Randomized Trial of a Yogurt-type Amino Acid-based Formula in Infants and Children With Severe Cow's Milk Allergy.

OBJECTIVES: Evaluation of a spoon-fed amino acid-based formula (AAF) with a yogurt-type texture compared to the reference oral liquid formula (Neocate). METHODS: Phase III/IV, prospective, randomized (1:1), open-label, multicenter study in infants/young children (6-36 months) with severe cow's milk protein allergy (CMA) who had consumed AAF for ≥1 month before the study. Patients received reference+test formula (Neocate with a yogurt-type texture for spoon-feeding: group 1) or reference formula (group 2) for 28 days. The study formulae were integrated into the patients' usual daily diet. Efficacy on Day 0, 14, and 28 was assessed primarily in terms of symptoms associated with CMA. The evolution of symptoms, amount of formula consumed, nutritional and energy intake, anthropometric data, and tolerability were also assessed. RESULTS: The incidence of CMA symptoms was similar in each group (P > 0.05) on day 0, 14, and 28. For specific symptoms, there was little change from day 0 and no significant difference between groups for incidence on day 0 or evolution at day 14 or 28. There was no difference in formula consumption (day 0-day 28) between groups (P = 0.90), but nutritional value was generally higher for group 1 and calcium intake was statistically higher for group 1 (P < 0.05). Weight-for-height, weight-for age, and body mass index-for-age z scores were higher for group 1 than group 2 (P < 0.05). Both formulae were well tolerated. CONCLUSIONS: There was no difference in efficacy, formula consumption, and tolerability between the new spoon-fed yogurt-type AAF formula and the reference formula, whereas significantly higher calcium intake was achieved with the new formula.

Effect of low-fat, fermented milk enriched with plant sterols on serum lipid profile and oxidative stress in moderate hypercholesterolemia.

BACKGROUND: Plant sterol (PS)-enriched foods have been shown to reduce plasma LDL-cholesterol concentrations. In most studies, however, PSs were incorporated into food products of high fat content. OBJECTIVE: We examined the effect of daily consumption of PS-supplemented low-fat fermented milk (FM) on the plasma lipid profile and on systemic oxidative stress in hypercholesterolemic subjects. DESIGN: Hypercholesterolemic subjects (LDL-cholesterol concentrations >or=130 and

Retinoic acid receptor-mediated induction of ABCA1 in macrophages.

ABCA1, the mutant molecule in Tangier Disease, mediates efflux of cellular cholesterol to apoA-I and is induced by liver X receptor (LXR)/retinoid X receptor (RXR) transcription factors. Retinoic acid receptor (RAR) activators (all-trans-retinoic acid [ATRA] and TTNPB) were found to increase ATP-binding cassette transporter 1 (ABCA1) mRNA and protein in macrophages. In cellular cotransfection assays, RARgamma/RXR activated the human ABCA1 promoter, via the same direct repeat 4 (DR4) promoter element as LXR/RXR. Chromatin immunoprecipitation analysis in macrophages confirmed the binding of RARgamma/RXR to the ABCA1 promoter DR4 element in the presence of ATRA, with weaker binding of RARalpha/RXR, and no binding of RARbeta/RXR. However, in macrophages from RARgamma(-/-) mice, TTNPB still induced ABCA1, in association with marked upregulation of RARalpha, suggesting that high levels of RARalpha can compensate for the absence of RARgamma. Dose-response experiments with ATRA in mouse primary macrophages showed that other LXR target genes were weakly induced (ABCG1 and SREBP-1c) or not induced (apoE and LXRalpha). The more specific RAR activator TTNPB did not induce SREBP-1c in mouse primary macrophages or liver. These studies indicate a direct role of RARgamma/RXR in induction of macrophage ABCA1.

Cellular and secreted pro-protein convertase subtilisin/kexin type 9 catalytic activity in hepatocytes.

