Regional variations in human milk oligosaccharides in Vietnam suggest FucTx activity besides FucT2 and FucT3.

Breastfeeding is the normal way of providing young infants with the nutrients they need for healthy growth and development (WHO). Human milk oligosaccharides (hMOS) constitute a highly important class of nutrients that are attracting strong attention in recent years. Several studies have indicated that hMOS have prebiotic properties, but also are effective in anti-adhesion of pathogens, modulating the immune system and providing nutrients for brain growth and development. Most of the latter functions seem to be linked to the presence of fucose-containing immunodeterminant epitopes, and Neu5Ac-bearing oligosaccharides. Analysis of hMOS isolated from 101 mothers' milk showed regional variation in Lewis- and Secretor based immunodeterminants. Lewis-negative milk groups could be sub-divided into two sub-groups, based on the activity of a third and hitherto unidentified fucosyltransferase enzyme. Analysis of hMOS remaining in faeces showed three sub-groups based on hMOS surviving passage through the gut, full consumption, specific partial consumption and non-specific partial consumption, fitting previous findings.

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids.

INTRODUCTION: The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS: Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS: RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION: The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.

Influence of a Concurrent Exercise Training Program During Pregnancy on Colostrum and Mature Human Milk Inflammatory Markers: Findings From the GESTAFIT Project.

BACKGROUND: Although exercise reduces systemic inflammation, information regarding its influence on human milk is scarce or inexistent. Research Aim: The aim of this study was to investigate the influence of an exercise intervention during pregnancy on colostrum and mature human milk inflammatory markers. METHODS: The authors conducted a pseudorandomized controlled trial. The exercise group followed a concurrent aerobic and strength training, three 60-minutes sessions per week, from the 17th gestational week until delivery. For the specific aims of this study, only women able to produce enough milk were included for data analyses, resulting in 24 exercise and 23 control women. Colostrum and mature human milk proinflammatory and anti-inflammatory cytokines (fractalkine, interleukin [IL]-1ß, IL-6, IL-8, IL-10, interferon [IFN]-γ, and tumor necrosis factor [TNF]-α) were measured using Luminex xMAP technology. RESULTS: The mothers who followed the exercise program had 36% lower IL-8 and 27% lower TNF-α concentrations in their colostrum than those in the control group ( p < .05 and p < .01, respectively). The colostrum from mothers who followed the exercise program also presented borderline significant 22% lower IL-6 ( p < .100). The mature milk from mothers who followed the exercise program had 30% greater fractalkine ( p = .05) and borderline significant 20% higher IL-10 ( p = .100). The exercise intervention did not affect IFN-γ concentrations. CONCLUSIONS: This concurrent exercise program promoted a less proinflammatory profile in human milk, especially in colostrum. Moreover, it might increase mature human milk fractalkine, which could induce a greater neurodevelopment and neuroprotection in the newborn. This trial was registered at ClinicalTrials.gov (NCT02582567) on October 20, 2015.

Relation between polyphenols, malondialdehyde, antioxidant capacity, lactate dehydrogenase and toxic elements in human colostrum milk.

The present study investigated the interrelationships between the concentration of total polyphenols (TP), malondialdehyde (MDA), total antioxidant capacity (TAC), lactate dehydrogenase activity (LDH) and content of toxic elements (Al, As, Cd, Ni, Pb) in human colostrum milk (n = 75), and further assessed their potential association with maternal lifestyle characteristics. As and Cd were always below detection limits while Al, Ni and Pb were found at the level of 89.7, 6.2 and 1.3 µg L-1, respectively. Concentrations of TP and MDA, in the studied group were 46.91 ± 21.25 mg GAE L-1 and 0.66 ± 0.27 nmol mL-1, respectively, and were inversely correlated (Rs = -0.32; p < 0.01). TP and TAC increased significantly with maternal consumption of vegetables (Rs = 0.25 and Rs = 0.37, respectively; p < 0.05). Concentration of Al was positively correlated with MDA (Rs = 0.21; p < 0.01) and negatively with TP (Rs = -0.28; p < 0.01). Positive correlation was also found between Pb and MDA (Rs = 0.32; p < 0.01). No association with place of living (urban/rural), women's age and former smoking were found for any studied milk parameter. The results add to the general understanding of factors influencing redox balance in milk and potentially affecting its quality.

