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Background: Little is known about the impact of nutrition on the development of skin structure and function in infants. Methods: We investigated epidermal, dermal, and subcutis parameters of aged-matched well-nourished and moderately undernourished infants in this single-center, cross-sectional, noninterventional study using noninvasive methods (skin caliper, 20-MHz sonography, transepidermal water loss, skin pH, and corneometry). Plasma fatty acids were determined as an indicator of nutritional differences. 310 infants from different age groups, i.e., 1 week, 4 weeks, and 6, 9, 12, 24, and 36 months were included. Approximately half of each age group was well-nourished (WHO reference values weight-for-height/length Zscore: –0.75 ≤ Z ≤ 0.75) and the other half was moderately undernourished (–3 ≤ Z < –2). Results: Structural maturational differences in the deeper dermis and subcutis regions of the skin and subtle functional changes in the epidermis were observed in moderately undernourished infants without notable clinical symptoms. Reduced skin barrier function or skin hydration were not observed in the undernourished infants, and skin pH shifted to more acidic values in this group. Conclusion: These findings reveal a greater impact of moderate undernutrition on the development of the dermis and subcutis and suggest that critical epidermal functions such as skin barrier and pH are mostly maintained.

Background: Post-natal gut maturation in infants interrelates maturation of the morphology, digestive, and immunological functions and gut microbiota development. Here, we explored both microbiota development and markers of gut barrier and maturation in healthy term infants during their early life to assess the interconnection of gut functions during different infant formulae regimes. Methods: A total of 203 infants were enrolled in this randomized double-blind controlled trial including a breastfed reference group. Infants were fed starter formulae for the first four weeks of life, supplemented with different combination of nutrients (lactoferrin, probiotics (Bifidobacterium animal subsp. Lactis) and prebiotics (Bovine Milk-derived Oligosaccharides&mdash, BMOS)) and subsequently fed the control formula up to eight weeks of life. Stool microbiota profiles and biomarkers of early gut maturation, calprotectin (primary outcome), elastase, &alpha, 1 antitrypsin (AAT) and neopterin were measured in feces at one, two, four, and eight weeks. Results: Infants fed formula containing BMOS had lower mean calprotectin levels over the first two to four weeks compared to the other formula groups. Elastase and AAT levels were closer to levels observed in breastfed infants. No differences were observed for neopterin. Global differences between the bacterial communities of all groups were assessed by constrained multivariate analysis with hypothesis testing. The canonical correspondence analysis (CCA) at genus level showed overlap between microbiota profiles at one and four weeks of age in the BMOS supplemented formula group with the breastfed reference, dominated by bifidobacteria. Microbiota profiles of all groups at four weeks were significantly associated with the calprotectin levels at 4 (CCA, p = 0.018) and eight weeks of age (CCA, p = 0.026). Conclusion: A meaningful correlation was observed between changes in microbiota composition and gut maturation marker calprotectin. The supplementation with BMOS seems to favor gut maturation closer to that of breastfed infants.

Background In the absence of breast-feeding and its immunomodulatory factors, supplementation of starter infant formula (IF) with probiotics is currently used to support immune functions and gut development. Aim To assess whether immune-related beneficial effects of regular dose (10 7 CFU/g of powder) of the probiotic Bifidobacterium lactis CNCM I-3446 (hereafter named B. lactis) in starter IF supplementation can be maintained with starter IF containing a low dose (10 4 CFU/g of powder) of B. lactis. Method This trial was designed as a pilot, prospective, double-blind, randomized, single-center clinical trial of two parallel groups ( n = 77 infants/group) of C-section delivered infants receiving a starter IF containing either low dose or regular dose of the probiotic B. lactis from birth to six months of age. In addition, a reference group of infants breast-fed for a minimum of four months ( n = 44 infants), also born by C-section, were included. All groups were then provided follow-up formula without B. lactis up to 12 months of age. Occurrence of diarrhea, immune and gut maturation, responses to vaccinations, and growth were assessed from birth to 12 months. The effect of low-dose B. lactis formula was compared to regular-dose B. lactis formula, considered as reference for IF with probiotics, and both were further compared to breast-feeding as a physiological reference. Results Data showed that feeding low-dose B. lactis IF provides similar effects as feeding regular-dose B. lactis IF or breast milk. No consistent statistical differences regarding early life protection against gastrointestinal infections, immune and gut maturation, microbiota establishment, and growth were observed between randomized formula-fed groups as well as with the breast-fed reference group. Conclusion This pilot study suggests that supplementing C-section born neonates with low-dose B. lactis-containing starter formula may impact immune as well as gut maturation similarly to regular-dose B. lactis, close to the breast-feeding reference.

