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Human breast milk provides immunological protection for newborns and infants against common childhood diseases in addition to its well-documented nutritional value. During the last few decades there is increasing evidence of a possible relationship between high risk of developing autoimmune diseases and allergies and decreased rates and/or duration of breastfeeding. This review summarizes evidence in relation to the effects of breastfeeding on the development of the neonatal immune system and compares compositions of gut microflora and the development of the thymus between breastfed and non-breastfed infants. It also discusses protective mechanisms of those cells in HBM survival, such as high stomach pH and gut permeability of the newborn and the presence of antiproteases and fat globules in HBM.
*Abbreviations: HBM, human breast milk; sIgA, secretory IgA; NEC, necrotizing enterocolitis; GIT, gastrointestinal tract; IL-7, interleukin-7; TH2, T helper 2 cell; TNF-Î±, tumour necrosis factor- Î±; LOS, late-onset septicaemia; G-CSF, granulocyte colony-stimulating factor; MFGs, milk fat globules; MFGM, milk fat globule membrane.
Although formula milk attempts to duplicate the composition of HBM, HBM is a highly complex secretion and undergoes dynamic changes over the lactation period. It provides optimal nutrition and various immunological components including living immune cells and bioactive proteins for neonates and infants in the vulnerable early months of life. The cellular components in HBM are around 14,000 cells/ml, mostly epithelial and immune cells, and the bioactive proteins include hormones, enzymes, growth factors, anticancer, anti-inflammatory, immunomodulating and anti-infectious factors. A high proportion of specific and non-specific immunological components are passively transferred through HBM to compensate for the synthesis deficiency of the infant during the first year of life such as secretory IgA (sIgA). Such cells and bioactive proteins are not available in formula milk.
There are convincing arguments relating to infants who receive exclusive breastfeeding in the first 6 months of life that show they have better development and more protection against common childhood infectious and gastrointestinal diseases including sepsis, pneumonia, otitis media, diarrhoea and necrotizing enterocolitis (NEC), than non-breastfed infants. Kramer and Kakuma found in a total of 22 countries, that infants who were exclusively breastfed for up to 6 months had no deficits in their growth and development. Another Norwegian cohort of preterm infants showed that infants who had early establishment of enteral feeding with HBM within the second week of life remarkably reduced relative risk of 3.7 for late-onset septicaemia (LOS).
There are substantial arguments for the potential benefits of HBM, not only in infancy but also in later life. A largely inconclusive argument exists related to the benefits of exclusive breastfeeding as providing better protection for allergy and autoimmune diseases. In other words, infants who do receive enough HBM during infancy have less chance of developing these diseases in the future. Recently, another finding was that of multiple lines of undifferentiated stem cells identified in human lactating mammary glands and acquired via HBM, which indicates potential for positive health outcomes through the lifespan.
However, the question arises as to how viable cells and proteins from HBM survive digestion and reach the intestine of the infant. Do immunological components from HBM enhance the development of the immune system for the offspring? The migration pattern of cells in HBM from the mother to the infant is largely unknown. One concern in regard to this topic is the difficulty of providing solid evidence using human infants, due to practical and ethical considerations. Therefore, most information contributing to answering this question comes from experiments from animals and comparisons between exclusively breast-fed infants and formula-fed infants. This brief review focuses on the evidence of cell survival after digestion and hypothesizes the support mechanisms for survival of milk proteins and cellular components in the gastrointestinal tract (GIT).
Evidence of cell survival in the breastfed infant
There are animal studies to suggest that immunological cells from HBM can attach to and traverse the neonatal GIT, and can be transported via the lymph ducts to the mesenteric lymph nodes. Using an autoradiographic study of intestinal tissue in rats and lambs, Sheldrake and Husband found that radiolabelled milk lymphocytes are absorbed into the gut mucosa and taken up into the circulation. A similar result was also observed by Tuboly et al in pigs. Further, labelled lymphoid cells are effectively taken up into a newborn lamb's lymph circulation regardless of the route, including injection or consumption via the digestive tract.
