Milk osteopontin promotes intestinal development by up-regulating the expression of integrin αvß3 and CD44.

Osteopontin (OPN) is a pleiotropic protein involved in numerous biological processes such as cell proliferation and differentiation. Since OPN is abundantly present in milk and is known to be relatively resistant to in vitro gastrointestinal digestion, the current study aimed to investigate the roles of oral intake of milk OPN in intestinal development using an established OPN knockout (KO, OPN-/- ) mouse model, in which wild-type (WT, OPN+/+ ) mouse pups were nursed by either WT (OPN+/+ OPN+ group) or OPN KO dams (OPN+/+ OPN- group; +/+ indicates genotype and - indicates milk without OPN), receiving milk with or without OPN from postnatal days 0 to 21 (P0-P21). Our results showed that milk OPN is resistant to in vivo digestion. Compared to OPN+/+ OPN- pups, OPN+/+ OPN+ pups at P4 and P6 had significantly longer small intestines, at P10 and P20 had larger inner jejunum surfaces, and at P30 exhibited more mature/differentiated intestines, as revealed by higher activities of alkaline phosphatase in brush border and more goblet cells, enteroendocrine cells, and Paneth cells. qRT-PCR and immunoblotting results showed that milk OPN increased the expression of integrin αv, integrin ß3, and CD44 in jejunum of mouse pups (P10, P20, and P30). Immunohistochemistry analysis showed that both integrin αvß3 and CD44 are localized in jejunum crypts. In addition, milk OPN increased the phosphorylation/activation of the ERK, PI3K/Akt, Wnt, and FAK signaling pathways. In summary, oral intake of milk OPN in early life promotes intestinal proliferation and differentiation by upregulating the expression of integrin αvß3 and CD44 and thus regulates OPN-integrin αvß3 and OPN-CD44 mediated cellular signaling pathways.

Rapid and sensitive fluorescence determination of oxytocin using nitrogen-doped carbon dots as fluorophores.

In this work, a novel nitrogen (N)-doped carbon dots (N-CDs) was prepared with quercetin as the carbon source and o-phenylenediamine as the nitrogen source by hydrothermal synthesis, and their application as fluorophores for selective and sensitive determination of oxytocin were reported. The fluorescence quantum yield of the as-prepared N-CDs, which exhibited good water solubility and photostability, was about 6.45 % using rhodamine 6 G as reference substance, and the maximum excitation (Ex) and emission (Em) wavelength were 460 nm and 542 nm, respectively. The results illustrated that the direct fluorescence quenching of N-CDs fluorophore for the detection of oxytocin achieved good linearity in the range of 0.2-5.0 IU/mL and 5.0-10.0 IU/mL, the correlation coefficients were 0.9954 and 0.9909, respectively, and the detection limit was 0.0196 IU/mL (S/N = 3). The recovery rates were 98.8∼103.8 % with RSD= 0.93 %. The interference experiments showed that common metal ions, possible impurities introduced in production and coexisting excipients in the preparation had little adverse influence on selective detection of oxytocin by the developed N-CDs based fluorescent detection method. The mechanism study on the fluorescence quenching of N-CDs by oxytocin concentrations under the given experimental conditions demonstrated that there were internal filtration effect and static quenching in the system. The developed fluorescence analysis platform for the detection of oxytocin had been proved to be rapid, sensitive, specific and accurate, and to be used for the quality inspection of oxytocin.

Milk-Derived miR-22-3p Promotes Proliferation of Human Intestinal Epithelial Cells (HIECs) by Regulating Gene Expression.

