Mucosal immune activation and maturation of the small intestine at weaning in the hypothymic (nude) rat

Activation of the mucosal immune system peaks at weaning on days 21 and 22 of life in the rat. We have investigated activation in the gut associated lymphoid tissue and maturation of intestinal mucosa in hypothymic (nude) and in phenotypically normal heterozygous CBH rats at 22 days of life. Intestinal maturation, as assessed by villus area, crypt length, crypt cell production rate and disaccharidase activity, was similar in hypothymic and normal rats, and indices of mucosal immune function were elevated in both groups at this time. The proportion of mononuclear cells from the mesenteric lymph node expressing IL-2R was 11% in heterozygous and 14% in hypothymic rats as determined by flow cytometry. Immunoperoxidase staining of MLN sections confirmed the presence of IL-2R+ cells in the T-dependent interfollicular areas. However, the number of T-cells was considerably depleted in hypothymic rats. Intraepithelial lymphocyte counts and serum rat mucosal mast cell protease II concentrations were similar in the two groups, while counts of jejunal mucosal mast cells and eosinophils were paradoxically increased in hypothymic animals. As T lymphocyte function is thought to be impaired in hypothymic rats, the intact mucosal immune activity in hypothymic rats could be due to activation of intrinsic "thymic independent" T lymphocytes in hypothymic rats, or to engraftment with extrinsic maternal milk-derived lymphocytes and their activation in the infant rat gut.     

Ontogeny of the intestinal immune system in the premature infant

A variety of variables influence the development of secretory immunity and oral tolerance, two immune mechanisms that are of paramount importance for the mucosal barrier function. Epithelial permeability is likely a significant primary or secondary event in the pathogenesis of several intestinal diseases, including adverse immune reactions to foods. This variable is determined by the individual's age (e.g., preterm versus term infant), concurrent infections, and the shielding effect of secretory IgA (SIgA) antibodies provided by breast milk. The clinical consequences will depend on how fast “closure” of the epithelial barrier can be attained or re-established, which is influenced both by the age of the infant and by its successful mounting of adaptive intestinal SIgA responses as well as generation of oral tolerance towards innocuous antigens from the diet and from the normal indigenous microbiota. SIgA is the best defined effector component of the mucosal immune system; its enhancement, and the homeostatic mucosal immune regulation in general, induced by probiotic commensal bacteria is of considerable clinical interest. Importantly, the feeding and treatment regimen (e.g., antibiotics) to which the newborn is subjected, may disturb the balance of the developing mucosal immune system.     

Postnatal development of the mucosal plexus in the porcine small and large intestine

Knowledge regarding the foetal and postnatal development of the enteric nervous system is crucial for the understanding of congenital disorders. While lot of information exists regarding the myenteric and submucosal plexuses, the development of the mucosal plexus has not been previously studied. The mucosal innervation seems to play an important role in the local reflex activity of the gut. In this study, we examined the development of enteric mucosal innervation in the pig at various ages of life. Small and large bowel paraffin-embedded specimens were stained with PGP 9.5 and neurofilament protein in three piglets from six age groups (60 and 90 days gestation, newborn, 4 and 12 weeks old, and adult pigs). Small and large bowel demonstrated identical innervation patterns. Myenteric and submucosal plexuses were stained with PGP 9.5 at 60 days gestation. However, the mucosal staining was first noted clearly at the newborn period. By 4 weeks, PGP 9.5 staining was noted in small amounts within the mucosa. Inner proprial and villous fibres were seen ahead in time to the subepithelial fibres. Both inner proprial and villous staining became quiet prominent by 12 weeks of age and remained unchanged into adulthood. However, the subepithelial fibres appear to increase in adulthood. This study demonstrates for the first time that enteric mucosal innervation first appears only at birth. The immaturity of the mucosa generated reflex activity, and secretory functions may have implication in the management of functional intestinal obstruction in the premature infant.     

