A Mediterranean diet plan in lactating women with obesity reduces maternal energy intake and modulates human milk composition - a feasibility study.

Introduction: Maternal obesity is associated with increased concentrations of human milk (HM) obesogenic hormones, pro-inflammatory cytokines, and oligosaccharides (HMOs) that have been associated with infant growth and adiposity. The objective of this pilot study was to determine if adherence to a Mediterranean meal plan during lactation modulates macronutrients and bioactive molecules in human milk from mothers with obesity. Methods: Sixteen healthy, exclusively breastfeeding women with obesity (body mass index ≥30 kg/m2) enrolled between 4 and 5 months postpartum. The women followed a 4-week Mediterranean meal plan which was provided at no cost. Maternal and infant anthropometrics, HM composition, and infant intakes were measured at enrollment and at weeks 2 and 4 of the intervention. Thirteen mother-infant dyads completed the study. Additionally, participants from an adjacent, observational cohort who had obesity and who collected milk at 5 and 6 months postpartum were compared to this cohort. Results: Participants' healthy eating index scores improved (+27 units, p < 0.001), fat mass index decreased (-4.7%, p < 0.001), and daily energy and fat intake were lower (-423.5 kcal/day, p < 0.001 and-32.7 g/day, p < 0.001, respectively) following the intervention. While HM macronutrient concentrations did not change, HM leptin, total human milk oligosaccharides (HMOs), HMO-bound fucose, Lacto-N-fucopentaose (LNFP)-II, LNFP-III, and difucosyllacto-N-tetrose (DFLNT) concentrations were lower following the intervention. Infant intakes of leptin, tumor necrosis factor (TNF)-α, total HMOs, HMO-bound fucose, LNFP-III and DFLNT were lower following the intervention. Specific components of the maternal diet (protein and fat) and specific measures of maternal diet quality (protein, dairy, greens and beans, fruit and vegetables) were associated with infant intakes and growth. Discussion: Adherence to a Mediterranean meal plan increases dietary quality while reducing total fat and caloric intake. In effect, body composition in women with obesity improved, HM composition and infants' intakes were modulated. These findings provide, for the first time, evidence-based data that enhancing maternal dietary quality during lactation may promote both maternal and child health. Longer intervention studies examining the impact of maternal diet quality on HM composition, infant growth, and infant development are warranted.

Experience of Induced Lactation in a Transgender Woman: Analysis of Human Milk and a Suggested Protocol.

Background: A growing number of diverse familial structures wish to colactate their infant. For transgender and gender diverse (TGD) individuals, chestfeeding or breastfeeding may be within their goals of parenthood. There is limited evidence on how to induce lactation for a nongestational parent on gender affirming estrogen treatment. Case Presentation: We report the case of a transgender woman who successfully underwent lactation induction following a protocol using the galactogue domperidone plus use of a breast pump. The patient had modifications to her hormone therapy with estrogen and progesterone while remaining on antiandrogen therapy with spironolactone. A description of the protocol, medications, laboratory monitoring, human milk analysis including macronutrients, oligosaccharides, and hormones is presented. Discussion: This is the fourth case to date known in the literature of a transgender woman with successful lactation induction, and the third case to remain on antiandrogen therapy during this process. Our report is the second to demonstrate comparable macronutrients, and the first to report on human milk oligosaccharides and hormones in induced milk compared with term human milk of a gestational parent. Conclusions: The opportunity to chestfeed or breastfeed an infant can be profound for many parents. Further research is needed to meet the needs of TGD individuals who wish to induce lactation as part of their parental goals.

Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome.

Human milk is a complex mix of nutritional and bioactive components that provide complete nutrition for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we used multi-omic profiling to characterize interactions between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in 242 exclusively breastfeeding mother-infant pairs. We identified 487 genetic loci associated with milk gene expression unique to the lactating mammary gland, including loci that impacted breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with IL-6 concentration in milk and the abundance of Bifidobacteria in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

Human Milk Oligosaccharides in Cord Blood Are Altered in Gestational Diabetes and Stimulate Feto-Placental Angiogenesis In Vitro.