MicroRNA (miRNA) is small non-coding RNA involved in gene silencing and post-transcriptional regulation of gene expression. Milk exosomes are microvesicles containing microRNAs (miRNAs). miR-22-3p (miR-22) is plentiful in human milk exosomes and may contribute to intestinal development since milk exosomes and microRNAs are resistant to gastrointestinal digestion in infants. After miR-22 mimics were transfected to human intestinal crypt-like epithelial cells (HIECs) using Lipofectamine for 24 h, RNA was isolated for microarray assay. Microarray results show that miR-22 markedly regulates gene expression, and the roles of miR-22 include promotion of proliferation, regulation of immune functions, and inhibition of apoptosis. Based on the microarray results and miR-22 predicted target genes, CCAAT/enhancer-binding protein δ (C/EBPδ) may be an important direct target of miR-22. C/EBPδ is a transcription factor that regulates numerous biological processes including cell proliferation. In miR-22 transfected HIECs, expression of the C/EBPδ gene was significantly inhibited. Silencing of the C/EBPδ gene by siRNA resulted in increased proliferation of HIECs. A luciferase assay showed that miR-22 specifically binds to the 3'-untranslated region of C/EBPδ mRNA. In summary, milk-derived miR-22 promotes intestinal proliferation by modifying gene expression, and C/EBPδ may be an important target for miR-22 involved in this effect.

The Optimal Number of Oocytes Retrieved From PCOS Patients Receiving IVF to Obtain Associated With Maximum Cumulative Live Birth Rate and Live Birth After Fresh Embryo Transfer.

Aims: This study aims to determine the optimal number of oocytes retrieved so that patients with polycystic ovary syndrome (PCOS) receiving in vitro fertilization (IVF) can obtain the best cumulative live birth rate (CLBR) and live birth after fresh embryo transfer. Methods: This is a retrospective study of 1,419 patients with PCOS who underwent their first IVF cycle at the Second Hospital of Hebei Medical University from January 2014 to December 2021. Multivariable regression analysis was performed to adjust for factors known to independently affect cumulative live birth aspiration. The number of oocytes retrieved to obtain the best cumulative live birth rate was explored through curve fitting and threshold effect analysis. The decision tree method was used to explore the best number of oocytes retrieved to achieve live birth in the shortest time. Results: (1) The number of oocytes retrieved was found to be an independent protective factor for the cumulative live birth rate (OR = 1.09 (95% CI: 1.06, 1.12)). When the number of oocytes retrieved was less than 15, CLBR increased by 16% with each increase in the number of oocytes retrieved (OR = 1.16 (95% CI: 1.11, 1.22)); and when more than 15, CLBR tended to be stable. (2) Live birth after the first fresh embryo transfer was analyzed through a classification decision tree. For patients younger than 35 years old, those with less than 6 oocytes and those with 7-16 oocytes had a similar proportion of live births with fresh embryo transfer but higher than 16 oocytes (53.7% vs. 53.8% vs. 18.4%). Patients older than 35 years old had a similar proportion of live births with fresh embryo transfer (35.7% vs. 39.0%) to those younger than 35 years old, but the proportion of no live births after using up all embryos was higher than those younger than 35 years old (39.3% vs. 19.2%). Conclusions: In PCOS patients, high CLBR can be obtained when the number of oocytes retrieved was 15 or more. The number of oocytes retrieved from 7 to 16 could achieve more chance of live birth after fresh embryo transfer.

The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model.

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague-Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1-20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

[Concentration of osteopontin in human milk and associated factors in Chinese populations from 2011 to 2013].

