Immune factors in breast milk and the development of atopic disease.

Breast-feeding provides protection against infections and contains numerous factors that modulate and promote the development of the infant immune system. These factors include secretory IgA, antimicrobial proteins like CD14, cytokines, and fatty acids. Studies examining the role of breast-feeding in the development of allergic disease in infants demonstrate potentially protective as well as neutral or nonprotective effects, likely due to the heterogeneity in their study design. In this overview, we explore the potential role of immune factors in the breast milk, as well as selected probiotics, in the development of allergy.

Macromolecular absorption. Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine.

The immature small intestine of neonatal mammals is permeable to gamma globulins as a source of passive immunity. Allegedly, macromolecular absorption ceases when the epithelial cell membrane matures. However, some evidence exists that adult animals retain a limited capacity to transport antigenic and biologically active quantities of large molecules. In this study, the mechanism of absorption of the tracer protein, horseradish peroxidase (HRP), was tested in neonatal and adult rat gut sacs. Transport into serosal fluid was quantitated by enzymatic assay and monitored morphologically by histochemical techniques. A greater transport of HRP was noted in the adult jejunum compared to adult ileum and neonatal intestine. Morphologically, the uptake mechanism in adult intestine was similar to the endocytosis previously reported in neonatal animals Like other endocytotic processes, HRP uptake in adult rats is an energy-dependent process as determined by metabolic inhibitors and temperature-controlled studies. An understanding of the mechanism whereby macromolecules are bound to intestinal membranes and engulfed by them is necessary before the action of physiologic macromolecules such as enterotoxins can be appreciated.

Stimulation by immune complexes of mucus release from goblet cells of the rat small intestine.

Immune complexes (bovine serum albumin with rat antibodies to bovine serum albumin) formed in twofold antibody excess were injected into the duodenum of normal rats. In comparison to controls injected with antigen only, there was a marked increase in the percentage of disrupted goblet cells (an index of mucus release) in segments from the intestine of rats exposed for 3 hours to immune complexes in vivo. Similarly, there was a significant increase in 35S-labeled mucus recovered by filtration of intestinal wash, rinse, and mucosal homogenate fluids from rats exposed to immune complexes compared to those from rats exposed to bovine serum albumin or purified rat antiserum to bovine serum albumin alone. These findings suggest that certain immune complexes can stimulate mucus release from intact rat small intestine; enhanced mucus release may have a role in clearing the surface of complexes.

Intestinal uptake of macromolecules: effect of oral immunization.

Animals were orally immunized with horseradish peroxidase and bovine serum albumin, and absorption of these antigens was studied. In comparison with controls, a consistent and significant decrease in peroxidase uptake was noted in both germ-free and conventional rats immunized with peroxidase; a similar decrease in serum albumin uptake was also noted in animals immunized with serum albumin. There was no difference in the uptake of an unrelated macromolecule. These observations suggest that local immunization interferes specifically with the intestinal uptake of macromolecular antigens.

Clinical evidence for immunomodulatory effects of probiotic bacteria.

Close, tightly orchestrated interactions between the intestinal epithelium and the mucosa-associated immune system are critical for normal intestinal absorptive and immunological functions. Recent data indicate that commensal intestinal microbiota represents a major modulator of intestinal homeostasis. This review analyzes the process of intestinal colonization and the interaction of microbiota with the intestinal epithelium and mucosal immune system, with special reference to the first years of extrauterine life. Dysregulation of the symbiotic interaction between intestinal microbiota and the mucosa may result in a pathological condition with potential clinical repercussions. Based on the concept that there is a beneficial and symbiotic relation between the host and endogenous microbiota, strategies aimed at directly modulating intestinal microbiota with regard to disease prevention or treatment have been developed. One strategy involves administering viable probiotic bacteria. Clinical evidence for the beneficial effect of probiotics in the prevention and/or treatment of necrotizing enterocolitis, infectious and antibiotic-associated diarrhea, allergic diseases, and inflammatory bowel disorders is reviewed herein.

Helminth-induced alterations of the gut microbiota exacerbate bacterial colitis.

