Factors In Human Milk Research Articles

Milk from Mothers of Both Premature and Full-Term Infants Provides Better Antioxidant Protection than Does Infant Formula

We hypothesized that premature (PT) infants' mother's milk may provide antioxidant advantages compared with milk from mothers of full-term (FT) infants, and human milk may provide antioxidant properties not seen in infant formulas. We designed three experiments to test these hypotheses. Experiment 1 assessed resistance to oxidative stress of human milk and formulas designed for FT and PT infants. Experiment 2 determined differences in resistance to oxidative stress between milk from mothers of FT and PT infants, including analysis of catalase activity. Experiment 3 examined factors in human milk that may account for increased resistance to oxidative stress. In experiment 1, we induced physiologic oxidative stress in human milk (n = 5) and formula (n = 2) and measured ascorbate radical using electron paramagnetic resonance. Results indicated the following: 1) during oxidative stress, ascorbate may be spared in human milk compared with formula; 2) ascorbate radical production is more intense in formula compared with human milk, with or without oxidative stress; and 3) oxygen consumption in human milk is less than that in formula, with or without oxidative stress. In experiment 2, milk samples were collected from mothers of PT (n = 28) and FT (n = 17) infants at wk 1, 2, and 12 of lactation. No differences in oxygen consumption after oxidative stress appeared between PT and FT milk. Catalase levels in human milk increased with time. In experiment 3, addition of catalase, superoxide dismutase, and glutathione peroxidase to formulas (n = 4) increased resistance to oxidative stress. Denaturing endogenous enzymes did not decrease the ability of human milk to resist oxidative stress. Ferrous sulfate plus vitamin C added to human milk and formulas fortified with iron increased oxidative stress. Addition of iron chelators to formula reduced oxidative stress. In conclusion, human milk has better antioxidant protection than do formulas, perhaps because of the higher iron content of formulas. Milk from mothers of PT and FT infants has equal resistance to oxidative stress.

Identification of Activin A and Follistatin in Human Milk

Activin A is a dimeric protein member of the transforming growth factor- g (TGF- g ) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P <0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.

Breastfeeding and jaundice.

In the breastfed infant, prolongation of unconjugated hyperbilirubinemia into the third and later weeks of life in the healthy newborn is a normal and regularly occurring extension of physiologic jaundice. This is known as breastmilk jaundice. A factor in human milk increases the enterohepatic circulation of bilirubin. Insufficient caloric intake resulting from maternal and/or infant breastfeeding difficulties may also increase serum unconjugated bilirubin concentrations. This is the infantile equivalent of adult starvation jaundice. It is known as breastfeeding jaundice or "breast-nonfeeding jaundice." This increase in severity of physiologic jaundice of the newborn also results from increased enterohepatic circulation of bilirubin, but not because of a factor in human milk. In extreme cases, it may place the infant at risk for development of bilirubin encephalopathy. Optimal breastfeeding practices, which result in minimal initial weight loss and early onset of weight gain, are associated with both reduced breastfeeding jaundice and minimization of the intensity of breastmilk jaundice.

Bioactive Factors in Human Milk

This article reviews the bioactive components of human milk. Special emphasis is given to immune and nonimmune protective function of major and minor nutrients in human milk. Immune modulating components, such as cytokines, nucleotides, hormones, and growth factors, are discussed. Milk enzymes with digestive function in the newborn are reviewed.

Effect of antimicrobial factors in human milk on rhinoviruses and milk-borne cytomegalovirus in vitro.

Various antimicrobial factors present in human milk were tested for in-vitro antiviral activity against three rhinoviruses (two clinical isolates and rhinovirus 2) and an isolate of cytomegalovirus (CMV) from human milk. These factors included the gangliosides GM1, 2 and 3, sialyl-lactose, chondroitin sulphates A, B and C, prostaglandins E2 and F2alpha, monolaurin, vitamin A and the protein lactoferrin. All were tested for their ability to inhibit growth of the viruses in cell culture. Human milk was also tested for antiviral activity against these viruses. Only vitamin A, monolaurin and lactoferrin inhibited the growth of CMV, whereas both prostaglandins enhanced the growth of this virus at least four-fold. CMV infects infants from milk but, nevertheless, the milk-borne CMV isolate showed no special resistance to any of the antiviral factors tested. None of the compounds inhibited or enhanced the growth of the rhinoviruses. However, human milk decreased the growth of some of the rhinoviruses and specific secretory immunoglobulin A (sIgA) neutralised the virus.