OBJECTIVE: To analyze content of human milk osteopontin(OPN) and to explore associated factors in Chinese populations. METHODS: The samples and data were extracted from the database for human milk composition in China between 2011 and 2013. A sub-sample of 459 mothers was randomly selected after stratification according to lactation stage, and human milk OPN concentrations were determined by ultra performance liquid chromatography-tandem mass spectrometer(UPLC/MS). RESULTS: The average OPN concentration(M(P25, P75)) in breast milk was 44.0(30.1-72.0) mg/L within 0-330 days postpartum. OPN concentrations were independent of lactation stage, which were 45.6(31.8, 80.7) mg/L in colostrum, 41.3(29.2, 70.0) mg/L in transitional milk and 46.9(30.2, 71.9) mg/L in mature milk, corresponding to 0.40%、0.42% and 0.65% of the total milk protein content(OPN/protein%). The percentage of OPN to total protein in milk showed an increasing trend with lactation progression(r=0.21, P<0.001). Multivariate analysis showed that sleep quality of mothers within one week prior to milk collection was correlated with the breast milk OPN level(P=0.04). The OPN level in breast milk from mothers with good sleep quality was significantly higher than that from mothers with poor sleep quality(46.5 mg/L vs.34.7 mg/L). The median level of milk OPN concentration in mothers from Yunnan was higher than mothers from Beijing(50.5 mg/L vs.36.1 mg/L, P=0.03). Maternal age, mode of delivery, prepregnancy body mass index, weight gain during pregnancy, passive smoking and outdoor activities 24 hours prior to milk collection were not correlated with milk OPN concentration. OPN concentration in breast milk was not related to preterm birth. Also, milk OPN concentration did not correlate with diarrhoea, respiratory disease, or allergic disease in infants during two weeks before milk collection. CONCLUSION: The concentration of OPN in breast milk of Chinese woman may be similar among different lactation stages. Geographic region and sleep quality of mothers may be related to the milk OPN concentration.

Effects of follicular output rate on cumulative clinical pregnancy rate and cumulative live birth rate in PCOS patients with different characteristics.

Objective: We aim to explore the effects of follicular output rate (FORT) on cumulative clinical pregnancy rate (CCPR) and cumulative live birth rate (CLBR) in polycystic ovary syndrome (PCOS) patients with different characteristics undergoing in vitro fertilization (IVF) treatment. Methods: This retrospective study analyzed 454 patients with PCOS undergoing their first IVF cycle at our center from January 2016 to December 2020. FORT was calculated as pre-ovulatory follicle count (PFC) × 100/antral follicle count (AFC). Multivariate regression analyses were conducted to explore the relationships between FORT and CCPR and CLBR. Curve fitting and threshold effect analyses were established to find nonlinear relationships. Effect modification in different subgroups were examined by stratification analyses. Results: Based on the FORT values, individuals were classified into the following three groups: low-FORT group, middle-FORT group and high-FORT group. Multivariate regression analyses revealed that FORT was an independent factor affecting the CCPR and CLBR significantly (OR = 1.015, 95% CI: 1.001, 1.030 and OR = 1.010, 95% CI:1.001, 1.020). Curve fitting and threshold effect analyses showed that the CCPR and CLBR had a positive correlation with FORT when the FORT was less than 70% (OR = 1.039, 95% CI: 1.013, 1.065 and OR = 1.024, 95% CI: 1.004, 1.044). Stratification analyses showed that the CLBR increased by 1.3% with each additional unit of FORT for patients with hyperandrogenic manifestations (OR = 1.013, 95% CI: 1.001, 1.025). Compared with the low-FORT group, in the high-FORT group, CCPR increased 1.251 times for patients with polycystic ovarian morphology, while CCPR and CLBR increased 1.891 times and 0.99 times for those with ovulation disorder, respectively (OR = 2.251, 95% CI: 1.008, 5.028 and OR = 2.891, 95% CI: 1.332, 6.323 and OR = 1.990, 95% CI: 1.133, 3.494). Conclusion: In patients with PCOS, cumulative IVF outcomes have a positive correlation with FORT when the FORT is less than 70%. For PCOS patients with polycystic ovarian morphology, ovulation disorder or hyperandrogenic manifestations, a high FORT could be conductive to achieving better pregnancy outcomes.

Human milk cholesterol is associated with lactation stage and maternal plasma cholesterol in Chinese populations.