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.

Expression and developmental regulation of Na+,K+ adenosine triphosphatase in the rat small intestine.

The Na+,K(+)-ATPase ion pump plays a critical role in fluid and electrolyte physiology of the small intestine. Here we show that, of the three known alpha isotypes (alpha 1, alpha 2, and alpha 3) of the sodium pump found in the rat, only alpha 1 is expressed in the small intestine. The expression of this isotype, considered at the level of mRNA, is under developmental control, with the adult intestine exhibiting approximately a threefold increase in alpha 1 message over the neonate. Cortisone treatment of the neonate results in near-adult levels of alpha 1 mRNA expression. An increase in the abundance of alpha 1 isotype parallels the changes in its mRNA expression. beta subunit mRNA is expressed coordinately with the alpha 1 subunit mRNA. A four- to five-fold rise in the Na+,K(+)-ATPase activity is also developmentally induced.

Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is associated with decreased toxin A receptor.

Human infants are relatively resistant to Clostridium difficile-associated diarrhea and colitis compared to adults. In that toxin A is the major cause of intestinal damage with this organism, we compared toxin A receptor binding and biological effects in newborn vs adult rabbit ileum. Purified toxin A (M(r) 308 kD) was labeled with tritium or biotin with full retention of biologic activity. Appearance of specific toxin A brush border (BB) binding was strongly age dependent with minimal [3H]toxin A specific binding at 2 and 5 d of life, followed by gradual increase in binding to reach adult levels at 90 d. Absence of toxin A binding sites in newborn and presence in adult rabbits was confirmed by immunohistochemical studies using biotinylated toxin A. Toxin A (50 ng to 20 micrograms/ml) inhibited protein synthesis in 90-d-old rabbit ileal loops in a dose-dependent fashion. In contrast, inhibition of protein synthesis in 5-d-old rabbit ileum occurred only at the highest toxin A doses (5 and 20 micrograms/ml) and at all doses tested was significantly less than the adult rabbit ileum. In addition, toxin A (5 micrograms/ml) caused severe mucosal damage in adult rabbit ileal explants but had no discernable morphologic effect on 5-d-old rabbit intestine. Our data indicate that newborn rabbit intestine lacks BB receptors for toxin A. The absence of the high-affinity BB receptor for toxin A in the newborn period may explain lack of biologic responsiveness to purified toxin, and the absence of disease in human infants infected with this pathogen.

Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine.

To study an enzymatic basis for the postnatal changes in intestinal glycosylation, the activities of sialyl- and fucosyltransferases were determined in the particulate fraction of mucosal cells prepared from rat small intestine of various ages. The results show that sialyltransferase activity was present in increased levels compared to adults during the preweaning period (1-2 weeks) and subsequently declined 5-fold to adult levels after weaning, while fucosyltransferase activity was decreased compared to adults in the first 3 weeks of life, rapidly increased at 4 weeks, and reached adult levels (10-fold) by 5 weeks. The changes in both sialyl- and fucosyltransferase activities were reflected by the membranous content of glycosidic-bound sialic acid and fucose, respectively. Cortisone injection precociously induced a decreased sialyltransferase activity and an increased fucosyltransferase activity in 2-week-old suckling rats. This study indicates that the activities of sialyl- and fucosyltransferases were reciprocally related and modulated by cortisone action in the developing intestine. These enzyme changes may be responsible for the previously noted shift from sialylation to fucosylation of the intestinal mucosa during maturation.

myo-inositol action on gerbil intestine: alterations in alkaline phosphatase activity upon phosphatidylinositol depletion and repletion in vivo.