It is not clear whether cells from breast milk provide protection locally at the GIT level or whether they have an ability to confer systemic immune protection. Evidence from animal studies has suggested that cells from breast milk reach other organs of the offspring after absorption from the GIT. Jain et al confirmed in their study that labelled human milk leukocytes in premature neonatal baboons were found primarily in the stomach and intestinal lumen followed by lower levels in the spleen and liver, and the lowest levels in bone marrow. There was a similar result repeated by Liebler-Tenorio and colleagues from newborn calves. Labelled macrophages, after having been fed to newborn mice, remained alive for at least 4 hours in the GIT and localized in the mucosal tissue and in some cases in the spleen. These macrophages in HBM were hypothesized to perform an immunological supportive function as antigen-presenting cells in the local immune response of the gut.
The neonatal immune system is naive because it has not been exposed to antigens and as a result, has deficiencies in memory T cells. However, convincing studies demonstrate that the memory T cells from the mother may be transferred to the offspring via HBM to compensate passively immunological memory T cells of the offspring. Wirt et al found that activated T cells are the primary population of lymphocytes in HBM, including CD4+ and CD8+ T cells, that have a higher population in the HBM than in human blood. The authors speculate that activation of T cells is due to being stimulated by tumour necrosis factor (TNF)-Î± and other cytokines and it is conversely possible that these activated T cells may produce cytokines, including TNF-Î±, found in HBM. These memory T cells pass through the stomach and intestine of the infant, particularly prior to the development of high gastric acidity which occurs after the first several days of life.
Composition of microflora
The gut-associated lymphoid tissue (GALT) in the intestine is thought the largest immune organ in the body, homing about 80% of the body's immune cells such as the majority of lymphocytes and other immune effector cells. The neonatal immune system is immature, therefore the lining of the gut which is the first barrier against the entry of exogenous pathogens and allergens is ineffective. HBM can enhance the immune system by the inoculation of the gut with microbiota, a complex microbial ecosystem composed of various strains of bacteria, protozoa and fungi. HBM helps the normal intestinal flora get settled and it counteracts potential pathogens.
There is increasing evidence to show striking differences in the composition of gut microflora between breast-fed and formula-fed infants. The intestine of breastfed infants is primarily colonized by higher numbers of Bifidobacteria which are considered beneficial bacteria. This bacterium seems to be found in children's microbiota, with both breast-fed and formula-fed infants. However, research suggests that in breastfed infants over 95% of the flora are Lactobacillus bifidus and Bifidobacterium spp., compared to formula-fed infants where only 40 to 60% of the flora is Bifidobacteria. Instead, formula-fed infants acquire a higher percentage of Gram-negative coliform bacteria, Bacteroides and others including Enterobacter, Enterococcus, Escherichia coli, and Clostridia than do breastfed infants.
Lactic acid bacteria and Bifidobacteria, which are predominately in the gut of breastfed infants, are thought to reduce the pathogenic potential of other bacteria in the gut by changing pH, producing certain antibiotic-like substances and/or reducing the invasive ability of pathogens. Breastfeeding confers several other positive effects including the inactivation of carcinogens as Lactobacillus and Bifido bacteria reduce tumour development and mucosal inflammatory activity, whereas Bacteroides and Clostridium increase the incidence and growth rate of colon tumours in animals. Lactobacillus provides benefits in reducing the numbers of infections by stimulating the production of antibodies. These probiotic bacteria are thought to be established by breastfeeding.
A large study of 957 infants in the Netherlands found an association between colonization with gut bacteria and the development of atopy within the first 2 years of life. Infants colonized with Escherichia coli had high risk of eczema. The high risk of developing recurrent wheeze, eczema, and allergic sensitization presented in infants colonized with Clostridium difficile compared with uncolonised infants. On the other hand, there is no association between these atopic outcomes and colonisation with Bifidobacteria, Bacteroides fragilis and Lactobacilli. Another prospective study of children from birth to 17 years found that exclusive breastfeeding provides significant protection, but the mechanism is unknown, against food allergies and eczema for the first 1 to 3 years and later against respiratory tract allergies. The prevalence of respiratory allergy increased to 65% at 17 year of age in infants who breastfed for less than 1 month of age. Recent clinical trials, but no conclusive studies, support the possibility of benefits from live microflora such as Lactobacilli and Bifidobacteria, suggesting they could be beneficial for allergy prevention or treatment in the future.