BACKGROUND: Milk cholesterol concentrations throughout lactation were analyzed, and the relationship between maternal plasma cholesterol and milk cholesterol in various Chinese populations was examined. METHODS: A sub-sample of 1138 lactating women was randomly selected from a large cross-sectional study in China (n = 6481). Milk cholesterol concentrations were determined by HPLC, and concentrations of maternal plasma lipids were determined by an automated biochemical analyzer. RESULTS: The mean cholesterol concentrations were 200, 171, and 126 mg/L for colostrum, transitional milk, and mature milk, respectively. Cholesterol concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p < 0.001). Concentrations of maternal plasma total cholesterol (TC) (p = 0.02) and low-density lipoprotein cholesterol (LDL-C) (p = 0.03) were significantly associated with milk cholesterol. Milk cholesterol concentrations varied among different ethnicities (Tibetan vs. Hui: 164 vs. 131 mg/L, p = 0.027) but not among different geographic regions. CONCLUSIONS: The concentration of cholesterol in human milk changes dynamically throughout lactation. Milk cholesterol concentrations are significantly associated with maternal plasma concentrations of TC and LDL-C, and milk cholesterol concentrations vary across ethnicities in China. IMPACT: Concentrations of milk cholesterol were measured in various Chinese populations. Cholesterol concentrations differ significantly between stages of lactation. Maternal plasma total cholesterol and low-density lipoprotein cholesterol are associated with milk cholesterol. Milk cholesterol concentrations vary across ethnicities in China.

Recombinant Bovine and Human Osteopontin Generated by Chlamydomonas reinhardtii Exhibit Bioactivities Similar to Bovine Milk Osteopontin When Assessed in Mouse Pups Fed Osteopontin-Deficient Milk.

SCOPE: Osteopontin (OPN), a highly phosphorylated and glycosylated protein, is present in most body fluids, including milk. OPN appears at a high concentration in human milk (130-180 mg L-1 ), but not bovine milk (≈18 mg mL-1 ). It is previously shown that milk OPN is involved in various biological processes and therefore may be a valuable infant formula additive. METHODS AND RESULTS: In the present study, recombinant bovine OPN (rbOPN) and recombinant human OPN (rhOPN) are generated in a Chlamydomonas reinhardtii (C. reinhardtii) algal expression system. The rbOPN and rhOPN are phosphorylated but not glycosylated. To assess the bioactivities of rbOPN and rhOPN and compare their bioactivities to those of bovine milk OPN (bmOPN), wild-type (WT) mouse pups nursed by OPN knock-out (KO) dams are orally fed bmOPN, rbOPN, and rhOPN daily from postnatal days 1-21 (P1-21). Effects of these OPNs on development of the brain, intestine, and immune function are evaluated. The results show that rbOPN and rhOPN exhibit effects similar to those of bmOPN as well as mouse milk OPN on stimulating proliferation of the small intestine, increasing brain myelination and cognitive development, and enhancing development of immune function. CONCLUSION: rbOPN and rhOPN are likely to provide beneficial bioactivities when added to infant diets.

Biological activities of commercial bovine lactoferrin sources.

Lactoferrin (Lf) samples from several manufacturers were evaluated in vitro. The purity and protein form of each Lf were examined by SDS-PAGE, Western blot, and proteomics analysis. Assays were conducted to evaluate uptake of Lfs and iron from Lfs by enterocytes as well as Lf bioactivities, including effects on intestinal cell proliferation and differentiation, IL-18 secretion, TGF-ß1 transcription, and growth of enteropathogenic Escherichia coli (EPEC). Composition of the Lfs varies; some only contain a major Lf band (∼80 kDa), and some also contain minor forms. All Lfs and iron from the Lfs were absorbed by Caco-2 cells, with various efficiencies. The bioactivities of the Lfs varied considerably, but there was no consistent trend. All Lfs promoted intestinal cell proliferation, secretion of IL-18, and transcription of TGF-ß1. Some Lfs exhibited pro-differentiation effects on Caco-2 cells. Effects of pasteurization (62.5 °C for 30 min, 72 °C for 15 s, or 121 °C for 5 min) on integrity, uptake, and bioactivities were examined using Dicofarm, Tatua, and native bovine Lfs. Results show that pasteurization did not affect protein integrity, but variously affected uptake of Lf and its effects on intestinal proliferation, differentiation, and EPEC growth. To choose a Lf source for a clinical trial, assessment of bioactivities is recommended.

Evaluation of Bioactivities of the Bovine Milk Lactoferrin-Osteopontin Complex in Infant Formulas.