OBJECTIVES: Pro-protein convertase subtilisin/kexin type 9 (PCSK9) impairs the low density lipoprotein receptor (LDLr) recyling. To reach the LDLr, the pro-protein must cleave itself in the endoplasmic reticulum. Using a fluorogenic peptide corresponding to the cleavage site, we directly monitored for the first time the cleavage activity of purified human PCSK9 and that of endogenous human wild-type PCSK9 and naturally occurring variants in hepatocytes. METHODS: Validation of the assay was performed with wild type or PCSK9 deficient primary mouse hepatocytes and immortalized human hepatocytes transfected with antiPCSK9 siRNA. An analysis of the cleaved peptide was performed using mass spectrometry. Pharmacological regulation of the enzyme was studied in human hepatocytes. Expression vectors coding for the variants S127R, D374Y, F216L, S386A were transfected in primary hepatocytes from PCSK9 deficient mice. RESULTS: PCSK9 activity was measured in cell lysates and media, at levels 100 times higher than with the human purified recombinant protein. The assay is highly specific for PCSK9 in cell lysate and cell culture media but not in plasma. Pharmacological up- or down-regulation of PCSK9 expression produced paralleled effects on the activity. The catalytic activity of gain-of-function variants S127R, D374Y recapitulated roughly the maturation efficiency estimated by western blots, in contrast with the F216L variant that presented with a 54% lower catalytic activity than the wild-type protein, despite similar proPCSK9 to PCSK9 ratios. Thus, other factors might be involved in the maturation of PCSK9. CONCLUSION: All together, these results shed a new light on PCSK9 enzymatic activity and could help identifying proPCSK9 inhibitors.

The lactotripeptides isoleucine-proline-proline and valine-proline-proline do not inhibit the N-terminal or C-terminal angiotensin converting enzyme active sites in humans.

BACKGROUND: The potential blood pressure lowering effect of fermented milk may involve inhibition of angiotensin-converting enzyme (ACE) by dairy lactotripeptides generated during milk fermentation, such as isoleucine-proline-proline (IPP) and valine-proline-proline (VPP). These peptides are weak ACE inhibitors in vitro but it remains unclear whether they inhibit ACE in vivo in humans. OBJECTIVE: To assess in vivo ACE inhibition in individuals given fermented milk over a 7-day period. DESIGN AND METHODS: Twelve healthy normotensive men were given 330 ml of fermented milk once daily from day 1 to day 7 (IPP: 4.5 mg and VPP: 6.6 mg) and a single dose of 50 mg captopril on day 8. ACE inhibition was assessed in vivo by measuring plasma and urine AcSDKP and plasma active renin and in vitro by measuring plasma ACE activity using hippuryl-histidine-leucine. Plasma IPP/VPP concentrations were measured by LC/MS/MS. RESULTS: Plasma IPP concentrations increased slightly and very transiently after fermented milk administration. Plasma VPP concentrations were below the limit of quantification. Fermented milk had no effect on plasma AcSDKP, ACE activity or active renin concentrations on days 1 or 7. Urine AcSDKP excretion underwent a small transient increase. In contrast, plasma and urine AcSDKP increased 7.7-fold and 70-fold, respectively, and plasma ACE activity decreased by 82.3 +/- 16.1% following captopril administration; plasma active renin concentration increased four-fold. CONCLUSION: IPP and VPP were poorly absorbed and rapidly eliminated. They did not inhibit plasma or endothelial ACE in vivo at the selected doses and had no specific effect on the N-terminal or C-terminal ACE domains.

Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c.

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4-5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 microm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.

Human mucosa/submucosa interactions during intestinal inflammation: involvement of the enteric nervous system in interleukin-8 secretion.

Interleukin-8 (IL-8) is a key chemokine upregulated in various forms of intestinal inflammation, especially those induced by bacteria such as Clostridium difficile (C. difficile). Although interactions between different mucosal and submucosal cellular components have been reported, whether such interactions are involved in the regulation of IL-8 secretion during C. difficile infection is unknown. Moreover, whether the enteric nervous system, a major component of the submucosa, is involved in IL-8 secretion during an inflammatory challenge remains to be determined. In order to investigate mucosa/submucosa interactions that regulate IL-8 secretion, we co-cultured human intestinal mucosa and submucosa. In control condition, IL-8 secretion in co-culture was lower than the sum of the IL-8 secretion of both tissue layers cultured alone. Contrastingly, IL-8 secretion increased in co-culture after mucosal challenge with toxin B of C. difficile through an IL-1 beta-dependent pathway. Moreover, we observed that toxin B of C. difficile increased IL-8 immunoreactivity in submucosal enteric neurones in co-culture and in intact preparations of mucosa/submucosa, through an IL-1 beta-dependent pathway. IL-1 beta also increased IL-8 secretion and IL-8 mRNA expression in human neuronal cell lines (NT2-N and SH-SY5Y), through p38 and ERK1/2 MAP kinase-dependent pathways. Our results demonstrate that mucosa/submucosa interactions regulate IL-8 secretion during inflammatory processes in human through IL-1 beta-dependent pathways. Finally we observed that human submucosal neurones synthesize IL-8, whose production in neurones is induced by IL-1 beta via MAPK-dependent pathways.

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells.

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.

Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E.

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an anti-atherogenic state. To determine whether PLTP regulates lipoprotein vitamin E content in vivo, we measured alpha-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoE-deficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of anti-oxidants in lipoproteins, independent of dietary supplementation.