A human milk factor susceptible to cathepsin D inhibitors enhances human immunodeficiency virus type 1 infectivity and allows virus entry into a mammary epithelial cell line.

Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells.

Hepatocyte growth factor in human milk and reproductive tract fluids.

Despite evidence indicating a role for hepatocyte growth factor (HGF) in gastrointestinal and reproductive physiology, the concentration and distribution of HGF in human breast milk (BM) and reproductive tract fluids remain unknown. Using enzyme-linked immunosorbent assay (ELISA), the HGF concentrations were determined in human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), seminal plasma (SP), and colostrum/milk samples, and expression of HGF mRNA by milk cells and AF cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). HGF is present at nearly 70-fold normal serum (0.85+/-0.15 ng/mL) concentration in FF (n = 3; x = 57+/-16 ng/mL) and AF (n = 17; x = 57+/-26 ng/mL), and is also present in hOF (n = 3; x = 4.8+/-2.3 ng/mL) and CVL (n = 8; x = 0.7+/-1.1 ng/mL) varying throughout the menstrual cycle. HGF is found at 3-times serum concentration in BM (n = 24; x = 2.3+/-1.3 ng/mL) with no significant difference between premature and full term or stage of lactation (colostrum, transitional, mature milk). HGF mRNA was detected in BM cells but not in AF cells. HGF is present in sufficient amounts to profoundly affect gastrointestinal maturation in the fetus via swallowed AF and neonate via BM, and helps to explain the increased rate of necrotizing enterocolitis (NEC) in infants of premature rupture of membrane (PROM)-complicated pregnancies, and the decreased rate in breast fed neonates. HGF in FF may be necessary for the development and maturation of the oocyte. HGF in hOF, SP, and cervicovaginal lavage (CVL) is likely to enhances epithelial cell integrity and the mucosal barrier. Thus, HGF is widely available in the reproductive tract with functions that remain to be fully elucidated.

Concentrations of Granulocyte Colony-Stimulating Factor in Human Milk after in Vitro Simulations of Digestion

Human milk contains proteins that survive digestion in the neonatal gastrointestinal tract. Our group and others have reported that granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine that influences neutrophil proliferation and differentiation, is present in human milk. We also reported that specific receptors for G-CSF are expressed on the villous enterocytes of neonates. However, the physiologic role of milk-borne G-CSF is not known. Thus, we sought to evaluate the capacity of human milk to protect G-CSF against proteolytic degradation after exposure to gastric secretions obtained from preterm (PT) and term (T) neonates at pH concentrations of 3.2, 5.8, and 7.4. Specifically, we examined degradation of 1) endogenous G-CSF in PT (n = 15) and T (n = 15) human milk; 2) recombinant human G-CSF (rhG-CSF) added to a protein-free buffer (n = 10, 5 PT and 5 T); and 3) rhG-CSF added to human milk (n = 12, 6 PT and 6 T), various commercially prepared infant formulas (n = 15), and cow's milk (n = 5). Endogenous G-CSF and rhG-CSF added to human milk resisted degradation at 1 and 2 h. However, when rhG-CSF was added to commercial formulas, >95% was degraded at 1 and 2 h at each pH level. Similarly, approximately 60% of rhG-CSF added to cow's milk was degraded at I and 2 h. We conclude that 1) endogenous G-CSF and rhG-CSF added to human milk are protected from degradation after exposure to gastric secretions at physiologic pH levels, 2) rhG-CSF added to infant formulas is not protected from degradation, and 3) it is likely that the G-CSF present in human milk is biologically available to the neonate.