Breast Milk from Smokers Contains Less Cholesterol and Protein and Smaller Size of Apolipoprotein A-I Resulting in Lower Zebrafish Embryo Survivability.

BACKGROUND: To determine the quality of breast milk (BM), we compared the functions of BM from ex-smokers and nonsmokers. SUBJECTS AND METHODS: We analyzed the contents of lipids, glucose, and protein in BM from ex-smokers (10 cigarettes/day for 13 ± 3 years) as well as infant formula. RESULTS: Nonsmokers' BM showed 2.4- and 1.4-fold higher cholesterol and protein contents, respectively, than BM from smokers. Infant formula contained almost no cholesterol, but did show remarkably higher glucose and triglyceride levels than BM. Microinjection of BM (50 nL) from nonsmokers and smokers into zebrafish embryos resulted in 59% and 44% survival, respectively, whereas formula injection resulted in 31% survival. The higher cholesterol and protein contents of BM were directly correlated with higher embryo survivability, suggesting that cholesterol content is directly and critically associated with growth of neonate infants. Smokers' BM contained smaller-sized apolipoproteinA-I (apoA-I) (24.4 ± 0.2 kDa) than BM from nonsmokers (26.7 ± 0.4 kDa), suggesting that putative modification and cleavage occurred in apoA-I. BM containing higher molecular weight apoA-I resulted in higher embryo survivability. CONCLUSIONS: Smoking before pregnancy can affect the composition and quality of BM, resulting in almost complete loss of cholesterol and protein, especially lactoferrin, lactalbumin, and apoA-I, accompanied by proteolytic degradation. These impairment effects of BM are associated with elevation of oxidative stress and lower embryo survivability.

Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs.

Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants.Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM.Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL · kg-1 · d-1) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL · kg-1 · d-1) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated.Results: A high bacterial load in the UP (6×105 colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidant markers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal health was also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs fed UVC-treated milk than in pigs fed UP and those fed HP-treated milk in both cecum contents (20% and 10%) and distal intestinal mucosa (24% and 20%) (all P < 0.05).Conclusions: UVC is better than HP treatment in preserving bioactive factors in DM. UVC-treated milk may induce better weight gain, intestinal health, and resistance against bacterial infections as shown in preterm pigs as a model for DM-fed preterm infants.

[Not Available]. / Antioxidantes, especies reactivas de oxígeno y daño oxidativo asociado a la presencia de plaguicidas organoclorados en la leche materna.

Introducción: los plaguicidas organoclorados (POC) pueden incrementar la producción de especies reactivas de oxígeno (ERO). Tales efectos pueden ser contrarrestados por el sistema antioxidante, el cual se encuentra también en la leche materna. Objetivo: evaluar los indicadores de estrés oxidativo en leche materna asociados a la presencia de POC y su relación con la ingesta de alimentos marinos. Métodos: la leche materna fue colectada de 108 mujeres lactantes. Los indicadores de estrés oxidativo (actividad enzimática antioxidante, concentración de glutatión, producción de radical superóxido [O 2•- ], concentración de peroxidación de lípidos y carbonilos proteicos) se analizaron por espectrofotometría. Las concentraciones de POC se midieron por cromatografía de gases. Resultados y discusión: la producción de O 2•- no presentó relación significativa con las concentraciones de POC. Se encontraron correlaciones significativas entre las concentraciones de POC y la actividad de las enzimas antioxidantes (actividad de glutatión reductasa [GR] y concentración de aldrín [r = - 0,5], actividad de superóxido dismutasa [SOD] y concentración de α-HCH [r = 0,45]). El daño oxidativo mostró baja correlación con el contenido de POC (r < 0,30, p < 0,05). Es posible que los niveles de POC no sean suficientes para incrementar la producción de O 2•- , ya sea que el incremento en la producción de ERO se deba a especies reactivas diferentes a O 2•- o debido a que la capacidad antioxidante es suficiente para evitar el daño oxidativo en leche materna. Conclusión: los resultados de este estudio sugieren que la dieta marina no es un factor determinante en el nivel de contaminación por POC, ni en el daño oxidativo presente en leche materna.