The role of breastfeeding in prevention of neonatal infection

The immune system of the human newborn is of very limited size. It expands rapidly, especially due to the exposure to the gut microflora. Normally the newborn is colonized with microbes from the mother's intestinal flora at and after delivery. The many defence factors of the mother's milk include large amounts of secretory IgA antibodies produced by lymphocytes which have migrated from the mother's gut to the mammary glands. Therefore the SIgA antibodies are mainly directed against the mother's previous and recent gut microflora. Thus breastfeeding modulates the early exposure of the neonate's intestinal mucosa to microbes and limits bacterial translocation through the gut mucosa. This may be a major reason why breastfeeding protects efficiently against neonatal septicaemia, as well as several other infections. The defence factors of the milk prevent infections already at the mucosal level. The transplacentally obtained maternal IgG antibodies protect primarily in tissues and do so at the cost of cytokine-induced clinical symptoms, tissue engagement and high energy consumption.     

Development of mucosal immune function in man: potential for Gl disease states

The human mucosal immune system is structurally mature and has all the necessary cellular components to generate an immune response at birth. However, in the absence of dietary antigens and bacterial flora, there are no secondary follicles in the Peyer's patches and virtually no immunoglobulin A plasma cells in the lamina propria. Reactive follicle centers develop after birth but it takes 2 years for mucosal IgA plasma cell density to reach adult levels. T cells are present in the epithelium and lamina propria at birth, albeit at a lower frequency than later in life and there are major differences in phenotype between T cells in fetal intestine and postnatal intestine. There is no information on the impact of the massive antigenic challenge at birth on the mucosal immune system. Well-documented deficiencies in the ability of the blood T cells of the neonate to produce interleukin-4 and interferon-gamma may also occur in the intestine. It is still an open question whether it is better to try to prevent immunological sensitization of the newborn by avoiding potential allergens (i.e. cow's milk), or whether early exposure (as happens when premature infants are given formula feeds) might tolerize the infant. Hydrolysed cow's milk formulae are probably less antigenic than whole cow's milk and have been widely used in the treatment of cow's milk allergy. Some thought is now being given as to whether the prophylactic use of hydrolysates can reduce cow's milk allergy in ‘at-risk’ infants.     

Functional foods and paediatric gastro-intestinal health and disease

The application of molecular methods to gastro-intestinal diseases is giving insight into the way in which the resident intestinal microbiota interacts with the mucosal immune system. Using traditional culture techniques, the importance of mucosally-associated bacterial biofilms in maintaining mucosal integrity has been demonstrated in ways previously impossible. Changes in the balance of organisms at initiation of and during disease provide a rationale for interventions with functional foods which facilitate re-establishment of the homeostasis of healthy gut.     

A human flora-associated rat model of the breast-fed infant gut

OBJECTIVES: Bacterial colonization of the infant gut may have important influences on the development of gastrointestinal, respiratory, and allergic disease. Early diet is a major determinant of the gut microflora. It is very difficult to carry out studies in human infants that can investigate the interaction of diet, flora, and mucosa. In this study we have developed an infant human flora-associated (IHFA) rat model to allow such investigation. METHODS: Germ-free infant rats were infected with fecal bacteria from exclusively breast-fed infants and were maintained on a modified infant formula for 8 weeks. The fecal and cecal contents were collected and compared with feces of breast-fed infants for bacterial populations, bacterial metabolites, and enzymes and for the ability to inhibit adhesion of pathogenic bacteria to human mucosal cells. RESULTS: The IHFA cecum and feces were dominated by lactic acid bacteria, Bifidobacterium, and lactobacilli, which were representative of the infant feces. The fecal short-chain fatty acid profile was dominated by acetic and lactic acid in a similar manner to human infant feces. Other bacterial metabolites were similar to those of the human infant. Rat intestinal samples were able to inhibit the adhesion of pathogens to mucosal cells, but to a lesser extent than the human samples. CONCLUSIONS: This IHFA infant model of the intestinal flora of the breast-fed infant is considered valid for studying the effect of diet on bacterial colonization and metabolism.     

