 Apr;119(4):1019-21. 152. Osborn DA, Sinn JK. Probiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database Syst Rev. 2007(4):CD006475. 153. Lee J, Seto D, Bielory L. Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. J Allergy Clin Immunol. 2008 Jan;121(1):116-21 e11. 154. Kramer MS, Kakuma R. Maternal dietary antigen avoidance during pregnancy or lactation, or both, for preventing or treating atopic disease in the child. Cochrane Database Syst Rev. 2006;3:CD000133. 155. Greer FR, Sicherer SH, Burks AW. Effects of early nutritional interventions on the development of atopic disease in infants and children: the role of maternal dietary restriction, breastfeeding, timing of introduction of complementary foods, and hydrolyzed formulas. Pediatrics. 2008 Jan;121(1):183-91. 156. Borish LC, Steinke JW. 2. Cytokines and chemokines. J Allergy Clin Immunol. 2003 Feb;111(2 Suppl):S460-75. 157. Böttcher MF, Jenmalm MC, Garofalo RP, Björkstén B. Cytokines in breast milk from allergic and nonallergic mothers. Pediatr Res. 2000 Jan;47(1):157-62. 158. Hamelmann E, Beyer K, Gruber C, Lau S, Matricardi PM, Nickel R, et al. Primary prevention of allergy: avoiding risk or providing protection? Clin Exp Allergy. 2008 Feb;38(2):233-45. 159. Matsuzaki T, Yamazaki R, Hashimoto S, Yokokura T. The effect of oral feeding of Lactobacillus casei strain Shirota on immunoglobulin E production in mice. J Dairy Sci. 1998 Jan;81(1):48-53. 160. Niers LE, Timmerman HM, Rijkers GT, van Bleek GM, van Uden NO, Knol EF, et al. Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines. Clin Exp Allergy. 2005 Nov;35(11):1481-9. 161. Kulig M, Tacke U, Forster J, Edenharter G, Bergmann R, Lau S, et al. Serum IgE levels during the first 6 years of life. J Pediatr. 1999 Apr;134(4):453-8. 162. Kulig M, Bergmann R, Klettke U, Wahn V, Tacke U, Wahn U. Natural course of sensitization to food and inhalant allergens during the first 6 years of life. J Allergy Clin Immunol. 1999 Jun;103(6):1173-9. 163. Tjellström B, Stenhammar L, Högberg L, Fälth-Magnusson K, Magnusson KE, Midtvedt T, et al. Gut microflora associated characteristics in children with celiac disease. Am J Gastroenterol. 2005 Dec;100(12):2784-8. 164. Isolauri E, Majamaa H, Arvola T, Rantala I, Virtanen E, Arvilommi H. Lactobacillus casei strain GG reverses increased intestinal permeability induced by cow milk in suckling rats. Gastroenterology. 1993 Dec;105(6):1643-50. 165. Rosenfeldt V, Benfeldt E, Valerius NH, Paerregaard A, Michaelsen KF. Effect of probiotics on gastrointestinal symptoms and small intestinal permeability in children with atopic dermatitis. J Pediatr. 2004 Nov;145(5):612-6.