Proteolytic Systems in Milk: Perspectives on the Evolutionary Function within the Mammary Gland and the Infant.

Milk contains elements of numerous proteolytic systems (zymogens, active proteases, protease inhibitors and protease activators) produced in part from blood, in part by mammary epithelial cells and in part by immune cell secretion. Researchers have examined milk proteases for decades, as they can cause major defects in milk quality and cheese production. Most previous research has examined these proteases with the aim to eliminate or control their actions. However, our recent peptidomics research demonstrates that these milk proteases produce specific peptides in healthy milk and continue to function within the infant's gastrointestinal tract. These findings suggest that milk proteases have an evolutionary function in aiding the infant's digestion or releasing functional peptides. In other words, the mother provides the infant with not only dietary proteins but also the means to digest them. However, proteolysis in the milk is controlled by a balance of protease inhibitors and protease activators so that only a small portion of milk proteins are digested within the mammary gland. This regulation presents a question: If proteolysis is beneficial to the infant, what benefits are gained by preventing complete proteolysis through the presence of protease inhibitors? In addition to summarizing what is known about milk proteolytic systems, we explore possible evolutionary explanations for this proteolytic balance.

Transforming growth factor-ß2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.

Human milk contains biologically important amounts of transforming growth factor-ß2 isoform (TGF-ß2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-ß2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-ß bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-ß2 (rTGF-ß2) to milk prior to feeding. Milk-borne TGF-ß bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-ß2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-ß2 (20-40 nM) to human preterm milk samples failed to increase TGF-ß bioactivity in milk. Milk-borne TGF-ß2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-ß2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-ß2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.

Purine nucleoside phosphorylase and the enzymatic antioxidant defense system in breast milk from women with different levels of arsenic exposure.

Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk.

Purina nucleósido fosforilasa (PNP) es una enzima ubicua que desempeña un papel importante en la desintoxicación del arsénico (As). As es un metaloide tóxico presente en el aire, el suelo y el agua; es abundante en el medio ambiente y se transfiere fácilmente a lo largo de la cadena trófica, encontrándose incluso en la leche materna humana. Información sobre la síntesis de la leche materna y su potencial mecanismo de defensa contra tóxicos es escasa. En este estudio, se cuantificó la actividad de PNP y de las enzimas antioxidantes así como la concentración de glutatión (GSH) y de arsénico total ([TAs]) en muestras de leche materna. La actividad de PNP, superóxido dismutasa (SOD), catalasa (CAT), glutatión S-transferasa (GST), glutatión peroxidasa (GPx), glutatión reductasa (GR) y la concentración de GSH se determinaron por espectrofotometría; la [TAs] se midió por espectrometría de absorción atómica. Los datos sugieren un incremento en la actividad de PNP (mediana= 0.034 U mg proteína-1) con la presencia de TAs (mediana= 1.16 g L-1). Para explicar la posible asociación de la actividad de las enzimas antioxidantes y la concentración de GSH, así como [TAs], con la actividad de PNP en la leche materna, se construyeron modelos lineales generalizados. En el modelo ajustado, la actividad de GPx y GR presentó una asociación estadística (p.

Following the digestion of milk proteins from mother to baby.

Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are digested, enzyme activity was compared between intact human milk samples from three mothers and the gastric samples from each of their 4-12 day postpartum infants, 2 h after breast milk ingestion. The activities of 7 distinct enzymes are predicted in the infant stomach based on their observed cleavage pattern in peptidomics data. We found that the same patterns of cleavage were evident in both intact human milk and gastric milk samples, demonstrating that the enzyme activities that begin in milk persist in the infant stomach. However, the extent of enzyme activity is found to vary greatly between the intact milk and gastric samples. Overall, we observe that milk-specific proteins are cleaved at higher levels in the stomach compared to human milk. Notably, the enzymes we predict here only explain 78% of the cleavages uniquely observed in the gastric samples, highlighting that further investigation of the specific enzyme activities associated with digestion in infants is warranted.

Synthesis, antioxidant, and xanthine oxidase inhibitory activities of 5-[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-2,4-thiazolidinedione derivatives.

Xanthine oxidase (XO) is a complex metalloflavoprotein, the overproduction of which usually leads to a pathological condition called gout. The XO inhibitors may prove to be promising antigout agents. The XO generates superoxide anions and H2O2 for the self-defense system of the organism. Abnormal production of this superoxide (reactive oxygen species) is responsible for a number of complications including inflammation, metabolic disorder, cellular aging, reperfusion damage, atherosclerosis, and carcinogenesis. In this paper, we report the synthesis of N-substituted analogs of thiazolidinedione derivatives as effective and new class of XO inhibitors and also as antioxidant agents. Among all the compounds in the series, compound 2i produced relatively better activity against human milk XO (72% inhibition), which was also supported by docking studies.

Inactivation of hepatitis C virus infectivity by human breast milk.

BACKGROUND: Hepatitis C virus (HCV) is spread through direct contact with blood, although alternative routes of transmission may contribute to the global burden. Perinatal infection occurs in up to 5% of HCV-infected mothers, and presence of HCV RNA in breast milk has been reported. We investigated the influence of breast milk on HCV infectiousness. METHODS/RESULTS: Human breast milk reduced HCV infectivity in a dose-dependent manner. This effect was species-specific because milk from various animals did not inhibit HCV infection. Treatment of HCV with human breast milk did not compromise integrity of viral RNA or capsids but destroyed the lipid envelope. Fractionation of breast milk revealed that the antiviral activity is present in the cream fraction containing the fat. Proteolytic digestion of milk proteins had no influence on its antiviral activity, whereas prolonged storage at 4°C increased antiviral activity. Notably, pretreatment with a lipase inhibitor ablated the antiviral activity and specific free fatty acids of breast milk were antiviral. CONCLUSIONS: The antiviral activity of breast milk is linked to endogenous lipase-dependent generation of free fatty acids, which destroy the viral lipid envelope. Therefore, nursing by HCV-positive mothers is unlikely to play a major role in vertical transmission.

Bifidobacterial α-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen.

Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different ß-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22:361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galß1-R], but not in a linear xenotransplantation antigen (Galα1-3Galß1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.

EnzymePredictor: a tool for predicting and visualizing enzymatic cleavages of digested proteins.