The thymus is a special organ for maturation of T lymphocytes and its differentiation is completed by 18 to 20 weeks of fetal life. In general, less than 10% of T lymphocytes can be permitted from the thymus to the circulation. For the last several decades, there is substantial evidence to suggest that breastfeeding increases the size of the thymus. A longitudinal study by Hasselbalch et al found that the thymus of exclusively breastfed infants was twice as large as the size of formula-fed infants at age 4 months when the thymus is growing. An additional finding was the correlation between birth weight and the size of the thymus. Low birth weight could decrease the relative size index of the thymus, which may cause infectious disease in infancy. Further, they found not only a strong connection between thymus sizes with breastfeeding, but also found a correlation between increased number of CD8+ cells in peripheral blood and the size of thymus at 10 months of age. The result clearly indicates that breastfed infants who have large thymuses obtain a high percentage of T lymphocytes in the blood circulation, more so than non-breastfed infants. While our knowledge regarding the possible role of an enlarge thymus has grown, it still remains incomplete.
The size of the thymus is significant for infants at 6 months of age because a small thymic size could be related to a higher risk of mortality. A study in Guinea-Bissau in West Africa has shown that the strong association between the small thymic size and the high rate of mortality during the last 6 months of the first year of life. The authors speculate that thymus size has an influence on immunocompetence during the first year of life. Another African study in Gambia suggests that the rates of mortality in young adulthood in the region could be predicted by season of birth. Infants born during the "hungry" season presented with smaller thymuses, with the risk of mortality being ten times higher than infants born in the "harvest" season. In relation to the immune system in the Gambian study, those born in the hungry season had lower T cell receptor-rearrangement excision counts, and HBM from their mothers had significantly lower cytokine interleukin-7 (IL-7) compared with those mothers whose infants were born in the harvest season. However, the relationship between increased neonatal mortality and breastfeeding is unclear in developed countries.
IL-7 is essential for lymphocyte homeostasis and development, particularly in the very first stage of Th2 subset development and the primary activation of naive CD4+ cells. IL-7 sends crucial signals to lymphoid cells at early stages of development which is required for lymphopoiesis, as IL-7 knockout mice became significantly lymphopenic into its peripheral blood and lymphoid organs. The total T cell population decreased about 10 to 20 fold and thymic cellularity reduced 20 fold. In addition, Aspinall et al found that IL-7 knock-out mice presented higher thymocyte subsets and peripheral T cell populations when they were fostered onto normal mothers compared to IL-7 knock-out mothers. IL-7 labelled in IL-7 knock-out mice milk shows that it not only crosses the intestinal mucosa but also enters the lymphoid tissues of the offspring. The permeability of the neonatal gut can allow bioactive proteins, even large peptides such as insulin or epidermal growth factor, to transfer to internal tissues.
Hypotheses regarding cell survival in the gastrointestinal tract
Some bioactive proteins must undergo hydrolytic processing to assist in providing adequate nutrition to breastfed infants. However, some milk proteins such as immunoglobulins, Îº-casein, lysozyme, lactoferrin, haptocorrin, Î±-lactalbumin and lactoperoxidase must survive the process of proteolysis in an intact or partially intact form. The digestion limitation of certain enzymes turns to a protective mechanism that prevents degradation of some immune cells and bioactive proteins in the GIT. In addition, other evidence exists to support the hypothesis regarding a mechanism of survival of milk components in the GIT, enhanced because of 1) high stomach pH in the newborn with antiproteolytic antivity, 2) a wide range of antiproteases in HBM, 3) protection of proteins in milk fat globules, and 4) the intestinal permeability turning into a beneficial mechanism for the offspring by transferring necessary bioactive constitutes.
High stomach pH & antiproteolysis
There are some reports which explain high stomach pH in newborn infants. The stomachs of newborn infants have an elevated gastric pH immediately after birth due to aspirated alkaline amniotic fluid in the stomach. This gastric pH is measured at around pH 6 to 8 compared to 2 to 3 in normal children and adults. Another possible reason for high pH value in infants' stomachs is due to less gastric acid production by parietal cells as cell mass increases with growth and feeding. The early study by Polacek and Ellison detected that the increase in parietal cell mass per unit area is about 2 to 3 times in the stomachs of full term newborn infants compared to preterm infants. This indicates that although the optimal proteolytic activity is at pH below 3, the pH value of infants is more alkaline, so proteins encounter limitations of pepsin activity and gastric peptic hydrolysis.
