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Human milk plays a significant role in postnatal gut maturation, and may serve as a vehicle for transmitting developmental signals from mother to neonate. Various growth factors, cytokines, and gut peptides have been isolated from human milk, often in quantities that exceed maternal serum levels. There exists an interrelated system in which compailisientation of milk’s components lead to controlled release of nutrients and metabolites to the breastfed infant. Various proteins are differentially sequestered within the compaitrnents of human milk. These proteins become equentially processed within the gastrointestinal tract. The majority of studies on human milk have focused on the aqueous (defatted) fraction of whole milk rather than the various compartments that include fat. Yet, the fat compartment composed of the milk fat globule with its associated plasma membrane appears to provide a unique delivery system of bioactive substances to the newborn gut. A growth factor that is present in all compartments of human milk is transforming growth factor-alpha (TGFα), a member of the epidermal growth factor (EGF) family. TGFα has biological activity in its transmembrane as well as its mature, cleaved form. As a gut peptide, TGFa contributes to the growth-promoting properties of human milk through activation of EGF receptors (EGFr) of human fetal small intestinal cells in vitro. Higher concentrations of TGFα are present in the fat compartment of human milk. It is unknown if TGFa in human milk fat is associated with the milk fat globule membrane (MFGM). Based on the transmembrane structure of TGFα, we hypothesized that TGFα would be present in the milk fat globule (MFG), and with further isolation, associated with the MFGM.

Objective: To determine if the addition of human milk fortifier (HMF) affects the distribution, immunoreactivity, or molecular mass profile of transforming growth factor-α (TGF-α) within the compartments of human milk. Methods: Fifteen milk samples were obtained. Each sample was divided into two aliquots; a powdered HMF was added to the first aliquot. TGF-α concentration was measured via radioimmunoassay in whole milk and its aqueous and fat fractions ± HMF. TGF-α molecular mass profiles of the samples (v/v) were measured via Western blotting. Results: TGF-α concentration (mean ± SD) in fortified whole milk (15.7 ± 7.1 pg/100 μl) vs. nonfortified whole milk (14.8 ± 8.0 pg/100 μl) and in the aqueous fraction of fortified (14.0 ± 2.7 pg/100 μl) vs. nonfortified (14.0 ± 3.5 pg/100 μl) did not differ statistically. There was, however, a marked decrease in the concentration of TGF-α in the fat fraction of fortified (30.6 ± 2.8 pg/100 μl) vs. nonfortified (98.0 ± 6.9 pg/100 μl) milk samples. Western blot for TGF-α in whole milk and its separated fractions revealed characteristic bands at 6.5, 12–16, 22, 26–30 and 46 kD. HMF alone and HMF with sodium taurocholate had a prominent band at 18 kD and fainter bands at 6.5, 26–30, and 46 kD. While whole and aqueous milk samples with HMF also consistently showed the 18-kD band, in 8/15 fat fraction samples with HMF the 18-kD band was nondetectable and was only faintly detectable in the remaining 7/15 samples. Conclusions: It appears that HMF differentially alters the biochemical profile of human milk with regard to TGF-α concentration and molecular mass profile. What effect this alteration in human milk biochemistry has on neonatal gut function remains unknown.

Objective: An in vitro model was devised to compare the relative effects of recombinant transforming growth factor-alpha (TGFα), aqueous human milk, and human milk macrophage (HMM) medium on human fetal small intestinal cell (FHs-74) proliferation. Methods: Recombinant TGFα at increasing concentrations (range 0.01–1,000 ng/ml media), the aqueous fraction of human milk (AHM), or HMM medium was added to FHs-74 cells in the presence or absence of a neutralizing TGFα antibody (1 µg/ml medium). At 24 h, cell proliferation was measured and expressed as percent control. The experimental variables were (1) activators of cell growth (TGFα, AHM, and HMM medium); (2) increasing concentrations of TGFα, and (3) neutralizing antibody to TGFα. The dependent variable for all experiments was cell proliferation. Results: Significant effects for growth stimulators and TGFα concentration as measured by cell proliferation were found. Specifically, there was a dose-dependent effect of TGFα on cell proliferation to the 5-ng/ml concentration, with a plateau reached in cell proliferation at higher concentrations. The stimulatory effect of TGFα was decreased in the presence of TGFα antibody (mean ± SD 22 ± 7.1% decline, p < 0.001). In the presence of TGFα antibody, there was a 25 ± 3.1% decline in HM-stimulated growth (p < 0.004), and a 27.6 ± 3.2% decline in HMM medium-stimulated growth (p < 0.001). Conclusions: Neutralization of recombinant TGFα and that present in human milk and HMM medium by TGFα antibody led to a consistent decrease in in vitro human fetal small intestine epithelial proliferation without affecting cell viability. These results support the hypothesis that TGFα, whether derived from human recombinant sources, human milk or HMM medium has a measurable, trophic effect on in vitro human gut epithelial cells.