The influence of phosphatidylinositol (PI) on intestinal alkaline phosphatase activity was studied in myo-inositol deficient gerbils. A reduction of membrane PI in intestinal mucosa to 30-40% of the control was produced by feeding female gerbils a myo-inositol-deficient diet containing coconut oil for 2 weeks. As expected, the animals developed typical intestinal lipodystrophy with abnormal fat accumulation. In the PI-depleted animal, intestinal alkaline phosphatase activity was reduced to 20-30% of the control group. The levels of both membranous and soluble enzymes in intestinal mucosa were affected, but there were no changes in liver, kidney and plasma levels. When the lipodystrophic gerbils were given dietary myo-inositol, the complete repletion of intestinal membrane PI to the control level occurred 36 h later, whereas membrane-bound alkaline phosphatase activity in intestine was not restored to the control level until 72 h later. Administration of cycloheximide or actinomycin D did not block this enzyme induction. Lymphatic output of triacylglycerol into the bloodstream was stimulated 10-fold at 18 h of myo-inositol repletion, but there was no parallel increase in the activity of alkaline phosphatase in plasma during this early phase of intestinal recovery. Thus, these data suggest a possible regulatory role of PI in the processing and/or turnover of alkaline phosphatase in vivo, but a negative role of alkaline phosphatase in lipid transport across gerbil intestine.

Developmental changes in galactosyltransferase activity in the rat small intestine.

During postnatal development, UDP-Gal: GlcNAc(beta 1-4)-galactosyltransferase (4 beta-GT) and UDP-Gal:GalNAc(beta 1-3)-galactosyltransferase (3 beta-GT) activities were increased by 17- and 24-fold, respectively, in the rat small intestine. The injection of cortisone into suckling rats resulted in precocious induction of distal 4 beta- and 3 beta-GT activities by 2.7- and 1.8-fold, respectively. Injection of phorbol-12-myristate-13-acetate (PMA) resulted in precocious induction of distal 3 beta-GT by 2.7-fold. These results suggest that intestinal galactosyltransferase activities are under developmental regulation and can be modified by cortisone and PMA.

Development of the gastrointestinal mucosal barrier. Evidence for structural differences in microvillus membranes from newborn and adult rabbits.

Electron spin resonance (ESR) and spin label methods with 5-doxylstearic acid as a probe were used to investigate the structure of microvillus membrane from the small intestine of adult and newborn rabbits. The spin label in microvillus membrane of newborns appeared to be in a more disordered environment than spin label in microvillus membrane of adult animals in the temperature range from 4 to 56 degrees C. In addition, a temperature transition at 39.6 +/- 0.3 degrees C was observed in the temperature dependence of the hyperfine splitting parameter for microvillus membrane from adult animals whereas a linear temperature dependence of the hyperfine splitting parameter was found for microvillus membrane from newborns. Cholera toxin was used as an external stimulus to test for the structural response in these two membrane preparations. Cholera toxin at 6 pM caused a decrease in the hyperfine splitting parameter at temperatures below 40 degrees C and a shift in the temperature break from 39.6 degrees C to 30.7 degrees C in microvillus membrane from adults. Using microvillus membrane from newborns, the temperature dependence of the hyperfine splitting parameter remained linear with a cholera toxin stimulus and the disordering effect of cholera toxin was only observed below 30 degrees C. These studies suggested that microvillus membrane from newborns were inherently more disordered than microvillus membrane from adult animals and that this difference in membrane organization might in part account for the increased attachment and penetration of macromolecules noted during the perinatal period.

Preliminary characterization of enterooxyntic activity on the guinea pig oxyntic cell.

Stimuli originating in the small intestine enhance gastric acid secretion, an effect termed the intestinal phase of gastric secretion. A humoral agent, enterooxyntin (EO) may mediate this effect. Mechanical distension of an ex vivo-perfused segment of canine jejunum caused the release of EO measured by cytochemical quantification of hydroxyl ion production (HIP) in guinea pig gastric oxyntic cells, an index of acid secretion. The H2 receptor antagonist, cimetidine (10(-5) M), and diamine oxidase, which hydrolyzes endogenous histamine, reduced HIP by 60% and 48%, respectively. Atropine (10(-5) M), the muscarinic cholinergic antagonist, reduced HIP by 75%. EO-induced HIP was also inhibited partially by the Ca2+ antagonist EGTA (10(-6) M) and by blockade of calcium channels with LaCl3 (10(-6) M). EO does not appear to operate through any single pathway. EO may be a single substance, different from gastrin, or a mixture of substances that have stimulatory effects on the oxyntic cell. Its action on the oxyntic cell is apparently mediated by both histaminergic and cholinergic pathways. Since neither cimetidine nor atropine completely inhibited EO-induced HIP, a direct effect of EO on the oxyntic cell seems likely and appears to depend on Ca2+. Isolation and purification of EO will be necessary to better assess the potency, efficacy, and the detailed cellular mechanisms of EO action.

Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium.

Communication between microorganisms and the gastrointestinal epithelium, ie, bacterial-epithelial "crosstalk," is examined. Because most basic research on the molecular interaction of bacteria with the gut epithelium relates to pathogen-enterocyte interaction, crosstalk with pathologic bacterial is considered in detail. Through their interactions with the intestinal epithelium, pathogens can modify epithelium function to enhance their penetration across the epithelial barrier and to exploit mucosal host defenses for their own benefit. Three representative pathogens are used to illustrate the various adaptive techniques used to colonize and penetrate the mucosal barrier. Salmonella enterica typhimurium interacts with the physiologic receptor for epidermal growth factor to co-opt the receptor's signal transduction mechanisms. Enteropathic Escherichia coli secretes a receptor (type III secretion) into the microvillus surface of enterocytes that disrupts the microvillus and alters its actin structure to form a dome-like anchoring site. Shigella flexneri is used to illustrate how pathogens use the follicular epithelial cell (M cell), the physiologic conduit for antigens to reach gut associated-lymphoid tissues, for penetration of the epithelial barrier. Shigella organisms attached to M cells use their endocytotic properties to enter the cell. Once inside the cell, the organism lyses the endocytic vacuole and co-opts actin and myosin to form a propelling tail for further penetration of the epithelium through the basolateral surface. Probiotics can protect the intestine by competing with pathogens for attachment, strengthening tight junctions between enterocytes, and enhancing the mucosal immune response to pathogens. However, additional molecular studies are needed to define more precisely the mechanism of probiotic-epithelial crosstalk.

Trichloroacetic acid (TCA) precipitability of 125I in the blood of mice fed 125I.

A recent study showed that after feeding 125I-labelled proteins or free 125I to mice, as much as 40% of total radioactivity in the circulation precipitated upon mixing whole blood with 10% trichloroacetic acid. We examined this potential limitation to the use of radiolabelled tracers for studies on intestinal digestion of proteins and protein uptake, and identified its mechanism. BALB/c mice were gavage-fed or injected intravenously (i.v.) with Na125I. Blood obtained at 15 min was added directly to 10% trichloroacetic acid (TCA) or was processed to obtain serum or plasma. On mixing with 10% TCA, 25-33% of the radioactivity in whole blood was precipitated; less than 2% of the radioactivity in plasma or serum was precipitated. In vitro studies identified hemoglobin as the primary carrier protein participating in this reaction. If hemoglobin was replaced by methemoglobin or cyanomethemoglobin, then the reaction with 125I did not occur, suggesting that iron in the heme group may be the site for 125I binding and that iron must be in its reduced or ferrous form (Fe2+). The administration of non-radioactive NaI in vivo or its addition to reaction mixtures in vitro completely inhibited the precipitation of 125I by hemoglobin in the presence of TCA. Thus the addition of non-radioactive iodide to TCA stock solutions may effectively prevent non-specific binding of 125I to free hemoglobin released unintentionally during venipuncture or at other stages of blood processing.

Absorption of protein and protein fragments in the developing intestine: role in immunologic/allergic reactions.

