W A Walker

PROTEIN UPTAKE BY THE INTESTINE: EVIDENCE FOR ABSORPTION OF INTACT MACROMOLECULES

The ability of the mature small intestine to absorb intact protein was studied in adult rats prepared with duodenal cannulas, mesenteric lymph fistulae, and portal vein cannulas. After infusion of tritiumlabeled bovine serum albumin (3H-BSA) into the duodenum, approximately 2% of the 3H-BSA was transmitted in macromolecular form into the lymph and blood. The amount of 3H-BSA was directly proportional to the amount administered. The molecular weight of the absorbed macromolecule was shown to exceed 50,000 by Sephadex gel filtration. Absorbed protein was identified immunologically as BSA on Ouchterlony plates with monospecific rabbit anti-BSA. The findings provide additional evidence for the concept that intact protein can traverse the mature intestinal epithelium under physiologic conditions.

Development of Gastrointestinal Mucosal Barrier. I. The Effect of Age on Intestinal Permeability to Macromolecules

Summary: Indirect evidence has suggested that increased quantities of antigen may penetrate the intestinal mucosa and enter the systemic circulation during the newborn period compared to adult life. However, no direct measurement of macromolecular transport has been reported as a function of perinatal age. To study this process, we administered 100 mg of tritiated bovine serum albumin ([3H]BSA) by gavage to rabbits at birth, one wk, 2 wk, 6 wk, and one year of age and measured plasma radioactivity 4 hr after gavage. Plasma concentration of trichloroacetic acid insoluble radioactivity and immunoreactive bovine serum albumin radioactivity decreased significantly after one wk of age. When adult animals were gavaged with the same amount of [3H]BSA per body weight as the one-wk-old animals, they failed to transport as much of the antigen as the younger animals. This study, therefore, provides objective evidence that the intestinal mucosal barrier of newborns may be incompletely developed at birth and allow increased intestinal transport of antigens into the circulation.Speculation: The development of an animal model for the study of gastrointestinal host defense during the neonatal period may ultimately provide the basis for a better understanding of the mechanisms responsible for intestinal uptake of antigenic molecules and their contribution, if any, to the pathogenesis of human disease. Of particular importance is the accurate quantitation of immunologically reactive antigen absorbed by newborn animals. Using immunologic techniques to quantitate macromolecular transport, it can be determined whether conditions (enteric delivery of nutrients, growth factors in natural milk, etc.) thought to stimulate intestinal epithelial cell turnover can also accelerate the development of the intestinal mucosal barrier and thereby contribute to the protection of the infant from potentially harmful luminal antigens.

Human milk as a carrier of biochemical messages.

Not only does breast milk provides an ideal nutrient composition for the newborn, but it also contains a variety of substances that may actively influence growth and development of the infant and stimulate neonatal protection against gastrointestinal diseases. Hormones, growth factors, cytokines and even whole cells are present in breast milk and act to establish biochemical and immunological communication between mother and child. In addition, milk nutrients such as nucleotides, glutamine and lactoferrin have been shown to influence gastrointestinal development and host defense. The unique properties of milk as a mediator of biochemical messages will be presented and the clinical significance of breastfeeding in the prevention of neonatal gastrointestinal diseases will be discussed.

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.

Vascular Endothelial Growth Factor (VEGF) Is Present in Human Breast Milk and Its Receptor Is Present on Intestinal Epithelial Cells

VEGF (vascular endothelial growth factor) is a multifunctional cytokine active on blood vessel cells. The present study measured VEGF in the aqueous phase of human milk and examined how the concentration varied with gestational age and the duration of lactation after birth. We hypothesized that VEGF-specific receptors were present on the apical surface of intestinal epithelial cells. The concentration of monomeric VEGF (containing 165 residues) measured by ELISA in the breast milk was 2 orders of magnitude greater than that measured in the serum of normal adults. The VEGF165 concentration in the first week of lactation was greater in the breast milk of mothers of full-term than in preterm babies (p < 0.05). The concentration in the breast milk of mothers of full-term infants decreased (p < 0.01) after the first week of lactation. Scatchard analysis of radioligand-receptor binding showed the presence of specific receptors for 125I-VEGF165 on the surface of Caco-2, an intestinal epithelial cell line, with a kd of 2.85 to 4 nM. Reverse transcriptase PCR of Caco-2 cell RNA showed mRNA for the VEGF receptor flt-1. In conclusion, VEGF is present in high concentrations in breast milk and binds to specific receptors on cells derived from intestinal epithelium.

