The amniotic fluid proteome changes across gestation in humans and rhesus macaques.

Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC-MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies.

Impaired placental hemodynamics and function in a non-human primate model of gestational protein restriction.

Maternal malnutrition increases fetal and neonatal morbidity, partly by affecting placental function and morphology, but its impact on placental hemodynamics are unknown. Our objective was to define the impact of maternal malnutrition on placental oxygen reserve and perfusion in vivo in a rhesus macaque model of protein restriction (PR) using advanced imaging. Animals were fed control (CON, 26% protein), 33% PR diet (17% protein), or a 50% PR diet (13% protein, n = 8/group) preconception and throughout pregnancy. Animals underwent Doppler ultrasound and fetal biometry followed by MRI at gestational days 85 (G85) and 135 (G135; term is G168). Pregnancy loss rates were 0/8 in CON, 1/8 in 33% PR, and 3/8 in 50% PR animals. Fetuses of animals fed a 50% PR diet had a smaller abdominal circumference (G135, p < 0.01). On MRI, placental blood flow was decreased at G135 (p < 0.05) and placental oxygen reserve was reduced (G85, p = 0.05; G135, p = 0.01) in animals fed a 50% PR diet vs. CON. These data demonstrate that a 50% PR diet reduces maternal placental perfusion, decreases fetal oxygen availability, and increases fetal mortality. These alterations in placental hemodynamics may partly explain human growth restriction and stillbirth seen with severe PR diets in the developing world.

Peptides Derived from In Vitro and In Vivo Digestion of Human Milk Are Immunomodulatory in THP-1 Human Macrophages.

BACKGROUND: Milk proteins contain many encrypted bioactive peptides. Whether these bioactive peptides are released in the infant intestine and exert immunomodulatory activity remains unknown. OBJECTIVE: This study examined in vitro immunomodulatory activities of peptides from in vitro- and in vivo-digested human milk. METHODS: Peptides were extracted from in vitro-digested human milk and pooled intestinal samples from 8 infants fed human milk. Peptides extracted from in vitro-digested samples were fractionated. The in vitro effects of these peptides and fractions on the secretion of TNF-α and IL-8 in LPS-treated human immune THP-1 macrophages were evaluated. The significance of differences between in vitro peptide fraction treatment and control on cytokine production was analyzed by t test. LC-MS/MS-based peptidomics was conducted to identify the peptides. The peptides were screened for potential bioactivity using a sequence homology search using the Milk Bioactive Peptide Database (MBPDB). RESULTS: Six fractions of the peptide mixture extracted from the in vitro-digested human milk significantly inhibited TNF-α production by LPS-challenged THP-1 macrophages. Fractions F4, F8, F11, F14, and F17 attenuated IL-8 secretion, and F6/7 and F18 increased IL-8 secretion. Peptides extracted from the pooled in vivo intestinal samples attenuated both TNF-α and IL-8 secretion. There were 266 and 418 peptides identified in the in vitro and in vivo samples, respectively. Among the peptides, 34 and 50 in the in vitro and in vivo samples, respectively, had >80% sequence similarity to bioactive peptides in the MBPDB. CONCLUSIONS: Peptides released by in vitro and in vivo infant digestion of human milk were immunomodulatory in human immune cells; fractions F4, F8, and F11 were anti-inflammatory; and F6/7 and F18 were proinflammatory. Thirteen peptides were present in all fractions with anti-inflammatory activity, and 38 peptides were present in all fractions with proinflammatory activity. These peptides potentially contributed to the observed immunomodulatory activity of the peptide mixtures.

Congenital Diaphragmatic Hernia Management: A Systematic Review and Care Pathway Description Including Volume-Targeted Ventilation.

BACKGROUND: Although it is well established that standardized treatment protocols improve outcomes for infants with congenital diaphragmatic hernia (CDH), there remains variance between existing protocols. PURPOSE: The purpose of this article was to review current literature on protocols for CDH management in the preoperative period and to describe a care pathway integrating best practice elements from existing literature with volume-targeted ventilation strategies previously in place at a major tertiary care center in the Pacific Northwestern United States. METHODS/SEARCH STRATEGY: A systematic review of literature was performed according to PRISMA guidelines to identify current publications on CDH protocols and examine them for similarities and differences, particularly regarding ventilation strategies. FINDINGS/RESULTS: Although existing protocols from multiple regions worldwide shared common goals of reducing barotrauma and delaying surgery until a period of clinical stabilization was achieved, their strategies varied. None included volume-targeted ventilation with pressure limitation as a method of avoiding ventilation-induced lung injury (VILI). IMPLICATIONS FOR PRACTICE: Institutions that routinely manage infants with CDH should have a standardized treatment protocol in place, as this is shown to improve outcomes. This may include volume-targeted ventilation with pressure limitation as a successful VILI-limiting strategy. IMPLICATIONS FOR RESEARCH: While standardized protocols have been shown to increase survival rate for infants with CDH, more research is needed to determine what these protocols should include. Specifically, there is a need for future study on the most appropriate ventilation mode for this population.

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity.

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.

Effect of digestion on stability of palivizumab IgG1 in the infant gastrointestinal tract.