Expression of Functional Immunomodulatory and Anti-Inflammatory Factors in Human Milk

In addition to well-recognized antimicrobial substances, a growing body of evidence has accrued during the last decade regarding the presence and function of immunomodulatory and anti-inflammatory factors present in human milk and their role in protecting the mature newborn as well as the premature infant against infections. In addition, it is now appreciated that a number of these factors present in human milk may actively modulate the synthesis and maturation of the recipient immune system. This complex and interactive system of bioactive substances in human milk appears ideally to be designed to function by noninflammatory mechanisms, to operate often in a complementary or synergistic manner, to resist the digestive process in the recipient gastrointestinal tract, and to supplement developmentally delayed immune factors of the infant. The in vivo fate and effects of these immune factors in human milk, however, are still poorly understood. Clinical studies in conjunction with a broader use of experimental animal models and basic research are needed in the future to address these questions.

Modulation Of Calcium Fluxes In Human Platelets By Human Milk in vitro † 21

The effect of adding human milk (HM) to washed fura-2 loaded human platelets was investigated in vitro. The effect of thrombin (0.01 U/ml) to increase intracellular free calcium ([Ca2+]i), when calcium was present in the medium, was inhibited approx. 60% by a brief (10 second) treatment with HM (6% produced the maximum inhibition). Similar inhibition of the ability of vasopressin to increase ([Ca2+]i) was also observed. The effect of thrombin to mobilize intracellular calcium in platelets (5 mM EGTA in the medium) was inhibited approx. 70% by HM (4% produced the maximum inhibition). This result suggests the HM contains a factor or factors that prevent thrombin from increasing([Ca2+]i) in platelets, perhaps by either stimulating calcium efflux from the platelet, inhibiting intracellular calcium release or stimulating calcium uptake into the intracellular stores. When calcium influx into platelets was measured using barium ions as a calcium surrogate, HM blocked the influx by approx. 70% (4% produced the maximum inhibition). This result also implies that HM also contains a factor that inhibits calcium influx across the plasma membrane. Significant effects could be observed with 1% HM on all three parameters. The inhibitory activity did not pass through a 10 KDa molecular weight cut off filter. The inhibitory effect of HM was substantially reduced by heating the milk for 15 min. at 100 C. Treating HM with 0.3 M HCl or 0.5M NaOH at room temperature for 10 min. had no effect on activity. It is concluded that HM contains a factors or factors that reduce the ability of thrombin to increase [Ca2+]i in platelets by both blocking calcium influx and preventing mobilization from internal calcium stores, alternatively the plasma membrane calcium ATPase pump may be activated. The factor is stable to mild acid and base treatment is heat labile and has a molecular weight in excess of 10 KDa. Because platelets participate in propagation of the acute inflammatory response, human milk factors that modify intracellular signalling in platelets might contribute to the anti-inflammatory character of human milk.

Evolution of immunologic functions of the mammary gland and the postnatal development of immunity.

Physiologic delays in production of immune factors occur in mammals including Homo sapiens. This finding is counter to a basic tenet of biologic evolution, because such delays increase the risk of infections. The disadvantage is, however, offset by defense factors in milk of the species in whom the developmental delay occurs. Reciprocal relationships between the production of immune factors by the lactating mammary gland and the production of those defense agents during early infancy are found in all investigated mammalian species. Thus, the evolution of these processes is closely related. Certain immunologic components of milk are highly conserved, whereas others vary according to the species. The variations most likely evolved by genetic mutations and natural selection. In addition, the immune composition of mammalian milks is associated with developmental delays in the same immunologic agents. Furthermore, most closely related mammals, such as humans and chimpanzees, are most similar in the defense agents in their milks and the corresponding developmental delays in their immune systems. Defense factors in human milk include antimicrobial agents (secretory IgA, lactoferrin, lysozyme, glycoconjugates, oligosaccharides, and digestive products of milk lipids), antiinflammatory factors (antioxidants, epithelial growth factors, cellular protective agents, and enzymes that degrade mediators of inflammation), immunomodulators (nucleotides, cytokines, and antiidiotypic antibodies), and leukocytes (neutrophils, macrophages, and lymphocytes). Because of a lack of geographic/ethnic variation in the immunologic composition of human milk and corresponding immunologic delays in infants, these evolutionary processes seem stable. This is supported by investigations of diverse populations that indicate that this evolutionary outcome is highly beneficial to human infants.