Iron saturation alters the effect of lactoferrin on the proliferation and differentiation of human enterocytes (Caco-2 cells)

Recent studies have indicated that lactoferrin may act as a cell mitogen. The effect of human and bovine lactoferrins on the proliferation and differentiation of a human intestinal epithelial cell line (Caco-2) was investigated and compared with that of human transferrin. Caco-2 cells were cultured in serum-free media supplemented with both iron-unsaturated and -saturated forms of the iron-binding proteins. Cell proliferation and differentiation were evaluated by examining growth curves and measuring sucrase and alkaline phosphatase activities of brush border membrane fractions, respectively. The iron-binding status of lactoferrins and transferrin affected the proliferation of Caco-2 cells. The iron-saturated forms of human (S-hLf), bovine (S-bLf) lactoferrins and human transferrin (S-hTf) enhanced cell proliferation, while iron-unsaturated forms (U-hLf, U-bLf, and U-hTf) suppressed it. Iron-binding status also determined the effect of lactoferrin and transferrin on cellular differentiation, but this effect differed for different brush border enzymes. S-hTf enhanced sucrase activity more than S-hLF or S-bLf. Both U-hLf and U-bLf markedly suppressed sucrase activity. U-hTf suppressed alkaline phosphatase activity appreciably, while the other iron-binding proteins showed no significant effect on it. Lactoferrin and transferrin may modulate the proliferation and differentiation of intestinal epithelial cells, but their efficacy depends on their saturation with iron.

Breast Milk-Transforming Growth Factor-β2 Specifically Attenuates IL-1β-Induced Inflammatory Responses in the Immature Human Intestine via an SMAD6- and ERK-Dependent Mechanism

Background: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. Objectives: We hypothesized that transforming growth factor-β2 (TGF-β2), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). Methods: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1β (IL-1β) with or without TGF-β2. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. Results: TGF-β2 significantly attenuated IL-1β-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-β2 reduced the IL-1β-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-β2 markedly inhibited IL-1β-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-β2 on IL-1β-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. Conclusions: TGF-β2 attenuates IL-1β-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-β2 is dependent on SMAD6. Breast milk TGF-β2 may provide the neonate with important immunoregulatory support. TGF-β2 might provide a novel means to improve intestinal immunophysiology in premature neonates.

Food Proteins and Gut Mucosal Barrier. II. Differential Interaction of Cow's Milk Proteins with the Mucous Coat and the Surface Membrane of Adult and Immature Rat Jejunum

ABSTRACT: Two in vitro intestinal models were used to investigate postnatal maturational changes of the gut barrier functions. Microvillus membrane (MVM) preparations were studied for surface binding, and everted gut sacs were studied for mucous coat binding, breakdown and uptake of radioiodinated bovine serum albumin (BSA), and β-lactoglobulin (β-LG). Surface binding of these proteins to MVM was weak and nonspecific. There was more binding of both proteins to immature MVM (BSA: newborns, 2.74 ± 0.52%, adults, 1.08 ± 0.17%, p < 0.001; β LG: newborns, 6.30 ± 0.54%; adults, 2.05 ± 0.07, p < 0.001). In contrast to MVM binding characteristics, mu-cous coat binding of the cows milk proteins to immature gut sacs was significantly less (BSA: preweanlings, 0.94 ± 0.30 μg 125-I-protein Eq/mg mucosal protein; adults, 3.06 ± 0.74, p < 0.001; β-LG: preweanlings, 5.61 ± 1.48; adults, 9.83 ± 1.33, p < 0.001). Protein binding and uptake were correlated in the immature animals (r = 0.76, p < 0.001 for BSA and r = 0.85, p < 0.001 for β-LG). More β-LG was bound and taken up than BSA in the preweanlings (p < 0.001). Trichloroacetic acid precipitation studies showed that, even in the immature rats, β-LG was much more readily broken down by mucosa-associated enzymes than BSA. Immature animals showed less protein breakdown than adult controls. Decreased protein breakdown and mucous coat binding as well as increased MVM binding may account for the increased uptake of intact food antigens in the newborn. Differences between increased MVM binding and decreased mucous coat binding of cows milk proteins are attributed to protective elements such as the mucus layer which is present in the gut sac model but lacking in MVM.

Development of the Gastrointestinal Mucosal Barrier

Prior to weaning, young rabbits take up increased amounts of macromolecules across the intestine and into the circulation. The increased macromolecular uptake may be due in part to decreased intestina

Development of intestinal host defense: an increased sensitivity in the adenylate cyclase response to cholera toxin in suckling rats.

ABSTRACT: To determine if developmental variations existed in the second messenger system that mediates cholera toxin (CT) action, the adenylate cyclase (AC) response was studied in 2-wk-old suckling and 6-wk-old weaned rats. AC was assayed in the proximal small intestine 4 h after intraduodenal administration of various doses of CT. Dose-effect analysis showed a 9-fold increase in the sensitivity of the CT-activated cyclase response in suckling rats when measured by the EDso, expressed as ng CT/g body wt (0.03 for 2 wk, 0.27 for 6 wk). When the CT dose was expressed as µ/animal, suckling rats were 50 times more sensitive than 6-wk-old rats. In addition, the CTinduced fluid secretion was closely correlated with the elevated cyclase activities (correlation coefficient: 0.83 for 2 wk, 0.93 for 6 wk). Furthermore, more fluid seemed to be secreted/unit wt of gut in the sucklings, even when the same level of enzyme activity was compared. A maximum of 3- to 4-fold rise in AC activation occurred at 0.5 ng CT/ g body wt, but both the basal and the maximal stimulated levels of AC were not developmentally different. This study demonstrates an in vivo increase in AC responsiveness to CT that may be in part responsible for the increased incidence of toxigenic diarrhea in neonates.