BACKGROUND: Potentially, orally administered antibodies specific to enteric pathogens could be administered to infants to prevent diarrheal infections, particularly in developing countries where diarrhea is a major problem. However, to prevent infection, such antibodies would need to resist degradation within the gastrointestinal tract. METHODS: Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used in this study as a model for examining the digestion of neutralizing antibodies to enteric pathogens in infants. The survival of this recombinant IgG1 across digestion in 11 infants was assayed via an anti-idiotype ELISA and RSV F protein-specific ELISA. Concentrations were controlled for any dilution or concentration that occurred in the digestive system using mass spectrometry-based quantification of co-administered, orally supplemented, indigestible polyethylene glycol (PEG-28). RESULTS: Binding activity of Palivizumab IgG1 decreased (26-99%) across each phase of in vivo digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. CONCLUSION: Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. IMPACT: Binding activity of palivizumab IgG1 decreased (26-99%) across each phase of in vivo infant digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. Palivizumab was likely degraded by proteases and changes in pH introduced in the gut. Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. The monoclonal antibody IgG1 tested was not stable across the infant gastrointestinal tract. The observation of palivizumab reduction was unlikely due to dilution in the gastrointestinal tract. The results of this work hint that provision of antibody could be effective in preventing enteric pathogen infection in infants. Orally delivered recombinant antibodies will need to either be dosed at high levels to compensate for digestive losses or be engineered to better resist digestion. Provision of enteric pathogen-specific recombinant antibodies to at-risk infants could provide a new and previously unexplored pathway to reducing the infection in infants. The strategy of enteric recombinant antibodies deserves more investigation throughout medicine as a novel means for treatment of enteric disease targets.

Survival of recombinant monoclonal and naturally-occurring human milk immunoglobulins A and G specific to respiratory syncytial virus F protein across simulated human infant gastrointestinal digestion.

To help rationally design an antibody for oral administration, we examined how different isotypes (IgG, IgA and sIgA) with the same variable sequence affect antibody stability across digestion. We compared the degradation of recombinant palivizumab (IgG1), and recombinant IgA and sIgA versions of palivizumab spiked in human milk to the degradation of naturally-occurring anti-respiratory syncytial virus (RSV) sIgA/IgA and IgG in human milk from four donors across gastric and intestinal phases of an in vitro model of infant digestion via a validated RSV F protein ELISA. Palivizumab IgG and IgA formats were less stable than the sIgA version after complete simulated gastrointestinal digestion: palivizumab IgG, IgA and sIgA decreased across complete simulated gastrointestinal digestion by 55%, 48% and 28%, respectively. Naturally-occurring RSV F protein-specific IgG was stable across digestion, whereas naturally-occurring sIgA/IgA was stable in the gastric phase but decreased 33% in the intestinal phase of simulated digestion.

Binding and Neutralizing Capacity of Respiratory Syncytial Virus (RSV)-Specific Recombinant IgG Against RSV in Human Milk, Gastric and Intestinal Fluids from Infants.

Oral administration of pathogen-specific recombinant antibodies may help to prevent infant gastrointestinal (GI) pathogen infection; however, to neutralize an infectious agent, these antibodies must resist degradation in the GI tract. Palivizumab, a recombinant antibody specific for the respiratory syncytial virus (RSV), was used as a model for pathogen-specific IgG in human milk. The aim was to compare the remaining binding capacity of palivizumab in milk between three mothers after exposure to an in vitro model of infant gastrointestinal digestion (gastric and duodenal fluids) using ELISA. The neutralizing capacity of palivizumab in pooled human milk, gastric contents, and stools from preterm infants was also evaluated for blocking RSV with green fluorescent protein (RSV-GFP) infection in Hep-2 cells using confocal and inverted microscopy and flow cytometry. The reduction of palivizumab binding capacity in human milk and digested samples was slightly different between mothers. Overall, palivizumab decreased 50% after simulated gastric digestion with pepsin and 62% after simulated intestinal digestion with pancreatin. Palivizumab (2-8 µg/mL) in human milk or stool samples blocked RSV (3.4 × 104 FFU/mL) infection (no syncytia formation on Hep-2 cells) by microscopy. Syncytia formation was detected on Hep-2 cells when RSV was incubated in gastric contents or virus medium with 2-4 µg/mL of palivizumab, but no infection was observed at 8 µg/mL. No fluorescence (absence of infected cells) was detected when palivizumab (100 µg/mL) was incubated in human milk or medium with RSV-GFP (1.1 × 105 FFU/mL), whereas fluorescence increased with the reduced concentration of palivizumab using flow cytometry. These results suggest that undigested and digested matrices could change the binding and neutralizing capacity of viral pathogen-specific antibodies.

Survival of Recombinant Monoclonal Antibodies (IgG, IgA and sIgA) Versus Naturally-Occurring Antibodies (IgG and sIgA/IgA) in an Ex Vivo Infant Digestion Model.

To prevent infectious diarrhea in infants, orally-supplemented enteric pathogen-specific recombinant antibodies would need to resist degradation in the gastrointestinal tract. Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used as a model to assess the digestion of neutralizing antibodies in infant digestion. The aim was to determine the remaining binding activity of RSV F protein-specific monoclonal and naturally-occurring immunoglobulins (Ig) in different isoforms (IgG, IgA, and sIgA) across an ex vivo model of infant digestion. RSV F protein-specific monoclonal immunoglobulins (IgG, IgA, and sIgA) and milk-derived naturally-occurring Ig (IgG and sIgA/IgA) were exposed to an ex vivo model of digestion using digestive samples from infants (gastric and intestinal samples). The survival of each antibody was tested via an RSV F protein-specific ELISA. Ex vivo gastric and intestinal digestion degraded palivizumab IgG, IgA, and sIgA (p < 0.05). However, the naturally-occurring RSV F protein-specific IgG and sIgA/IgA found in human milk were stable across gastric and intestinal ex vivo digestion. The structural differences between recombinant and naturally-occurring antibodies need to be closely examined to guide future design of recombinant antibodies with increased stability for use in the gastrointestinal tract.