Human milk contains several bioactive proteins, including lactoferrin (LF) and osteopontin (OPN). These two proteins have been shown to form a complex, which shows increased bioactivities. Bovine LF and OPN can also form such a complex. We assessed bioactivities of the bovine LF-OPN complex (at molar ratios of LF:OPN = 3:1, 5:1, or 8:1) in a formula protein matrix, including LF, OPN, bovine whey protein hydrolysate, and α-lactalbumin. Our results show that the bovine LF-OPN complex together with formula proteins is resistant to in vitro digestion, stimulates intestinal cell proliferation (by 15-50%) and differentiation (by 30-50%), increases antibacterial activity (by 25-50%), and enhances intestinal immunity. The 3:1 ratio of LF to OPN exhibits the most potent effects, as compared with the other two ratios. In conclusion, adding bovine LF and OPN to infant formulas may result in increased stability of the two components and enhanced bioactivities, possibly improving outcomes in formula-fed infants.

Effects of Milk Osteopontin on Intestine, Neurodevelopment, and Immunity.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein involved in a wide range of biological activities, such as cell proliferation and differentiation, as well as immunomodulatory functions. OPN contains integrin and CD44 binding sites, and it exerts its multiple functions by binding to its receptors on the cell membrane to trigger various cellular signaling pathways. It is generated by a variety of cell types, including epithelial cells and immune cells. OPN appears in most body fluids, such as milk and blood, and is present at a high concentration in human milk but not in bovine milk. Milk OPN is relatively resistant to digestion, and orally ingested OPN can enter the circulatory system. Milk OPN may, therefore, play essential roles in the development in early life. The impact of milk OPN on development has been investigated using cell models, animal models, and randomized clinical trials. Recent OPN studies strongly suggest that milk OPN plays important roles in intestinal proliferation and maturation, brain myelination, and neurodevelopment, as well as immune development.

Evaluation of Bioactivities of Bovine Milk Osteopontin Using a Knockout Mouse Model.

OBJECTIVES: Osteopontin (OPN) is a multifunctional protein present abundantly in human milk, but at low levels in bovine milk and infant formula. Bovine milk OPN (bmOPN) is commercially available, and may therefore, be added to formula. OPN exerts its multiple functions by binding to its receptors to activate cell signaling pathways. The OPN receptor (integrin)-binding site is conserved across species; therefore, bmOPN may exert bioactivities in humans and mice. The objective of the present study was to evaluate bioactivities of bmOPN using an established OPN knock-out (KO) mouse model. METHODS: We evaluated bioactivities of bmOPN, including effects on intestinal growth, immune response, and brain development. In the present study, wild-type (WT) pups were nursed by WT dams, KO dams, or KO dams with bmOPN supplementation from postnatal days 1 to 21 (P1--P21). RESULTS: Our results show that orally ingested bmOPN is partly resistant to in vivo gastrointestinal digestion, and supplemental bmOPN exhibited similar effects as mouse milk OPN (mmOPN) on promoting growth of the small intestine revealed by histological analysis of duodenum villus height and crypt depth at P10, on modifying TNF-α response against a LPS challenge at P30, as well as promoting brain myelination by increasing expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) and improving cognitive development. CONCLUSIONS: Our finding that bmOPN with an amino acid sequence different from mmOPN but with a conserved integrin binding site exerts bioactivities similar to mmOPN suggests that bmOPN may provide bioactivities to human infants when added to formula.

The bovine Lactoferrin-Osteopontin complex increases proliferation of human intestinal epithelial cells by activating the PI3K/Akt signaling pathway.