Human milk oligosaccharides and their potential benefits for the breast-fed neonate

Human milk oligosaccharides (HMO), unconjugated complex carbohydrates that are highly abundant in human milk but not in infant formula, have recently received much attention due to their potential benefits for the breast-fed neonate. While it is becoming evident that HMO structure determines their specific function, understanding the metabolic fate of ingested HMO is key in assessing their biological roles. Remarkably little is known about how, when and where they are metabolized. HMO have long been regarded as metabolically "inert" to the host, as significant amounts are excreted with the fe-ces. HMO reach the colon intact where their prebiotic effects promote healthy gut colonization. HMO can also function as soluble decoy receptors and block adhesion of microbial pathogens to epithelial surfaces. Local effects at the mucosal lining include differential cell responses or modulation of the innate immune system. A small percentage of HMO is believed to be absorbed intact in the small intestine and later excreted with the urine, which opens speculations on possible systemic effects, e.g. in the immune system or in the context of neuronal development. Oligosaccharides currently added to infant formula are structurally different from HMO and therefore most likely not functionally equivalent. Selected "authentic" HMO might soon become available for the supplementation of infant formula, but additional preclinical and clinical studies are required to demonstrate efficacy. This review provides an overview about the structural and functional properties of HMO with emphasis on recent findings in metabolism studies.     

Infant formulas supplemented with prebiotics: intestinal microbiota and immune responses

It is well known that the type of feeding influences the composition of the gut microflora after birth. Human milk favours the growth of a 'bifidus flora' which, according to several evidences, may activate the immune system and defend from pathogens. Breast milk oligosaccharides, which are involved in many functional effects both at local and systemic level, are thought to stimulate the growth of health promoting microbes, such as bifidobacteria, and may ultimately influence the immune system. In accordance with this current working hypothesis, dietary modulation of the gut microbiota to obtain a 'bifidus flora' also in bottle-fed infants may be a useful way to stimulate immunological functions and to harbour a biological barrier against pathogens. In several clinical trials prebiotic oligosaccharides have been used to mimic the beneficial effects of breast milk oligosaccharides. A mixture of oligosaccharides has shown its efficacy in stimulating the establishment of a 'bifidus flora', with stools closer to those found in breast-fed infants. Several experimental data also indicate that oligosaccharides might modulate the immune system and contribute to the improvement of the protective properties of infant formulas.     

Alterations of the intestinal mucosal block in ulcerative colitis and Crohn's disease--immunological and ultrastructural findings, and considerations of the pathogenesis

The intestinal mucosa is the very site of the chronic inflammatory lesion in ulcerative colitis (UC) and in Crohn's disease (CD). The lamina mucosa together with its own and associated cellular and humoral protective properties can be described as the mucosal block which is functionally related to the intestinal and systemic immune system. Deteriorations of this mucosal block probably are of pathogenetic significance in UC and CD. This paper will summarize observations on the systemic immune system and local immune phenomena and their possible relations to the tissue injury in the gut. An immunopathogenetic concept intends to explain the chronicity of the diseases: epithelial insufficiencies of the mucosal block are followed by strong antigenic intrusion and stimulation of the local immune system with disturbance of the local immune homeostasis and the formation of immune complexes. The interactions of phagocytes with immune complexes cause the release of degrading lysosomal enzymes, which leads to continuous injury of the mucosal block and therefore to a perpetuation of the disease.     

Prospects for clinical applications of butyrate-producing bacteria

As the major source of energy for colonic mucosal cells and as an important regulator of gene expression, inflammation, differentiation, and apoptosis in host cells, microbiota-derived butyrate can enhance the intestinal mucosal immune barrier, modulate systemic immune response, and prevent infections. Maintaining a certain level of butyrate production in the gut can help balance intestinal microbiota, regulate host immune response, and promote the development and maintenance of the intestinal mucosal barrier. Butyrate-producing bacteria act as probiotics and play important roles in a variety of normal biological functions. Bacteriotherapeutic supplementation by using fecal microbiota transplantation to restore butyrate-producing commensal bacteria in the gut has been very successful in the treatment of recurrent and refractory Clostridium difficile (C. difficile) infection or C. difficile-negative nosocomial diarrhea. Administration of probiotics that include butyrate-producing bacteria may have a role in the treatment of inflammatory bowel diseases and in the prevention of necrotizing enterocolitis and late-onset sepsis in premature infants. Furthermore, modulating gut microbiota with dietary approaches may improve intestinal dysbiosis commonly seen in patients with obesity-associated metabolic disorders. Supplementation with a butyrate-producing bacterial stain might be used to increase energy expenditure, improve insulin sensitivity, and to help control obesity and metabolic syndrome.     