An important adaptation of the gastrointestinal tract to the extrauterine environment is its development of a mucosal barrier against the penetration of proteins and protein fragments. To combat the potential danger of invasion across the mucosal barrier the newborn infant must develop within the lumen and on the luminal mucosal surface an elaborate system of defense mechanisms which act to control and maintain the epithelium as an impermeable barrier to the uptake of macromolecular antigens. As a result of a delay in the maturation of the mucosal barrier, newborn infants are particularly vulnerable to pathologic penetration by harmful intraluminal substances. The consequences of altered defense are susceptibility to infection and the potential for hypersensitivity reactions and the formation of immune complexes. With these reactions comes the potential for developing life-threatening diseases such as necrotizing enterocolitis, sepsis, and hepatitis. Fortunately, "nature" has provided a means for passively protecting the "vulnerable" newborn against the dangers of a deficient intestinal defense system, namely human milk. It is now increasingly apparent that human milk contains not only antibodies and viable leukocytes but many other substances that can interfere with bacterial colonization and prevent antigen penetration.

Chronic protracted diarrhea of infancy: a nutritional disease.

Diarrhea is an extremely common cause of morbidity in infancy. Occasionally, it becomes protracted, leading to a vicious cycle of malabsorption, malnutrition, and failure to thrive. A number of causes of chronic diarrhea in infancy are discussed, including postinfectious enteritis, celiac sprue, cow's milk allergy, and parasitic infection. Although many mechanisms may contribute to diarrhea, a similar pathophysiologic syndrome of mucosal atrophy, inflammation, and malabsorption results. Attention should be paid to recognition of malnutrition as well as etiologic diagnosis. Therapeutic efforts should concentrate on nutritional rehabilitation, through appropriate oral elemental formulas or total parenteral nutrition. However, encouragement of breast-feeding is probably a more effective way of preventing this difficult problem.

Effect of cow's milk on the gastrointestinal tract: a persistent dilemma for the pediatrician.

The confusing area of cow's milk intolerance is explored in an attempt to define the various mechanisms whereby milk affects gastrointestinal function, resulting in clinical symptoms (diarrhea, vomiting, gastrointestinal bleeding, etc.). The adverse reaction of infants to cow's milk ingestion may relate to lactose intolerance (enzymatic), a direct toxic reaction to the mucosal surface resulting in epithelial damage, or it may be immunologically mediated. Factors such as increased intestinal permeability to milk proteins during the newborn period may also contribute to susceptibility of young infants to milk sensitivity. The relative roles of systemic (milk agglutinins) and local immunity (SIgA antibodies) in milk intolerance are discussed and differential immunologic responses (IgE versus IgA/IgM) considered in the pathogenesis. It was concluded that new techniques such as organ culture of intestinal biopsy specimens are needed to establish the diagnosis of hypersensitivity and to begin to provide ways of adequately treating the condition.

Chronic nonspecific diarrhea: dietary relationships.

Chronic nonspecific diarrhea (CNSD) is the most common cause of prolonged diarrhea without failure to thrive. Although it is most commonly seen from ages 6 to 36 months, CNSD may persist until 54 months of age. Forty-four patients with this syndrome had complete dietary histories, and were divided into four groups on the basis of their intakes and responses to its modification. Each of the four groups had significantly less fat in their diet at the time of presentation than did ten non-CNSD patients (P less than .005) presenting similarly. In three of the groups, daily fat consumption was increased, irrespective of the adequacy of their initial intakes. In all 38 patients in these groups, this dietary modification was associated with the resolution of symptoms. The fourth group, with initially normal dietary fat ingestion, did not respond to dietary therapy. The overall success rate of the regimen in this patient population was 82%. Carbohydrate, fiber, and caloric contents of the diets did not appear to play as significant a role as fat intake.

Growth failure and inflammatory bowel disease: approach to treatment of a complicated adolescent problem.

Severe retardation of linear growth occurs in a minority of children with Crohn's disease. It appears to be associated with increased disease activity and decreased caloric intake. Why some children are affected and others are not is unknown, but some degree of growth retardation is probably more prevalent than is generally appreciated. The use of somatomedin-C levels may be of some future value in predicting which children will be affected. Growth failure is often difficult to treat and requires vigorous medical and nutritional support. No current treatment is without attendant problems. Proper and frequent assessment of growth and development will help ensure intervention while growth potential still exists in these children. Large cooperative studies are needed to compare the effects of various treatment plans on the growth velocity and ultimate stature of children with Crohn's disease-related growth retardation.