Decreased secretion of gastric acid and the high buffering capacity of milk may be important to provide protection for its cellular components in the gut, and promote the activity of immunoglobulins and antigen recognition molecules in the GIT. These proteins remain intact or partially intact throughout, or at least throughout part of, the GIT. Chatterton and colleagues confirmed that many milk proteins still remain intact in the stomach of term infants aged 8 and 28 days, after 1 and 3 hours of feeding respectively. The hydrolytic activity rapidly reduced between pH 6.5 and 4 compared to lower values of pH. Some milk proteins even resist degradation with gastric juice pH 2.0 such as sIgA. This is the reason why sIgA is considered the first marker of being resistant to proteolytic activity followed by lactoferrin, which are both easily found in the stool of breastfed infants. Around 10 to 85% of milk sIgA survive intact through to the feces of term infants in the first 3 to 4 months postpartum. This clearly demonstrates that infants who have high gastric pH could protect milk proteins from degradation in the stomach.
HBM, particularly the early colostrum milk, contains a wide range of proteases and antiproteases that gradually decreases throughout lactation. Protease may function to digest protein whereas antiproteases including primarily alpha1(Î±1)-antitrypsin and alpha1(Î±1)-antichymotrypsin provide partial inhibition of pancreatic proteases. Î±1-antichymotrypsin and Î±1-antitrypsin are produced by the mammary gland and protect HBM proteins from degradation in the GIT.
An interesting point is that those antiproteases from HBM influence the survival of other proteins. One example is Î±1-antitrypsin. Î±1-antitrypsin protects lactoferrin from proteolysis so that lactoferrin exerts a block to iron uptake in microorganisms to inhibit the metabolism and proliferation of organisms such as E. coli. It has been estimated that around 6 to 19% of lactoferrin escapes digestion, destroying harmful pathogens and reducing inflammation responses. Lönnerdal points out that after providing their biological properties, those survival proteins undergo degradation to some extent, possibly in the lower part of the small intestine.
Calhoun et al evaluated the capacity of HBM to protect granulocyte colony-stimulating factor (G-CSF) against proteolytic degradation after exposure to neonatal gastric secretions (pH 3 to 7) in vitro compared to the capacity of formula milk and cow's milk. Strikingly, endogenous G-CSF in HBM is protected from degradation after exposure to gastric secretions at physiologic pH levels, whereas endogenous G-CSF in formula milk and cow's milk were not protected from degradation in the same conditions. It should be noted that only breastfed infants could receive the biological effects of G-CSF, which influences hematopoietic functions such as enhancing neutrophil proliferation and differentiation to be able to reduce infection in the newborns. What is the difference in the protection between bovine milk and HBM even under the same conditions? One speculation could be related to protection by milk fat globules (MFGs).
HBM lipid exists as MFGs, which contain a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins and glycoproteins. Milk triglycerides which are 99% of total milk fat are hydrolyzed to free fatty acids and monoglyceride by lipoprotein lipase. After hydrolysis, the free fatty acids and monoglyceride provide potent detergent properties which are able to lyse enveloped viruses, bacteria and protozoa. In addition, HMFG glycoproteins prevent acute gastrointestinal infections in infants and young children. MUC-1 mucin, for instance, has the ability to bind S-fimbriated E. coli, and inhibit the replication of rotavirus. Mucin and lactadherin are the major components of milk fat globule membrane (MFGM) and resist degradation in the neonatal stomach up to 4 hours after feeding. They enable to maintain their properties even at high gastric acidity and in the presence of pepsin activity.
Some studies have found that the primary difference between HBM and bovine milk in relation to the capacity of HBM against proteolytic degradation in the neonatal gut was the surface of MFGs. The surface of the bovine globule is smooth whereas long filamentous structures extend more than 0.5-1 Î¼m from the surface of the fat globules in horse and HBM. These filaments have been shown to contain glycoprotein, particularly the MUC-1 mucin that is the most abundant mucin and resists digestion even at pH 2.0 in HBM. These glycoproteins in HBM are embedded in the MFGM and are maintained until its isolation from the globule.