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166. Prescott SL, Björkstén B. Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol. 2007 Aug;120(2):255-62. 167. Böttcher MF, Abrahamsson TR, Fredriksson M, Jakobsson T, Björkstén B. Low breast milk TGF-beta2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy. Pediatr Allergy Immunol. 2008 Jan 22, e-pub ahead of print. 168. Gueimonde M, Sakata S, Kalliomaki M, Isolauri E, Benno Y, Salminen S. Effect of maternal consumption of lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in neonates. J Pediatr Gastroenterol Nutr. 2006 Feb;42(2):166-70. 169. Kopp MV, Goldstein M, Dietschek A, Sofke J, Heinzmann A, Urbanek R. Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-gamma release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates. Clin Exp Allergy. 2007 Dec 20. 170. Shadid R, Haarman M, Knol J, Theis W, Beermann C, Rjosk-Dendorfer D, et al. Effects of galactooligosaccharide and long-chain fructooligosaccharide supplementation during pregnancy on maternal and neonatal microbiota and immunity--a randomized, double-blind, placebo-controlled study. Am J Clin Nutr. 2007 Nov;86(5):1426-37. 171. Marschan E, Honkanen J, Kukkonen K, Kuitunen M, Savilahti E, Vaarala O. Increased activation of GATA-3, IL-2 and IL-5 of cord blood mononuclear cells in infants with IgE sensitization. Pediatr Allergy Immunol. 2008 Mar;19(2):132-9. 172. Larsson AK, Nilsson C, Höglind A, Sverremark-Ekström E, Lilja G, Troye-Blomberg M. Relationship between maternal and child cytokine responses to allergen and phytohaemagglutinin 2 years after delivery. Clin Exp Immunol. 2006 Jun;144(3):401-8. 173. Rautava S, Kalliomäki M, Isolauri E. Probiotics during pregnancy and breast-feeding might confer immunomodulatory protection against atopic disease in the infant. J Allergy Clin Immunol. 2002 Jan;109(1):119-21. 174. Inoue R, Nishio A, Fukushima Y, Ushida K. Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga. Br J Dermatol. 2007 Mar;156(3):499-509. 175. Sad S, Mosmann TR. Single IL-2-secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype. J Immunol. 1994 Oct 15;153(8):3514-22. 176. Nakamura T, Kamogawa Y, Bottomly K, Flavell RA. Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells. J Immunol. 1997 Feb 1;158(3):1085-94. 177. Pohjavuori E, Viljanen M, Korpela R, Kuitunen M, Tiittanen M, Vaarala O, et al. Lactobacillus GG effect in increasing IFN-gamma production in infants with cow's milk allergy. J Allergy Clin Immunol. 2004 Jul;114(1):131-6. 178. Prescott SL, Dunstan JA, Hale J, Breckler L, Lehmann H, Weston S, et al. Clinical effects of probiotics are associated with increased interferon-gamma responses in very young children with atopic dermatitis. Clin Exp Allergy. 2005 Dec;35(12):1557-64. 179. Marschan E, Kuitunen M, Kukkonen K, Poussa T, Sarnesto A, Haahtela T, et al. Probiotics in infancy induce protective immune profiles that are characteristic for chronic low-grade inflammation. Clin Exp Allergy. 2008; 38:611-8.180. Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science. 2002 Apr 19;296(5567):490-4. 181. Di Giacinto C, Marinaro M, Sanchez M, Strober W, Boirivant M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. J Immunol. 2005 Mar 15;174(6):3237-46. 182. Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev. 2001 Aug;182:68-79. 183. Taylor AL, Hale J, Hales BJ, Dunstan JA, Thomas WR, Prescott SL. FOXP3 mRNA expression at 6 months of age is higher in infants who develop atopic dermatitis, but is not affected by giving probiotics from birth. Pediatr Allergy Immunol. 2007 Feb;18(1):10-9. 184. Taylor A, Hale J, Wiltschut J, Lehmann H, Dunstan JA, Prescott SL. Evaluation of the effects of probiotic supplementation from the neonatal period on innate immune development in infancy. Clin Exp Allergy. 2006 Oct;36(10):1218-26. 185. Böhme M, Lannerö E, Wickman M, Nordvall SL, Wahlgren CF. Atopic dermatitis and concomitant disease patterns in children up to two years of age. Acta Derm Venereol. 2002;82(2):98-103.