Mass spectrometric analysis of peptides contained in enzymatically digested hydrolysates of proteins is increasingly being used to characterize potentially bioactive or otherwise interesting hydrolysates. However, when preparations containing mixtures of enzymes are used, from either biological or experimental sources, it is unclear which of these enzymes have been most important in hydrolyzing the sample. We have developed a tool to rapidly evaluate the evidence for which enzymes are most likely to have cleaved the sample. EnzymePredictor, a web-based software, has been developed to (i) identify the protein sources of fragments found in the hydrolysates and map them back on it, (ii) identify enzymes that could yield such cleavages, and (iii) generate a colored visualization of the hydrolysate, the source proteins, the fragments, and the predicted enzymes. It tabulates the enzymes ranked according to their cleavage counts. The provision of odds ratio and standard error in the table permits users to evaluate how distinctively particular enzymes may be favored over other enzymes as the most likely cleavers of the samples. Finally, the method displays the cleavage not only according to peptides, but also according to proteins, permitting evaluation of whether the cleavage pattern is general across all proteins, or specific to a subset. We illustrate the application of this method using milk hydrolysates, and show how it can rapidly identify the enzymes or enzyme combinations used in generating the peptides. The approach developed here will accelerate the identification of enzymes most likely to have been used in hydrolyzing a set of mass spectrometrically identified peptides derived from proteins. This has utility not only in understanding the results of mass spectrometry experiments, but also in choosing enzymes likely to yield similar cleavage patterns. EnzymePredictor can be found at http://bioware.ucd.ie/∼enzpred/Enzpred.php.

Coenzyme A contained in mothers' milk is associated with the potential to induce atopic dermatitis.

T(h)2 adjuvant activity can be qualitatively and quantitatively evaluated using a mixed lymphocyte reaction and by changes in the intracellular cyclic adenosine 3',5'-monophosphate concentration, using human dendritic cells in vitro. The current study shows that mothers, whose children (n = 55) developed atopic dermatitis (AD) within 6 months after birth, often demonstrate a higher T(h)2 adjuvant activity in their milk, in comparison to those whose children did not develop such symptoms. Such an activity was recovered in a liquid phase of mothers' milk and was eluted as a single fraction by reversed-phase HPLC. Further analysis of this fraction by mass spectrometry showed that signals originating from a factor with a molecular weight of 767.53 are observed, exclusively in milk with a high T(h)2 adjuvant activity. The mass is exactly that of Coenzyme A (CoA), and indeed, a low concentration of CoA exhibited T(h)2 adjuvant activity both in vitro and in vivo. Moreover, mesenteric lymph node non-T cells obtained from mice that were orally treated with CoA led allogeneic naive CD4(+) T cells to differentiate into T(h)2. Furthermore, the oral administration of CoA induced rough skin, hyperplasia of the epidermis, hypergranulosis in the spinous layer and the thickening of the stratum in mice. These data collectively indicate that some of the patients with AD were exposed to mothers' milk carrying high T(h)2 adjuvant activity right after birth, which may be attributable to presence of CoA contained in the milk.

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-ß-strands 1-2 (H2-S12) or H2-ß-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Mutation of conserved cysteines in the Ly6 domain of GPIHBP1 in familial chylomicronemia.

We investigated a family from northern Sweden in which three of four siblings have congenital chylomicronemia. LPL activity and mass in pre- and postheparin plasma were low, and LPL release into plasma after heparin injection was delayed. LPL activity and mass in adipose tissue biopsies appeared normal. [(35)S]Methionine incorporation studies on adipose tissue showed that newly synthesized LPL was normal in size and normally glycosylated. Breast milk from the affected female subjects contained normal to elevated LPL mass and activity levels. The milk had a lower than normal milk lipid content, and the fatty acid composition was compatible with the milk lipids being derived from de novo lipogenesis, rather than from the plasma lipoproteins. Given the delayed release of LPL into the plasma after heparin, we suspected that the chylomicronemia might be caused by mutations in GPIHBP1. Indeed, all three affected siblings were compound heterozygotes for missense mutations involving highly conserved cysteines in the Ly6 domain of GPIHBP1 (C65S and C68G). The mutant GPIHBP1 proteins reached the surface of transfected Chinese hamster ovary cells but were defective in their ability to bind LPL (as judged by both cell-based and cell-free LPL binding assays). Thus, the conserved cysteines in the Ly6 domain are crucial for GPIHBP1 function.

Immunoglobulin A with protease activity secreted in human milk activates PAR-2 receptors, of intestinal epithelial cells HT-29, and promotes beta-defensin-2 expression.

Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.

Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.