Infant influenza.

Reports on the symptoms of infant influenza (less than 1 year of age) are very few. From 1989 to 1996, 105 infants with influenza were examined. The symptoms, nutrition and source of infection of the 105 patients were investigated in Sakuma Pediatric Clinic and viral studies were performed at the Kitakyushu City Institute of Environmental Sciences. The symptoms of patients under 6 months old are very mild, with a low maximum body temperature and a short duration of fever. Other complications are few. The symptoms gradually become more severe with each month of age. The ratio of patients displaying a diphasic fever with two peak temperatures increases with each month of age. Over 50% of patients over 9 months old have this diphasic fever with two peak temperatures. It is inconclusive whether an anti-infective factor in human milk exists or not. Of the 105 patients in this study, 28 were under 4 months of age: of these 28, all but 3 were infected by family members.

An IgA, IgG and IgM-containing human immunoglobulin for oral administration to high-risk bottle-fed newborns. I. Immunochemical characterisation and antibody activity.

Antibodies to a wide spectrum of infectious agents belonging to the IgA, IgM and IgG isotypes are thought to be one of the protective factors in human milk. Cow milk-fed newborns are at an increased risk of infections as well as of allergic diseases and of necrotising enterocolitis. A reasonable approach would be to add to the milk formula fed to them the immunoglobulins present in human milk. We developed a pasteurised immunoglobulin preparation from pooled donor plasma ('Orabulin') containing 75% IgG, 18% IgA and 6% IgM for feeding to high-risk bottle-fed babies. Its molecular composition was studied by HPLC and by SDS-PAGE. The levels of IgA, IgG and IgM antibodies in Orabulin were compared to these in the immunoglobulin fraction of human colostrum and an enrichment was found. It is suggested that the presence of a standardised amount of human IgM in an immunoglobulin preparation intended for feeding to newborns may bring an additional advantage because of the high opsonising and virus-neutralising activity of the antibodies of this isotype.

Granulocyte-macrophage colony-stimulating factor in human milk

Granulocyte-macrophage colony-stimulating factor in human milk

Trophic Factors for the Gastrointestinal Tract

GI trophic factors that influence the coordinated pre- and postnatal growth and development of the GI tract have been identified. Studies in animals and humans demonstrate that GI trophic factors can initiate cellular growth and expression of differentiated function, and they are important in adaptation and repair following injury. Systemically as well as enterally administered growth factors can stimulate GI growth and maturation, suggesting that trophic factors in the serum of neonates may modulate GI growth via receptors on the serosal membranes of enterocytes. GI trophic factors may be synthesized endogenously or provided postnatally in milk. GI trophic factors in human milk play an important role in regulating the adaptive functional changes that accompany the transition to postnatal enteral feedings. Although human milk growth factors do not appear to be essential for infant survival, the elevated risk of gastrointestinal-related illnesses in formula-fed as compared with breast-fed infants (diarrhea, necrotizing enterocolitis, colitis, Crohn's disease) suggest that bioactive compounds in human milk contribute to the apparent protective effect of breast feeding. GI trophic factors have the potential to be used therapeutically to enhance GI maturation and repair following injury. These applications may be particularly useful in the premature or postsurgical infant. Several issues require further research, including (1) the mechanism of action, (2) the efficacy of oral versus systemic administration, (3) characterization of the complex interactions between the various growth factors, because some appear to act synergistically, (4) the effect of exogenously administered growth factors on endogenous production of that factor, its receptor, or other growth factors, (5) the effect of growth factors upon tissues not directly associated with the GI tract, and (6) the determination of safe and effective upper limits.

Dermal Cylindroma

Thirteen dermal cylindromas (DC) have been studied immunohistochemically using a panel of antibodies that stain different portions of normal eccrine and apocrine glands. Distinct staining patterns were found in the different cell populations of the tumor. Although the expression of cytokeratins (CK) 19 and 1/10/11 in occasional duct structures could indicate excretory (ductal) differentiation, a link between DC and apocrine secretory coil is suggested by the expression of alpha-1-antichymotrypsin, lysozyme, human milk factor globulin 1, alpha smooth muscle actin (1A4), and CK 8 and 18. The presence of intermingled S-100 protein-, HLA DR-, and CD1a-positive cells argues for the existence of Langerhans cells within the neoplasm. DC shares epithelial membrane antigen, carcinoembryonic antigen, mucin-like carcinoma-associated antigen (B12), laminin, collagen IV, fibronectin, and CD34(QBEND/10) expression with both eccrine and apocrine glands.