Milk fat is thought to be an effective delivery system for some vitamins and hormones. Research suggests that microdispersion of vitamin E in milk with regards to the delivery mode is twice more effective than in orange juice or vitamin E capsules. Further, a study by German and Dillard72 introduced the concept of milk lipoprotein as a delivery vehicle, particularly for the fat-soluble vitamins A and D, and hormones. Singh argues that the unique composition of the MFGM phospholipid attempts to deliver and protect sensitive compounds in milk via the formation of liposomes with enhanced bioavailability properties. Liposomes, lipid vesicles, are found in HBM and carry essential nutrients to the offspring taken up by M cells on Payer's patches. It is available in a wide range of vesicles varying in size. There is speculation regarding delivery of other bioactive factors by milk fat, but more studies are needed to provide additional proof.
Maturation of the GIT is still continuing at birth and could be assessed by measuring intestinal permeability. Newborns, in particular preterm infants, have an immature gut mucosa with an increased permeability and less intact tight junctions between mucosal cells. Increased permeability in the gut reduces the effectiveness of the innate immune system, which is possibly more important for newborns in relation to an infectious challenge than active or adaptive immunity since the newborn has no immunological memory. However, Aspinall et al suggest that the intestinal permeability could turn into a beneficial mechanism for the offspring by transferring necessary immune cellular components and bioactive proteins such as IL-7 or other immunomodulating factors to develop and maintain immunological function. Convincing studies have suggested that preterm infants undergo a temporary period of enhanced intestinal permeability which promotes absorption of immunological and growth-promoting factors of a large size, such as lactulose, albumin and lactoglobulin, from HBM to compensate for the shortened gestation. The reduction of intestinal permeability normally takes about 3 to 4 weeks after a full term birth.
It is important to note that increased intestinal permeability could turn into positive outcomes if the infant receives HBM. Permitted bioactive proteins and cellular components in HBM provide numerous types of support for the infant's immune system and decreases intestinal permeability. Breastfed preterm infants showed more rapid gastric emptying with fewer gastric residuals in fewer hours compared with formula-fed infants, resulting in a shorter duration of hospitalization and less morbidity such as NEC and LOS. Similar health outcomes of preterm infants have been observed by Shulman et al and Taylor et al.
On the other hand, increased intestinal permeability for non-breastfed infants is not beneficial at all. Substantial studies have shown that formula-fed infants have a higher incidence of NEC approximately 6 to 10 times higher compared to breastfed infants. Strikingly, certain volumes of infant formula act as a toxic dose for the premature intestine. This is because preterm infants do not have the ability to fully digest carbohydrates and proteins which cause the production of organic acids, and these may be harmful to the developing intestine. To illustrate, undigested casein in the protein of formulas can attract neutrophils with stimulating inflammatory responses, opening the tight junctions between epithelial cells, interrupting the integrity of the epithelium barrier and allowing the harmful pathogens directly into the systemic circulation. In addition, intestinal hyperpermeability presents a possible association to food allergies or autoimmune diseases including celiac disease and type 1 diabetes, along with specific gene and environmental triggers. Further investigations of the correlation between early intestinal maturation and allergy and/or autoimmune disease in the later life are ongoing and more studies are required.
Conclusion and recommendations
Mounting evidence has indicated that cells and bioactive proteins from HBM are transported to the offspring and provide significant positive short and long-term health benefits for infants. Given the infants' unique physiological differences to adults, such as high pH in the stomach and more permeable intestine, these cells from HBM resist degradation in the GIT of the infant. Furthermore, HBM contains possible protective mechanisms from degradation including antiproteases, milk fat globules and perhaps other factors. A lot of information surrounding HBM is incomplete and more consistent research is required, for example, the correlation between breastfeeding and autoimmune diseases and allergies.
Questions relating to the quality of HBM versus formula milk have now moved to raw versus pasteurized donor milk; a factor which demands further research. To date, there is not a sufficiently sound explanation of the clinical consequences of using pasteurized donor milk as opposed to the mother's own raw milk. During pasteurization biological compositions in HBM become inactive, which could explain the poor weight gain in preterm infants who receive donor milk compared to raw milk. One speculation is the inactivation of lipase which affects fat absorption and subsequently the infant's growth. There is an obvious void in terms of consistent research in this area. A study from thirty years ago is currently referred to, with only few other intermittent studies having been done. More research needs to be carried out in order to identify the effect of pasteurized milk on preterm infants. There are also questions that need to be addressed in relation to the feeding of preterm infants with donor milk from mostly full-term mothers. It is necessary to analyze these factors with science based methodology, and to ensure implementing into practice the supply of HBM for infants, particularly for preterm and ill newborns. â€ƒ