Lactoferrin (LF) and osteopontin (OPN), multifunctional proteins involved in cell proliferation, can form a complex. LF binds iron, whereas OPN binds calcium. We investigated whether iron- and calcium-binding influences complex formation and the pro-proliferation property of the LF-OPN complex, and the mechanism behind this effect. LF-OPN complexes were prepared using bovine milk LF and OPN, and effects on proliferation of human intestinal epithelial cells (HIECs) were evaluated using a BrdU proliferation assay. Of the four complexes formed by apo- and holo-LF/OPN, the apo-Lf&holo-OPN complex (AH) exhibited the strongest pro-proliferative effect on HIECs, and we therefore focused on AH. AH was resistant to in vitro gastrointestinal digestion, co-localized with both LF and OPN receptors as revealed by confocal microscopy, and stimulated proliferation of HIECs by activating PI3K/Akt signaling. In conclusion, forming a LF-OPN complex may help both proteins to resist digestion and increase the capacity to promote intestinal development in infants.

Assessment of bioactivities of the human milk lactoferrin-osteopontin complex in vitro.

Lactoferrin (LF) and osteopontin (OPN) are both multi-functional whey proteins present at high levels in human milk. These two proteins have a high affinity for each other due to their opposite charges; LF is a basic glycoprotein while OPN is an acidic phosphorylated glycoprotein. LF and OPN were identified to bind to each other over a decade ago, but potential functions of their complex remain unexplored. In this work, we investigated the characteristics of the LF-OPN complex with a focus on its bioactivities. Our results reveal a stronger stability of the LF-OPN complex towards in vitro digestion and more effective binding and uptake by human intestinal cells (HIEC) than LF or OPN alone show. Moreover, the LF-OPN complex promotes proliferation and differentiation of intestinal cells significantly more than the individual proteins do and shows an effect on anti-bacterial function and immune-stimulatory activities intermediate between those of LF and OPN. Thus, by forming a complex in human milk, LF and OPN may protect each other against proteolysis and enhance their individual bioactivities.

Osteopontin in human milk and infant formula affects infant plasma osteopontin concentrations.

BACKGROUND: Osteopontin (OPN), a multifunctional protein, is present abundantly in human milk, but not in bovine milk and infant formulas. A recent randomized clinical trial showed that supplementing infant formula with bovine milk OPN (bOPN) resulted in better immune outcomes. METHODS: Human milk OPN (hOPN) concentrations were analyzed by ELISA. Plasma samples were obtained from infants receiving one of four treatments: breast milk (BF), unsupplemented formula (F0), formula supplemented with 65 mg/L bOPN (F65), or with 130 mg/L bOPN (F130). Plasma samples were analyzed for hOPN and bOPN by ELISA. RESULTS: The hOPN concentration was high in early lactation (D1 to D8), decreased gradually after D9, and deceased significantly after 1 month. At 4 and 6 months, higher levels of hOPN were found in plasma samples from the BF, F65, and F130 groups than in samples from the F0 group; the plasma bOPN concentration in the F130 group was greater than that in the F65 group. CONCLUSION: Dynamic changes in the concentration of milk OPN may reflect infant needs for different amounts of milk OPN for various functions at different developmental stages. Supplemental bOPN in infant formula may exert its beneficial effects by increasing endogenous OPN in plasma.

Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Osteopontin (OPN) is a pleiotropic protein and is abundantly present in milk. Its functions include immune modulation and cellular proliferation and differentiation. OPN is highly expressed in the brain. We investigated the effects of milk-derived OPN on brain development of mouse pups. Wild-type (WT) dams producing OPN+ milk and OPN knockout (KO) dams producing OPN- milk nursed WT pups (OPN+/+), yielding 2 pup treatment groups, OPN+ OPN+/+ and OPN- OPN+/+, for comparison. Preliminary studies supported use of this model by showing high concentrations of OPN in milk of WT dams and no OPN in milk of OPN KO dams, and production of similar amounts of milk by WT and KO dams. The ability of ingested milk OPN to enter the brain was revealed by appearance of orally gavaged [125I]-labeled and antibody-probed milk OPN in brains of pups. Brain OPN mRNA levels were similar in both nursed groups, but the brain OPN protein level was significantly lower in the OPN- OPN+/+ group at postnatal days 6 and 8. Behavior tests showed impaired memory and learning ability in OPN- OPN+/+ pups. In addition, our study revealed increased expression of myelination-related proteins and elevated proliferation and differentiation of NG-2 glia into oligodendrocytes in the brain of OPN+ OPN+/+ pups, accompanied by increased activation of ERK-1/2 and PI3K/Akt signaling. We concluded that milk OPN can play an important role in brain development and behavior in infancy by promoting myelination.-Jiang, R., Prell, C., Lönnerdal, B. Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Concentration of Lactoferrin in Human Milk and Its Variation during Lactation in Different Chinese Populations.