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166. Prescott SL, Björkstén B. Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol. 2007 Aug;120(2):255-62. 167. Böttcher MF, Abrahamsson TR, Fredriksson M, Jakobsson T, Björkstén B. Low breast milk TGF-beta2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy. Pediatr Allergy Immunol. 2008 Jan 22, e-pub ahead of print. 168. Gueimonde M, Sakata S, Kalliomaki M, Isolauri E, Benno Y, Salminen S. Effect of maternal consumption of lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in neonates. J Pediatr Gastroenterol Nutr. 2006 Feb;42(2):166-70. 169. Kopp MV, Goldstein M, Dietschek A, Sofke J, Heinzmann A, Urbanek R. Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-gamma release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates. Clin Exp Allergy. 2007 Dec 20. 170. Shadid R, Haarman M, Knol J, Theis W, Beermann C, Rjosk-Dendorfer D, et al. Effects of galactooligosaccharide and long-chain fructooligosaccharide supplementation during pregnancy on maternal and neonatal microbiota and immunity--a randomized, double-blind, placebo-controlled study. Am J Clin Nutr. 2007 Nov;86(5):1426-37. 171. Marschan E, Honkanen J, Kukkonen K, Kuitunen M, Savilahti E, Vaarala O. Increased activation of GATA-3, IL-2 and IL-5 of cord blood mononuclear cells in infants with IgE sensitization. Pediatr Allergy Immunol. 2008 Mar;19(2):132-9. 172. Larsson AK, Nilsson C, Höglind A, Sverremark-Ekström E, Lilja G, Troye-Blomberg M. Relationship between maternal and child cytokine responses to allergen and phytohaemagglutinin 2 years after delivery. Clin Exp Immunol. 2006 Jun;144(3):401-8. 173. Rautava S, Kalliomäki M, Isolauri E. Probiotics during pregnancy and breast-feeding might confer immunomodulatory protection against atopic disease in the infant. J Allergy Clin Immunol. 2002 Jan;109(1):119-21. 174. Inoue R, Nishio A, Fukushima Y, Ushida K. Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga. Br J Dermatol. 2007 Mar;156(3):499-509. 175. Sad S, Mosmann TR. Single IL-2-secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype. J Immunol. 1994 Oct 15;153(8):3514-22. 176. Nakamura T, Kamogawa Y, Bottomly K, Flavell RA. Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells. J Immunol. 1997 Feb 1;158(3):1085-94. 177. Pohjavuori E, Viljanen M, Korpela R, Kuitunen M, Tiittanen M, Vaarala O, et al. Lactobacillus GG effect in increasing IFN-gamma production in infants with cow's milk allergy. J Allergy Clin Immunol. 2004 Jul;114(1):131-6. 178. Prescott SL, Dunstan JA, Hale J, Breckler L, Lehmann H, Weston S, et al. Clinical effects of probiotics are associated with increased interferon-gamma responses in very young children with atopic dermatitis. Clin Exp Allergy. 2005 Dec;35(12):1557-64. 179. Marschan E, Kuitunen M, Kukkonen K, Poussa T, Sarnesto A, Haahtela T, et al. Probiotics in infancy induce protective immune profiles that are characteristic for chronic low-grade inflammation. Clin Exp Allergy. 2008; 38:611-8.180. Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science. 2002 Apr 19;296(5567):490-4. 181. Di Giacinto C, Marinaro M, Sanchez M, Strober W, Boirivant M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. J Immunol. 2005 Mar 15;174(6):3237-46. 182. Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev. 2001 Aug;182:68-79. 183. Taylor AL, Hale J, Hales BJ, Dunstan JA, Thomas WR, Prescott SL. FOXP3 mRNA expression at 6 months of age is higher in infants who develop atopic dermatitis, but is not affected by giving probiotics from birth. Pediatr Allergy Immunol. 2007 Feb;18(1):10-9. 184. Taylor A, Hale J, Wiltschut J, Lehmann H, Dunstan JA, Prescott SL. Evaluation of the effects of probiotic supplementation from the neonatal period on innate immune development in infancy. Clin Exp Allergy. 2006 Oct;36(10):1218-26. 185. Böhme M, Lannerö E, Wickman M, Nordvall SL, Wahlgren CF. Atopic dermatitis and concomitant disease patterns in children up to two years of age. Acta Derm Venereol. 2002;82(2):98-103.
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