Objective: Transforming growth factor-α (TGF-α), present in human milk, is thought to play a significant role in postnatal gut development. Macrophages, which abound in human milk, are known to secrete various isoforms of TGF-α in other areas of the body. It was hypothesized that human milk macrophages (HMM) secrete TGF-α, and HMM are a source of TGF-α isoforms present in human milk. We sought to measure in vitro HMM TGF-α secretion, and to compare the TGF-α isoform(s) present in human milk with those secreted by HMM. Study Design: Colostrum and mature milk samples, obtained from mothers (n = 48) on postpartum days 3-30, were centrifuged. HMM were isolated, placed in culture for 24 h, and cell-free media collected. The biochemical forms of TGF-α in media in human milk supernatant samples were identified by Western blot analysis under reducing conditions. The concentration of the mature (6-kD) TGF-α isoform in those samples was quantified using a radioimmunoassay. Results: Western blot analysis under reducing conditions identified a single 6-kD TGF-α isoform in all human milk supernatant samples tested, but variable (6- and 30- to 46-kD) TGF-α isoforms in HMM media. The mean (± SE) concentration of the 6-kD TGF-α isoform found in human milk supernatants was 706 ± 88 pg/ml and 17.6 ± 2.6 pg/ml in HMM media. Conclusion: These experiments show that HMM secrete TGF-α in biochemical forms both similar to and distinct from that found in human milk supernatant.

Human milk fortifier (HMF) is routinely added to human milk feedings to augment nutrient and caloric delivery to premature infants. We previously reported that the TGFα immunoreactive concentration in the fat, but not the aqueous compartment, of human milk is decreased with the addition of HMF[Ped Res 1997; 41(4):515A]. TGFα is detectable in HMF when reconstituted in sterile water (concentration 2 pg/mL), but not in commercially available formulas. It is unknown if HMF alters the biochemical properties, including the TGFα isoform profile, of human milk. To address this question, the TGFα isoform profiles of whole milk and its fractions were compared with the same fractions with a commercially available powered HMF (Mead-Johnson). Fifteen whole milk samples were collected from 12 mothers on postpartum days 4 through 90; gestational age of the infants ranged from 26-40 weeks. These samples were divided into 2 aliquots of 25 mL; 1 packet of HMF (0.96 g) was added to the first aliquot. Two cc of whole milk were retained for later analysis. The remainder of each sample was centrifuged at 1000 X g for 20 min at 4°C to achieve separation of the fat and aqueous fractions. Sodium taurocholate, a bile salt, was added 1:1 (v/v) to the fat for emulsion. Western blot analysis of the samples was performed with a monoclonal TGFα antibody (0.5 μg/mL; RDI) and a polyclonal secondary antibody (1:20,000; Pierce) using 15% SDS-PAGE electrophoresis- semi-dry transfer method. Isoform bands were visualized by enhanced chemilluminescence(Amersham). TGFα isoforms were detected in whole milk and its separated fractions at 6.5, 12-16, 22, 26, and 30 kD. HMF alone and HMF with sodium taurocholate had a single detectable band at 18 kD. While whole and aqueous milk samples with HMF present also showed the 18 kD band, the fat fraction samples with HMF did not have the 18 kD band. It appears that HMF differentially alters the biochemical profile of human milk with regard to TGFα concentration and the immunodetection of TGFα isoforms. What effect this alteration in human milk biochemistry has on neonatal gut function remains unknown.