BACKGROUND: Lactoferrin (Lf) is a multifunctional protein and one of the most abundant proteins in human milk. Various factors may affect its concentration in human milk, such as stage of lactation, ethnicity, and diet. OBJECTIVES: The objectives of the present study were to examine the dynamic change in milk Lf throughout the course of lactation and explore factors associated with milk Lf concentrations in various Chinese populations. METHODS: This investigation was a part of a large cross-sectional study conducted in 11 provinces/autonomous regions/municipalities (Beijing, Gansu, Guangdong, Guangxi, Heilongjiang, Inner Mongolia, Shandong, Shanghai, Xinjiang, Yunnan, and Zhejiang) across China between 2011 and 2013. Lactating women (n = 6481) within 0⁻330 days postpartum were recruited in the original study. A sub-sample of 824 women was randomly selected, and milk Lf concentrations were determined by UPLC/MS. RESULTS: The Lf concentration in milk from women delivering at term was 3.16 g/L, 1.73 g/L and 0.90 g/L for colostrum, transitional milk, and mature milk, respectively. Lf concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p < 0.001). Maternal BMI, age, mode of delivery, parturition, protein intake, and serum albumin concentration were not correlated with milk Lf concentration. However, milk Lf concentrations varied among different geographical regions (Guangdong (1.91 g/L) vs. Heilongjiang (1.44 g/L), p = 0.037; Guangdong (1.91 g/L) vs. Gansu (1.43 g/L), p = 0.041) and ethnicities (Dai (1.80 g/L) vs. Tibetan (0.99 g/L), p = 0.007; Han (1.62 g/L) vs. Tibetan (0.99 g/L), p = 0.002) in China. CONCLUSIONS: The concentration of Lf in human milk changes dynamically throughout lactation. Few maternal characteristics affect the milk Lf concentration, but it varies across different geographical regions and ethnicities in China.

Cloning and characterization of the human lactoferrin receptor gene promoter.

Lactoferrin (Lf) is a major protein in human milk. Multiple biological functions of Lf are postulated to be mediated by a Lf receptor (LfR). The Lf receptor (LfR) plays an important role in absorption of Lf and Lf-bound iron by intestinal epithelial cells. Here, we cloned and characterized the promoter from a ~ 3.1 kb 5'-flanking region of the human LfR gene. Neither a TATA box nor a CCAAT box is found at the typical positions. The transcription start site was identified as 298 bp upstream of the translation start codon (+ 1) by 5' RLM-RACE. A series of deletions of 5'-flanking sequences of the human LfR gene were cloned into a promoter-less pGL3 luciferase reporter and transiently transfected into an intestinal enterocyte model (Caco-2 cells). A fragment of - 299/+ 63 elicited the maximal promoter activity in transfected Caco-2 cells, suggesting that functional transcription factor binding sites appear in the region of - 299/+ 63. Bioinformatics analysis indicates that the - 299/+ 63 fragment contains two putative Sp1 binding sites. The promoter activity was significantly decreased when the Sp1 binding sites were mutated by site-directed mutagenesis. Additionally, the promoter activity was dramatically inhibited by treating cells with an Sp1 inhibitor. Binding of Sp1 to the promoter was confirmed by EMSA. Moreover, after Sp1 expression was significantly suppressed by RNA interference, LfR was significantly decreased at both RNA and protein levels. In conclusion, the LfR gene promoter contains downstream core promoter elements, and the Sp1 binding sites play critical roles in transcriptional regulation of the LfR gene.