(1) Background: Human milk oligosaccharides (HMOs) are present in maternal serum during pregnancy and their composition is altered in gestational diabetes (GDM). HMOs are also in fetal cord blood and in contact with the feto-placental endothelium, potentially affecting its functions, such as angiogenesis. We hypothesized that cord blood HMOs are changed in GDM and contribute to increased feto-placental angiogenesis, hallmark of GDM. (2) Methods: Using HPLC, we quantified HMOs in cord blood of women with normal glucose tolerance (NGT, n = 25) or GDM (n = 26). We investigated in vitro angiogenesis using primary feto-placental endothelial cells (fpECs) from term placentas after healthy pregnancy (n = 10), in presence or absence of HMOs (100 µg/mL) isolated from human milk, 3'-sialyllactose (3'SL, 30 µg/mL) and lactose (glycan control) and determined network formation (Matrigel assay), proliferation (MTT assays), actin organization (F-actin staining), tube formation (fibrin tube formation assay) and sprouting (spheroid sprouting assay). (3) Results: 3'SL was higher in GDM cord blood. HMOs increased network formation, HMOs and 3'SL increased proliferation and F-actin staining. In fibrin assays, HMOs and 3'SL increased total tube length by 24% and 25% (p < 0.05), in spheroid assays, by 32% (p < 0.05) and 21% (p = 0.056), respectively. Lactose had no effect. (4) Conclusions: Our study suggests a novel role of HMOs in feto-placental angiogenesis and indicates a contribution of HMO composition to altered feto-placental vascularization in GDM.

Human Milk Oligosaccharide Profile Variation Throughout Postpartum in Healthy Women in a Brazilian Cohort.

Human milk oligosaccharide (HMO) composition varies throughout lactation and can be influenced by maternal characteristics. This study describes HMO variation up to three months postpartum and explores the influences of maternal sociodemographic and anthropometric characteristics in a Brazilian prospective cohort. We followed 101 subjects from 28-35 gestational weeks (baseline) and throughout lactation at 2-8 (visit 1), 28-50 (visit 2) and 88-119 days postpartum (visit 3). Milk samples were collected at visits 1, 2 and 3, and 19 HMOs were quantified usinghigh-performance liquid chromatography with fluorescence detection (HPLC-FL). Friedman post-hoc test, Spearman rank correlation for maternal characteristics and HMOs and non-negative matrix factorization (NMF) were used to define the HMO profile. Most women were secretors (89.1%) and presented high proportion of 2'-fucosyllactose (2ꞌFL) at all three sample times, while lacto-N-tetraose (LNT, 2-8 days) and lacto-N-fucopentaose II (LNFPII, 28-50 and 88-119 days) were the most abundant HMOs in non-secretor women. Over the course of lactation, total HMO weight concentrations (g/L) decreased, but total HMO molar concentrations (mmol/L) increased, highlighting differential changes in HMO composition over time. In addition, maternal pre-pregnancy body mass index (BMI) and parity influence the HMO composition in healthy women in this Brazilian cohort.

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies.

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Bovine colostrum-driven modulation of intestinal epithelial cells for increased commensal colonisation.

Nutritional intake may influence the intestinal epithelial glycome and in turn the available attachment sites for bacteria. In this study, we tested the hypothesis that bovine colostrum may influence the intestinal cell surface and in turn the attachment of commensal organisms. Human HT-29 intestinal cells were exposed to a bovine colostrum fraction (BCF) rich in free oligosaccharides. The adherence of several commensal bacteria, comprising mainly bifidobacteria, to the intestinal cells was significantly enhanced (up to 52-fold) for all strains tested which spanned species that are found across the human lifespan. Importantly, the changes to the HT-29 cell surface did not support enhanced adhesion of the enteric pathogens tested. The gene expression profile of the HT-29 cells following treatment with the BCF was evaluated by microarray analysis. Many so called "glyco-genes" (glycosyltransferases and genes involved in the complex biosynthetic pathways of glycans) were found to be differentially regulated suggesting modulation of the enzymatic addition of sugars to glycoconjugate proteins. The microarray data was further validated by means of real-time PCR. The current findings provide an insight into how commensal microorganisms colonise the human gut and highlight the potential of colostrum and milk components as functional ingredients that can potentially increase commensal numbers in individuals with lower counts of health-promoting bacteria.

