Associations of α- and γ-tocopherol during early life with lung function in childhood.

BACKGROUND: Tocopherol isoforms may regulate child lung growth and spirometric measures. OBJECTIVE: Our aim was to determine the extent to which plasma α-tocopherol (α-T) or γ-tocopherol (γ-T) isoform levels in early childhood or in utero are associated with childhood lung function. METHODS: We included 622 participants in the Project Viva cohort who had lung function at a mid-childhood visit (age 6-10 years). Maternal and child tocopherol isoform levels were measured by HPLC at the second trimester and 3 years of age, respectively. Multivariable linear regression models (adjusted for mid-childhood body mass index z scores, maternal education, smoking in pregnancy, and prenatal particulate matter with diameter of <2.5 micrometers (PM2.5) particulate exposure) stratified by tertiles of child γ-T level were used to assess the association of α-T levels with FEV1 and forced vital capacity (FVC) percent predicted. Similarly, models stratified by child α-T tertile evaluated associations of γ-T levels with lung function. We performed similar analyses with maternal second trimester tocopherol isoform levels. RESULTS: The median maternal second trimester α-T level was 63 µM (interquartile range = 47-82). The median early-childhood level was 25 µM (interquartile range = 20-33 µM). In the lowest tertile of early-childhood γ-T, children with a higher α-T level (per 10 µM) had a higher mid-childhood FEV1 percent predicted (ß = 3.09; 95% CI = 0.58-5.59 and a higher FVC percent predicted (ß = 2.77; 95% CI = 0.47-5.06). This protective association of α-T was lost at higher γ-T levels. We did not see any consistent associations of second trimester levels of either α-T or γ-T with mid-childhood FEV1 or FVC. CONCLUSION: When γ-T levels were in the lowest tertile, a higher early-childhood α-T level was associated with better lung function at mid-childhood. Second trimester maternal plasma α-T concentration was 3-fold higher than in the adult nonpregnant female population.

Mechanism for initiation of food allergy: Dependence on skin barrier mutations and environmental allergen costimulation.

BACKGROUND: Mechanisms for the development of food allergy in neonates are unknown but clearly linked in patient populations to a genetic predisposition to skin barrier defects. Whether skin barrier defects contribute functionally to development of food allergy is unknown. OBJECTIVE: The purpose of the study was to determine whether skin barrier mutations, which are primarily heterozygous in patient populations, contribute to the development of food allergy. METHODS: Mice heterozygous for the filaggrin (Flg)ft and Tmem79ma mutations were skin sensitized with environmental and food allergens. After sensitization, mice received oral challenge with food allergen, and then inflammation, inflammatory mediators, and anaphylaxis were measured. RESULTS: We define development of inflammation, inflammatory mediators, and food allergen-induced anaphylaxis in neonatal mice with skin barrier mutations after brief concurrent cutaneous exposure to food and environmental allergens. Moreover, neonates of allergic mothers have increased responses to suboptimal sensitization with food allergens. Importantly, responses to food allergens by these neonatal mice were dependent on genetic defects in skin barrier function and on exposure to environmental allergens. ST2 blockade during skin sensitization inhibited the development of anaphylaxis, antigen-specific IgE, and inflammatory mediators. Neonatal anaphylactic responses and antigen-specific IgE were also inhibited by oral pre-exposure to food allergen, but interestingly, this was blunted by concurrent pre-exposure of the skin to environmental allergen. CONCLUSION: These studies uncover mechanisms for food allergy sensitization and anaphylaxis in neonatal mice that are consistent with features of human early-life exposures and genetics in patients with clinical food allergy and demonstrate that changes in barrier function drive development of anaphylaxis to food allergen.

Cytosolic Delivery of Bioactive Cyclic Peptide Cargo by Spontaneous Membrane Translocating Peptides.

Cyclic peptides that inhibit protein-protein interactions have significant advantages over linear peptides and small molecules for modulating cellular signaling networks in cancer and other diseases. However, the permeability barrier of the plasma membrane remains a formidable obstacle to the development of cyclic peptides into applicable drugs. Here, we test the ability of a family of synthetically evolved spontaneous membrane translocating peptides (SMTPs) to deliver phalloidin, a representative bioactive cyclic peptide, to the cytosol of human cells in culture. Phalloidin does not enter cells spontaneously, but if delivered to the cytosol, it inhibits actin depolymerization. We thus use a wound-healing cell mobility assay to assess the biological activity of phalloidin conjugated to three SMTPs that we previously discovered. All three SMTPs can deliver phalloidin to the cell cytosol, and one does so at concentrations as low as 3 µM. Delivery occurs despite the fact that the SMTPs were originally selected based on membrane translocation with no cargo other than a small fluorescent dye. These results show that SMTPs are viable delivery vehicles for cyclic peptides, although their efficiency is moderate. Further, these results suggest that one additional generation of synthetic molecular evolution could be used to optimize SMTPs for the efficient delivery of any bioactive cyclic peptide into cells.

5-HTP inhibits eosinophilia via intracellular endothelial 5-HTRs; SNPs in 5-HTRs associate with asthmatic lung function.