Identification of macrophage colony-stimulating factor in human milk and mammary gland epithelial cells.

Human milk contains cellular and soluble host-protective components. Macrophages in human milk constitute a major cellular component in contrast to those in the peripheral blood. We have investigated a mechanism of local expansion of macrophages in human milk. First, biologically active macrophage colony-stimulating factor (M-CSF) was identified in human milk. The maximal concentrations of M-CSF in human milk were 10- to 100-fold higher than those in the serum. The concentrations of M-CSF in the milk had no correlation with those in the serum. M-CSF was immunohistochemically detected in the epithelial cells of the ducts and alveoli of the mammary gland. In situ hybridization study confirmed the local synthesis of M-CSF in the mammary gland epithelial cells. A possible role of female sex steroids was discussed in the regulation of M-CSF production by mammary gland epithelial cells.

Immunoglobulin and non-immunoglobulin components of human milk inhibit Clostridium difficile toxin A-receptor binding.

Clostridium difficile is isolated from the intestinal tracts of > 50% of healthy infants. The mechanism by which intestinal colonisation of infants by toxigenic C. difficile is generally asymptomatic is unknown but may reflect the presence in human milk of neutralising activity against C. difficile toxin A. On this basis, the ability of human milk to inhibit the binding of toxin A to a purified hamster brush border membrane receptor was determined. Ten milk samples from healthy volunteers in various stages of lactation inhibited the binding of toxin A to the receptor by an average of 90%. Heating and dialysis did not significantly alter the inhibitory activity of any of the milk samples. Human milk protected adult hamsters against a lethal challenge with toxin A but had no effect on the cytotoxic activity of the toxin. SDS-PAGE and ligand blot analyses showed that there were at least four distinct factors in human milk that specifically bound toxin A. Thiophilic adsorption chromatography was used to separate immunoglobulin from non-immunoglobulin components of human milk. IgA was the only immunoglobulin detected in human milk and > 90% of this immunoglobulin was recovered after purification by thiophilic adsorption. Both the unbound non-immunoglobulin and bound immunoglobulin fractions of human milk inhibited the binding of toxin A to the purified receptor. These results suggest that human milk may be important in protecting infants against C. difficile-associated intestinal disease.

Defense factors in human milk

The newborn infant is more susceptible to infections than older children or adults. This is because all three lines of defense against microbial invasion, ie, barrier functions, innate immunity, and specific immunity, are to a greater or lesser extent immature at birth. Nature's response to the imma

The effect of nutritional additives on anti-infective factors in human milk.

It has become a common practice to supplement human milk with a variety of additives to improve the nutritive content of the feeding for the premature infant. Twenty-two freshly frozen human milk samples were measured for lysozyme activity, total IgA, and specific IgA to Escherichia coli serotypes 01, 04, and 06. One mL aliquots were mixed with the following: 1 mL of Similac, Similac Special Care, Enfamil, Enfamil Premature Formula, and sterile water; 33 mL of Poly-Vi-Sol, 33 mg of Moducal, and 38 mg of breast-milk fortifier, and then reanalyzed. Significant decreases (41% to 74%) in lysozyme activity were seen with the addition of all formulas; breast-milk fortifier reduced activity by 19%, while no differences were seen with Moducal, sterile water, or Poly-Vi-Sol. No differences were seen in total IgA content, but some decreases were seen in specific IgA to E. coli serotypes 04 and 06. E. coli growth was determined after 3 1/2 hours of incubation at 37 degrees C after mixing. All cow-milk formulas enhanced E. coli growth; soy formulas and other additives preserved inhibition of bacterial growth. Nutritional additives can impair anti-infective properties of human milk, and such interplay should be considered in the decision on the feeding regimen of premature infants.