A case study on breastfeeding education in Lebanon's public medical school: exploring the potential role of social networks in medical education.

BACKGROUND: Limited knowledge, negative beliefs, and lack of sufficient breastfeeding promotion and support by physicians contribute to global suboptimal breastfeeding rates. Formal medical education is well-known to influence future physicians' knowledge, beliefs, and medical practice. However, less understood is the influence of social networks and processes on the exchange and diffusion of knowledge and practices related to breastfeeding. OBJECTIVES: We selected the underserved and under-supported public medical school in Lebanon to examine the social side of medical education. Our objectives were to assess knowledge, beliefs, and self-efficacy related to breastfeeding promotion and support among interns and residents. We also examined the social ecosystem surrounding these students concerning the exchange of breastfeeding knowledge. DESIGN: All data were collected during one study visit per participant. First, an interview-administered structured survey was used to assess beliefs, perceived knowledge, basic breastfeeding knowledge, and self-efficacy related to breastfeeding among n = 70 medical interns and residents. Then, social network data were collected during a semi-structured interview and analyzed using an ego-network approach. All interviews were voice-recorded, transcribed, coded, and thematically analyzed. Descriptive statistics were used to analyze quantitative survey and social network results. RESULTS: Although interns and residents had positive beliefs about breastfeeding benefits, they had limited knowledge and low self-efficacy related to the psychosocial and clinical aspects of breastfeeding promotion and support. They did not seem to have a well-connected professional network around breastfeeding knowledge and practices. Several tended to rely on their informal/non-professional network, such as their mothers, partners, and sisters, for knowledge and practice. CONCLUSIONS: Our work using breastfeeding as an exemplary case suggests there is a role for better attending to the beliefs of medical students as well as to the social side of medical education. Future studies can use social network theory to help identify and address influences on medical education outcomes.

Human Milk Oligosaccharides Influence Intestinal Epithelial Cell Maturation In Vitro.

OBJECTIVES: Human milk oligosaccharides (HMOs) are reported to promote epithelial cell differentiation in vitro. The aim of the present study was to assess induction of epithelial cell differentiation by individual and combined administration of 3 HMOs. METHODS: An in vitro epithelial model of the crypt-villus axis consisting of preconfluent HT-29, preconfluent Caco-2Bbe, and postconfluent Caco-2Bbe cells was used. Cultures were randomized to 17 treatments for 72 hours of incubation: low- and high-dose HMOs (3'sialyllactose [3'SL] at 0.2 and 1.0 g/L, 6'siallylactose [6'SL] at 0.4 and 1.0 g/L, and 2'fucosyllactose at 0.2 and 2.0 g/L), HMO combinations at both low and high doses, and controls (culture medium, 4 g/L pooled HMO, and lipopolysaccharide). RESULTS: High doses of individual HMOs (P < 0.05), combined HMOs (P < 0.05), and pooled HMO decreased (P < 0.001) proliferation in preconfluent HT-29 cultures. Pooled means of individual low and high treatments with 3'SL and 6'SL, combinations of 2 or 3 high-dose HMOs, and total HMO significantly reduced (P < 0.05) proliferation in preconfluent Caco-2Bbe cells. HMOs increased differentiation in preconfluent HT-29 and Caco-2Bbe cells. 3'SL and 6'SL increased alkaline phosphatase activity but did not affect disaccharidase activity in postconfluent Caco-2Bbe cells. Apoptosis and necrosis were both decreased (P < 0.001) in postconfluent Caco-2Bbe cells treated with pooled HMO. CONCLUSIONS: HMO treatments inhibited proliferation with some associated enhancement of epithelial differentiation. Effects of HMOs were additive but no specific combinations of HMOs were especially potent. These results suggest that commercially viable individual HMOs and specific combinations may promote intestinal epithelial cell maturation.