Background: Previous research showed that 5-hydroxytryptophan (5HTP), a metabolic precursor of serotonin, reduces allergic lung inflammation by inhibiting eosinophil migration across endothelial monolayers. Objective: It is unknown if serotonin receptors are involved in mediating this 5HTP function or if serotonin receptor (HTR) single nucleotide polymorphisms (SNPs) associate with lung function in humans. Methods: Serotonin receptor subtypes were assessed by qPCR, western blot, confocal microscopy, pharmacological inhibitors and siRNA knockdown. HTR SNPs were assessed in two cohorts. Results: Pharmacological inhibition or siRNA knockdown of the serotonin receptors HTR1A or HTR1B in endothelial cells abrogated the inhibitory effects of 5HTP on eosinophil transendothelial migration. In contrast, eosinophil transendothelial migration was not inhibited by siRNA knockdown of HTR1A or HTR1B in eosinophils. Surprisingly, these HTRs were intracellular in endothelial cells and an extracellular supplementation with serotonin did not inhibit eosinophil transendothelial migration. This is consistent with the inability of serotonin to cross membranes, the lack of selective serotonin reuptake receptors on endothelial cells, and the studies showing minimal impact of selective serotonin reuptake inhibitors on asthma. To extend our HTR studies to humans with asthma, we examined the CHIRAH and GALA cohorts for HTR SNPs that affect HTR function or are associated with behavior disorders. A polygenic index of SNPs in HTRs was associated with lower lung function in asthmatics. Conclusions: Serotonin receptors mediate 5HTP inhibition of transendothelial migration and HTR SNPs associate with lower lung function. These results may serve to aid in design of novel interventions for allergic inflammation.

ß-Glucosylceramide From Allergic Mothers Enhances Offspring Responsiveness to Allergen.

In animals and humans, offspring of allergic mothers have increased responsiveness to allergen and the allergen-specificity of the offspring can be different than that of the mother. In our preclinical models, the mother's allergic responses influence development of the fetus and offspring by elevating numbers of cells in dendritic cell subsets. A major question is the identity of maternal factors of allergic mothers that alter offspring development of responsiveness to allergen. Lipids are altered during allergic responses and lipids are transported to the fetus for growth and formation of fetal membranes. We hypothesized that pro-inflammatory lipids, that are elevated in allergic mothers, are transported to the fetus and regulate fetal immune development. We demonstrate in this report that there was a significant 2-fold increase in ß-glucosylceramides (ßGlcCer) in allergic mothers, the fetal liver and her offspring. The ßGlcCer were transported from mother's plasma, across the placenta, to the fetus and in breastmilk to the offspring. Administration of ßGlcCer to non-allergic mothers was sufficient for offspring responses to allergen. Importantly, maternal administration of a clinically relevant pharmacological inhibitor of ßGlcCer synthase returned ßGlcCer to normal levels in the allergic mothers and her offspring and blocked the offspring increase in dendritic cell subsets and offspring allergen responsiveness. In summary, allergic mothers had increased ßGlcCer that was transported to offspring and mediated increases in offspring DCs and responsiveness to allergen. These data have a significant impact on our understanding of mechanisms for development of allergies in offspring of allergic mothers and have the potential to lead to novel interventions that significantly impact risk for allergic disease early in life.

Applications and evolution of melittin, the quintessential membrane active peptide.

Melittin, the main venom component of the European Honeybee, is a cationic linear peptide-amide of 26 amino acid residues with the sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2. Melittin binds to lipid bilayer membranes, folds into amphipathic α-helical secondary structure and disrupts the permeability barrier. Since melittin was first described, a remarkable array of activities and potential applications in biology and medicine have been described. Melittin is also a favorite model system for biophysicists to study the structure, folding and function of peptides and proteins in membranes. Melittin has also been used as a template for the evolution of new activities in membranes. Here we overview the rich history of scientific research into the many activities of melittin and outline exciting future applications.

A Fluorescence Quenching Analysis of the Binding of Fluoroquinolones to Humic Acid.

Fluorescence quenching was used to investigate the interaction of six fluoroquinolones with humic acid. Static quenching was observed for the binding of ciprofloxacin, enoxacin, fleroxacin, levofloxacin, norfloxacin, and ofloxacin to humic acid. The equilibrium binding constants were found from Stern-Volmer plots of the data. The quenching experiments were repeated over a temperature range of 25-45 ℃ and van't Hoff plots were generated. From these linear plots, thermodynamic values were calculated for Δ H, Δ G, and Δ S for each of the fluoroquinolones. The equilibrium binding constants were found to be <1 for all the antibiotics studied. The calculated ΔH values were all negative and ranged from -9.5 to -27.6 kJ/mol. The high water solubility of the antibiotics and low ΔH of binding suggests that the antibiotics will be transported easily through the environment. Finally, whether the fluoroquinolones are in a protonated, deprotonated, or partially protonated state is found to correlate to the strength of binding to humic acid.

Structural Basis for CD4+ T Cell Epitope Dominance in Arbo-Flavivirus Envelope Proteins: A Meta-Analysis.

A meta-analysis of CD4+ T cell epitope maps reveals clusters and gaps in envelope-protein (E protein) immunogenicity that can be explained by the likelihood of epitope processing, as determined by E protein three-dimensional structures. Differential processing may be at least partially responsible for variations in disease severity among arbo-flaviruses and points to structural features that modulate protection from disease.