The role of the gut in beta-cell autoimmunity and type 1 diabetes: a hypothesis

The origin of autoimmunity leading to the destruction of insulin-producing beta-cells is not known. Several studies suggest that a link exists between the gut immune system and the islets infiltrating lymphocytes. Inflamed pancreatic islets express the same adhesion molecules involved with the homing of gut-associated lymphocytes. The manifestation of autoimmune diabetes in the animal models can be modified by dietary factors, which cause changes in the cytokine production by islet-infiltrating lymphocytes. Increased risk of type 1 diabetes has been associated with an early introduction of cows' milk formula in infancy, indicating that triggering of the gut immune system in early infancy may contribute to the later development of beta-cell autoimmunity. Enhanced immune reactivity to cow milk (CM) proteins in the patients with type 1 diabetes suggests aberrant regulation of the gut immune system in this disease. In the patients with newly diagnosed type 1 diabetes, anti-glutamate decarboxylase (GAD)-reactivity was found in the subpopulation of lymphocytes expressing gut-associated homing receptor alpha 4 beta 7. Based on these findings, the hypothesis that aberrant function of the gut immune system would lead to the development of beta-cell autoimmunity and type 1 diabetes has recently received a lot of attention. The possibility that regulation of the gut immune system is not normal in subjects at risk of autoimmune diabetes should be considered when treatments interfering with mucosal immunity for the prevention of type 1 diabetes are planned.     

Immunopathogenesis of chronic inflammatory bowel disease

The aetiology and pathogenesis of chronic inflammatory bowel disease are unknown. However, there is circumstantial evidence that immune mechanisms may play a significant role in mediating the gut lesion and various systemic manifestations. The role of the cellular immune system, soluble mediators, including proinflammatory and immunoregulatory cytokines, humoral immunity and mucosal complement activation will be discussed.     

Breastmilk cell trafficking induces microchimerism‐mediated immune system maturation in the infant

Initiating breastfeeding within the first hour of life confers an important benefit in terms of child mortality and severe morbidity. Intestinal permeability to ingested macromolecules and immunoglobulins is limited to the first days of human life. These exchanges cease in the very early post‐partum period but may increase beyond the neonatal period in response to local inflammation or introduction of a weaning food. From animal‐ and limited human‐based observations, compelling evidence points out to breastmilk cells also trafficking from mother to infant mucosal tissues and participating to the maternal microchimerism. The precise nature of breastmilk cells that are involved is presently not known but likely includes progenitor/stem cells—representing up to 6% of breastmilk cells—with possible contribution of mature immune cells. Stem cell microchimerism may induce tolerance to non‐inherited maternal antigens (NIMAs), breastfeeding generating regulatory T cells (T_reg) that suppress antimaternal immunity. Therefore, in complement to pregnancy‐induced microchimerism, breastfeeding‐induced microchimerism may be pivotal in infant immune development, intestinal tissue repair/growth and protection against infectious diseases. As a continuum of the gestational period, the neonatal gut may be considered as a temporary, but important developmental extension of the role played by the placenta during intrauterine life; breastmilk playing the role of maternal blood by delivering maternal soluble factors (macromolecules, Ig, cytokines) and immunologically active milk cells. A better understanding of breastfeeding‐induced maternal microchimerism would provide further evidence in support of public health messages that reinforce the importance of early initiation of breastfeeding.     

Breast-feeding, a complex support system for the offspring

The newborn has an immune system, very limited in size at birth and its postnatal expansion and maturation takes time. In the meantime the transplacental IgG antibodies from the mother play an important role for the protection of the infant. However, these antibodies act in tissues and induce inflammation and are energy-consuming. In contrast, the milk secretory IgA antibodies stop microbes already on the mucosa preventing infection, tissue engagement and energy loss. In addition, the milk contains many protective factors such as lactoferrin and oligosacharides functioning as analogues for microbial receptors preventing mucosal attachment, the initial step of most infections. As a result, breast-feeding significantly reduces the risk of neonatal septicemia, respiratory tract infections, otitis media, diarrhea, urinary tract infections, infection-induced wheezing and necrotizing enterocolitis. Via several mechanisms it seems that human milk can actively stimulate the immune system of the breast-fed infant. This reduces the risk of infections like otitis media, respiratory tract infections, diarrhea and infection-induced wheezing for several years after the termination of breast-feeding. Furthermore, it seems that breast-feeding decreases the risk of attracting celiac disease and allergic diseases. The latter has been much debated, but a recent critical review of published reports gives good support for long-term protection of allergic diseases, especially in high-risk children.     