The functional biology of human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a group of complex sugars that are highly abundant in human milk, but currently not present in infant formula. More than a hundred different HMOs have been identified so far. The amount and composition of HMOs are highly variable between women, and each structurally defined HMO might have a distinct functionality. HMOs are not digested by the infant and serve as metabolic substrates for select microbes, contributing to shape the infant gut microbiome. HMOs act as soluble decoy receptors that block the attachment of viral, bacterial or protozoan parasite pathogens to epithelial cell surface sugars, which may help prevent infectious diseases in the gut and also the respiratory and urinary tracts. HMOs are also antimicrobials that act as bacteriostatic or bacteriocidal agents. In addition, HMOs alter host epithelial and immune cell responses with potential benefits for the neonate. The article reviews current knowledge as well as future challenges and opportunities related to the functional biology of HMOs.

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells.

BACKGROUND: Human milk oligosaccharides (HMOs) are a highly abundant, diverse group of unique glycans that are postulated to promote the development of a protective bacterial microbiota in the intestine and prevent adhesive and invasive interactions of pathogenic bacteria with mucosal epithelia. Candida albicans, a prevalent fungal colonizer of the neonatal gut, causes the majority of fungal disease in premature infants and is highly associated with life-threatening intestinal disorders. OBJECTIVE: The objective of the current study was to test the hypothesis that HMOs protect human premature intestinal epithelial cells (pIECs) from invasion by C. albicans. METHODS: To study fungal invasion, a quantitative immunocytochemical assay was used to distinguish invading from noninvading C. albicans cells in the presence and absence of HMOs. To understand how HMOs affect C. albicans invasion of pIECs, the expression of C. albicans virulence traits that are important for invasiveness (hyphal morphogenesis and ability to associate with host cells) were quantified. RESULTS: Treatment with HMOs reduced invasion of pIECs by C. albicans in a dose-dependent manner by 14-67%, with a physiologic concentration (15mg/mL) of HMOs causing a 52% reduction in invasion (P < 0.05). The decreased invasive ability of C. albicans was associated with hyphal lengths that were ∼30% shorter (P < 0.05), likely because of a delay in the induction of hyphal morphogenesis after inoculation of yeast onto pIECs, which correlated with a 23% reduction in the combined expression level of hyphal-specific genes (P < 0.05). In addition, HMOs caused a 40% decrease in the number of C. albicans cells able to associate with pIECs at the time of hyphal induction (P < 0.05). CONCLUSIONS: These results, obtained with the use of a primary pIEC model, indicate that HMOs reduce virulence characteristics of C. albicans and suggest a role for HMOs in protecting the premature infant intestine from invasion and damage by C. albicans hyphae.

Human milk oligosaccharides protect bladder epithelial cells against uropathogenic Escherichia coli invasion and cytotoxicity.

The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.

Human milk galectin-3 binding protein and breast-feeding-associated HIV transmission.

Analysis of milk from 247 HIV-infected Zambian mothers showed that galectin-3 binding protein concentrations were significantly higher among HIV-infected mothers who transmitted HIV through breast-feeding (6.51 ± 2.12 µg/mL) than among nontransmitters but were also correlated with higher milk and plasma HIV RNA copies/mL and lower CD4+ cell counts. The association between galectin-3 binding protein and postnatal transmission was attenuated after adjustment for milk and plasma HIV load and CD4+ cell counts. This suggests that although milk galectin-3 binding protein is a marker of advanced maternal disease, it does not independently modify transmission risk.

Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP(+). Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC(50) values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC(50) values. The most potent hG6PD inhibitors presented IC(50) values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC(50) ~25 µM) more strongly than that of normal MCF10-A cells (IC(50) >50 µM).

Human milk oligosaccharide concentration and risk of postnatal transmission of HIV through breastfeeding.

