A Partially Hydrolyzed Whey Infant Formula Supports Appropriate Growth: A Randomized Controlled Non-Inferiority Trial.

The aim of the current study was to investigate the effects of a partially hydrolyzed whey infant formula (PHF) on growth in healthy term infants as compared to a standard infant formula with intact protein (IPF). In a double-blind, non-inferiority, randomized controlled trial, a total of 163 healthy formula-fed infants, 55-80 days old, were recruited and randomly allocated to either the PHF (test) or the IPF (control) group. They were followed up for three months during which they were evaluated monthly on growth and development. In total, 21 infants discontinued the study, while 142 infants completed the study (test n = 72, control n = 70). The primary outcome was daily weight gain during the three months. Secondary outcomes included additional anthropometric indices at every timepoint over the intervention period. Daily weight gain during the three-month intervention period was similar in both groups with the lower bound of 95% confidence interval (CI) above the non-inferiority margin of -3 g/day [mean difference (95% CI) test vs. control: -0.474 (-2.460, 1.512) g/day]. Regarding secondary outcomes, i.e., infants' weight, length, head circumference, body mass index (BMI), and their Z-scores, no differences were observed between the two groups at any time point. The PHF resulted in similar infant growth outcomes as the standard IPF. Based on these results, it can be concluded that the partially hydrolyzed whey infant formula supports adequate growth in healthy term infants.

The epithelial barrier-protecting properties of a soy hydrolysate.

Enhancing the epithelial barrier function could be a possible strategy to prevent food allergy or reduce its symptoms. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187-induced TEER reduction were studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify the underlying pathways involved, the barrier disruptor specificity of the effect was studied by comparing the protective effects on TEER and Lucifer Yellow flux after the exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and deoxynivalenol (DON). Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 (restored to 105% of the starting point, while A23187 alone decreased to 53% of the starting value) and mellitin (restored to 11% of the starting point, while mellitin alone decreased to 3.8% of the starting value). This soy hydrolysate was found to increase claudin-1 and decrease claudin-2 expression. The protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin, but was not observed for DON. This observation suggests that the soy hydrolysate may act via PKC isoforms, which are known to lead to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in the management of the barrier function in individuals at risk of developing food allergy.

Toll-like receptor mediated activation is possibly involved in immunoregulating properties of cow's milk hydrolysates.

Immunomodulating proteins and peptides are formed during the hydrolysis of cow's milk proteins. These proteins are potential ingredients in functional foods used for the management of a range of immune related problems, both in infants and adults. However, the mechanism behind these effects is unknown. We hypothesize that the interaction of peptides with Toll-like receptors (TLRs) can induce immune effects, since these receptors are known to sample many dietary molecules in the intestine in order to regulate immune effects. To investigate this, we compared the immune effects and TLR activation and inhibition by whey and casein hydrolysates with different hydrolysis levels. We first measured cytokine production in primary peripheral blood mononuclear cells stimulated with either whey, casein, or their hydrolysates. IL-10 and TNFα were induced by whey hydrolysates (decreasing with increasing hydrolysis level), but not by casein hydrolysates. Next, the activation of TLR 2, 3, 5 and 9 receptors were observed by intact and mildly hydrolysed whey proteins only and not by casein hydrolysates in TLR reporter cell lines. Many casein hydrolysates inhibited TLR signaling (mainly TLR 5 and 9). These results demonstrate that the effects of cow's milk proteins on the immune system are protein type and hydrolysis dependent. TLR signaling is suggested as a possible mechanism for differences in effect. This knowledge contributes to a better understanding of the immune effects of hydrolysates and the design of infant formula, and nutrition in general, with specific immunoregulatory effects.

Intra- and inter-laboratory validation of an innovative huFcεRIα-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates.

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.

Metabolic imprinting, programming and epigenetics - a review of present priorities and future opportunities.

Metabolic programming and metabolic imprinting describe early life events, which impact upon on later physiological outcomes. Despite the increasing numbers of papers and studies, the distinction between metabolic programming and metabolic imprinting remains confusing. The former can be defined as a dynamic process whose effects are dependent upon a critical window(s) while the latter can be more strictly associated with imprinting at the genomic level. The clinical end points associated with these phenomena can sometimes be mechanistically explicable in terms of gene expression mediated by epigenetics. The predictivity of outcomes depends on determining if there is causality or association in the context of both early dietary exposure and future health parameters. The use of biomarkers is a key aspect of determining the predictability of later outcome, and the strengths of particular types of biomarkers need to be determined. It has become clear that several important health endpoints are impacted upon by metabolic programming/imprinting. These include the link between perinatal nutrition, nutritional epigenetics and programming at an early developmental stage and its link to a range of future health risks such as CVD and diabetes. In some cases, the evidence base remains patchy and associative, while in others, a more direct causality between early nutrition and later health is clear. In addition, it is also essential to acknowledge the communication to consumers, industry, health care providers, policy-making bodies as well as to the scientific community. In this way, both programming and, eventually, reprogramming can become effective tools to improve health through dietary intervention at specific developmental points.

Asymmetrical labeling of giant phospholipid vesicles.

The method for labeling of inner membrane leaflet in unilamellar giant POPC vesicles was developed and characterised. Symmetrically NBD-PC labeled vesicles were treated by sodium dithionite, which undergoes an irreversible chemical reaction with NBD-PC molecule making it non-fluorescent. After the addition of dithionite the fluorescence on single vesicles as well as on vesicle suspension showed a 50 % decrease of its initial value corresponding to marker quenching in the outer leaflet. Hence, flourimetry as well as flourescence microscopy prove that dithionite quenching is a suitable method to induce an asymmetrical labeling of the NBD-PC marked giant vesicles.

Effect of pH on red blood cell deformability.

The effect of pH on the red blood cell (RBC) deformability, which is a consequence of a change of cell membrane elastic properties is studied experimentally. With the intention to reduce the effects on deformability of cell geometry and cytoplasmic viscosity, we measured the deformability of the cells with the same volume at various pH of cell suspension from 6.2 to 8.0. Constant cell volume was achieved by varying osmolarity. Deformability was quantified by measuring the elongation of RBCs subjected to velocity gradient in a transparent cone-plate rheoscope. Observed significant decrease of deformability at lower pH leads to the conclusion that membrane elastic properties could be affected by pH changes in the range from 6.2 to 8.0.