Modulation of type 1 and type 2 diabetes risk by the intestinal microbiome

The prevalence of type 1 and type 2 diabetes have both risen dramatically over the last 50 years. Recent findings point towards the gut microbiota as a potential contributor to these trends. The hundred trillion bacteria residing in the mammalian gut have established a symbiotic relation with their host and influence many aspects of host metabolism, physiology, and immunity. In this review, we examine recent data linking gut microbiome composition and function to anti‐pancreatic immunity, insulin‐resistance, and obesity. Studies in rodents and human longitudinal studies suggest that an altered gut microbiome characterized by lower diversity and resilience is associated with type 1 and type 2 diabetes. Through its metabolites and enzymatic arsenal, the microbiota shape host metabolism, energy extracted from the diet and contribute to the normal development of the immune system and to tissue inflammation. Increasing evidence underscores the importance of the maternal microbiome, the gestational environment and the conditions of newborn delivery in establishing the gut microbiota of the offspring. Perturbations of the maternal microbiome during gestation, or that of the offspring during early infant development may promote a pro‐inflammatory environment conducive to the development of autoimmunity and metabolic disturbance. Collectively the findings reviewed herein underscore the need for mechanistic investigations in rodent models and in human studies to better define the relationships between microbial and host inflammatory activity in diabetes, and to evaluate the potential of microbe‐derived therapeutics in the prevention and treatment of both forms of diabetes.     

Presacral neuroenteric fistula in a newborn presenting with an epidural abscess: case report and review of the literature

We describe a newborn infant (<24 hours of age) who presented with mild swelling on the back and buttocks attributable to a neuroenteric fistula complicated by an epidural parasacral abscess infected with mixed coliforms. Epidural abscesses in infancy are extremely rare, and one has not been observed previously in the newborn period. The infant was surprisingly mildly affected. Prompt intervention led to an excellent outcome. Coliforms may colonize the infant gut in <24 hours, even in the developed world. Unexplained swellings on the backs of infants should lead to a search for underlying malformations and an early surgical review, which is best conducted with a combined pediatric surgical and neurosurgical approach.     

The ontogeny of the gut mucosal immune system and the susceptibility to infections in infants of developing countries

In this review we summarize data on the human gut mucosa associated lymphatic tissues as part of the common mucosal immune system. Its embryonal-fetal and post-natal ontogeny becomes severely distorted and compromised by mal-/undernutrition which is so prevalent in developing countries. Pathogenetic interdependencies exist between maternal-fetal undernutrition, the ontogeny of the immune system, constant antigenic stimulation of the mucosal immune system post-natally, and the 14 million deaths annually from infections in children below the age of 5 years in developing countries. A detailed knowledge of these interdependencies is required for effective prevention and treatment in an attempt to reduce the high morbidity and mortality rates of children in developing countries.     

Scientific rationale and benefits of nucleotide supplementation of infant formula

The present review examines the role of dietary nucleotides in infants, and the scientific rationale and benefits of nucleotide supplementation of infant formula. The immunoprotective benefits of human milk, the biology of human milk nucleotides, and the immunological and gastrointestinal effects of dietary nucleotides in animal studies and in vitro experiments are examined. Clinical studies are reviewed, especially those examining the efficacy of nucleotide-supplemented infant formula in enhancing immunity and reducing the risk of sepsis. The presence of human milk cells, and a variety of immunoactive and trophic components of human milk, can explain the reduced incidence of sepsis in breastfed term and preterm infants. Nucleotides, believed to play an immunomodulatory role, are found in lower concentrations in infant formula. Animal studies have shown that dietary nucleotides enhance a number of immune responses and the growth, differentiation and repair of the gut. Several clinical studies have reported beneficial effects of nucleotide supplementation on gut microflora, diarrhoea and immune function, and one study has reported better catch-up growth in term infants with severe intrauterine growth retardation. More basic research studying the metabolism of nucleotides in neonates is encouraged. Additional randomized controlled trials are necessary to demonstrate the clinical benefits of nucleotide supplementation of infant formula, as it cannot be presumed that nucleotides produce the same benefits for the infant as human milk. Studies are especially necessary in high-risk neonatal situations, such as extreme prematurity, significant suboptimal nutrient intake before and after birth, and recovery from gut injury.