BACKGROUND: The inefficiency of HIV breast-milk transmission may be caused by the presence of immunologically active factors, including human milk oligosaccharides (HMOs). OBJECTIVE: We investigated whether HMO concentrations are associated with a reduced risk of postnatal HIV transmission. DESIGN: A nested case-control study was conducted within a larger cohort study of HIV-infected women and their infants followed from birth to 24 mo in Lusaka, Zambia. Breast-milk samples collected at 1 mo from 81 HIV-infected women who transmitted via breastfeeding, a random sample of 86 HIV-infected women who did not transmit despite breastfeeding, and 36 uninfected breastfeeding women were selected. Total and specific HMO concentrations were measured by HPLC and compared between groups with adjustment for confounders by using logistic regression. RESULTS: HIV-infected women with total HMOs above the median (1.87 g/L) were less likely to transmit via breastfeeding (OR: 0.45; 95% CI: 0.21, 0.97; P = 0.04) after adjustment for CD4 count and breast-milk HIV RNA concentrations; a trend toward higher concentrations of lacto-N-neotetraose being associated with reduced transmission (OR: 0.49; 95% CI: 0.23, 1.04; P = 0.06) was also observed. The proportion of 3'-sialyllactose (3'-SL) per total HMOs was higher among transmitting than among nontransmitting women (P = 0.003) and correlated with higher plasma and breast-milk HIV RNA and lower CD4 counts. Neither Secretor nor Lewis status distinguished between transmitting and nontransmitting women. CONCLUSIONS: Higher concentrations of non-3'-SL HMOs were associated with protection against postnatal HIV transmission independent of other known risk factors. Further study of these novel, potentially anti-HIV components of breast milk is warranted.

Human milk oligosaccharides reduce Entamoeba histolytica attachment and cytotoxicity in vitro.

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

Human milk oligosaccharides reduce HIV-1-gp120 binding to dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN).

Breast-feeding is the predominant postnatal transmission route for HIV-1 infection in children. However, a majority of breast-fed infants do not become HIV-infected despite continuous exposure to the virus through their mothers' milk over many months. What protects some breast-fed infants from HIV-1 infection? HIV-1 entry across the infant's mucosal barrier is partially mediated through binding of the HIV-1 surface glycoprotein gp120 to dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN) on human dendritic cells. Lewis antigen glycans, present in human milk, bind to DC-SIGN and inhibit HIV-1 transfer to CD4 + T lymphocytes. Human milk contains a high amount of unbound, complex oligosaccharides (5-10 g/l) that carry one or more Lewis antigen glycans, and we hypothesized that they compete with gp120 for DC-SIGN binding. Here, we show in two independent assays that physiological concentrations of human milk oligosaccharides significantly reduce gp120 binding to DC-SIGN by more than 80%. These results may provide an additional explanation for the inhibitory effects of human milk on HIV-1 mother-to-child-transmission. Identifying the specific milk oligosaccharides that interact with DC-SIGN may guide the development of glycan-based drugs that prevent transmission of HIV-1 and other pathogens that use DC-SIGN as an entry point. However, blocking DC-SIGN may be a two-edged sword.

Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function.

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Heparan sulfate plays a central role in a dynamic in vitro model of protein-losing enteropathy.

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a symptom in ostensibly unrelated diseases. Emerging commonalities indicate that genetic insufficiencies predispose for PLE and environmental insults, e.g. viral infections and inflammation, trigger PLE onset. The specific loss of heparan sulfate (HS) from the basolateral surface of intestinal epithelial cells only during episodes of PLE suggests a possible mechanistic link. In the first tissue culture model of PLE using a monolayer of intestinal epithelial HT29 cells, we proved that HS loss directly causes protein leakage and amplifies the effects of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). Here, we extend our in vitro model to assess the individual and combined effects of HS loss, interferon gamma (IFNgamma), TNFalpha, and increased pressure, and find that HS plays a central role in the patho-mechanisms underlying PLE. Increased pressure, mimicking venous hypertension seen in post-Fontan PLE patients, substantially increased protein leakage, but HS loss, IFNgamma, or TNFalpha alone had only minor effects. However, IFNgamma up-regulated TNFR1 expression and amplified TNFalpha-induced protein leakage. IFNgamma and TNFalpha compromised the integrity of the HT29 monolayer and made it more susceptible to increased pressure. HS loss itself compromises the integrity of the monolayer, amplifying the effects of pressure, but also amplifies the effects of both cytokines. In the absence of HS a combination of increased pressure, IFNgamma, and TNFalpha caused maximum protein leakage. Soluble heparin fully compensated for HS loss, providing a reasonable explanation for patient favorable response to heparin therapy.