Non-digestible milk oligosaccharides were proposed as receptor decoys for pathogens and as nutrients for beneficial gut commensals like bifidobacteria. Bovine milk contains oligosaccharides, some of which are structurally identical or similar to those found in human milk. In a controlled, randomized double-blinded clinical trial we tested the effect of feeding a formula supplemented with a mixture of bovine milk-derived oligosaccharides (BMOS) generated from whey permeate, containing galacto-oligosaccharides and 3'- and 6'-sialyllactose, and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446. Breastfed infants served as reference group. Compared with a non-supplemented control formula, the test formula showed a similar tolerability and supported a similar growth in healthy newborns followed for 12 weeks. The control, but not the test group, differed from the breast-fed reference group by a higher faecal pH and a significantly higher diversity of the faecal microbiota. In the test group the probiotic B. lactis increased by 100-fold in the stool and was detected in all supplemented infants. BMOS stimulated a marked shift to a bifidobacterium-dominated faecal microbiota via increases in endogenous bifidobacteria (B. longum, B. breve, B. bifidum, B. pseudocatenulatum).

Limited evidence is available regarding the effect of partially hydrolyzed whey-based formula (pHF-W) on growth and atopic dermatitis (AD) risk reduction in infants within the general infant population, and without a familial history of allergy as an inclusion or exclusion criterion. We reviewed the current evidence available from studies using pHF-W in the general population and summarized the data on safety (growth) and efficacy outcomes (reduction of AD), comparing the studies side by side. A total of 8 clinical trials were identified from the literature search, 7 of which used the same pHF-W. Six out of 8 studies indicated a reduction of atopic manifestations using a specific pHF-W versus cow’s milk formula (CMF) in the first years of life. Data were summarized and compared side by side for growth (3 studies) and efficacy (5 studies). In these diverse general populations, the results on growth and AD were consistent with the previous findings reported on infants with a family history of allergy, but numerous limitations to these studies were identified. This literature review confirms that pHF-W supports normal growth in infants, and suggests that the risk of AD may be reduced in not-fully breastfed infants from the general population when supplemented with a specific pHF-W when compared to CMF during the first 4–6 months of life. Further studies are warranted to confirm these results.

Background: Prebiotics and probiotics exert beneficial effects by modulating gut microbiota and immune system. This study evaluates efficacy and safety of an infant formula containing bovine milk-derived oligosaccharides and Bifidobacterium animalis ssp lactis (B. lactis) (CNCM I-3446) on incidence of diarrhea and febrile infections during the first year of life (primary outcome). Methods: Full-term infants receiving Test or Control (without bovine milk-derived oligosaccharide and B. lactis) formulae were enrolled in a multicenter, randomized, controlled, and double-blind trial with a reference breastfeeding group. . Results: 413 infants were assigned between Test (n = 206) and Control (n = 207) formula. There was no significant difference for diarrhea and febrile infections incidence between groups at 6 (odds ratio (95% confidence interval) = 0.56 (0.26–1.15), P = 0.096) and 12 mo (odds ratio = 0.66 (0.38–1.14), P = 0.119). Test formula was well tolerated, anthropometrics parameters were not significantly different between groups and aligned with WHO growth standards up to 12 mo. Data from test group showed that gut microbiota pattern, fecal IgA and stool pH were brought to be closer to those of breastfed infants. Conclusion: An infant formula enriched with bovine milk-derived oligosaccharide and B. lactis supports normal infant growth, is well tolerated and improves intestinal health markers. No differences in diarrhea and febrile infection incidence were found in the population studied.

ObjectiveThis multicenter non-inferiority study evaluated the safety of infant formulas enriched with bovine milk fat globule membrane (MFGM) fractions.MethodsHealthy, full-term infants ( n = 119) age ≤14 days were randomized to standard infant formula (control), standard formula enriched with a lipid-rich MFGM fraction (MFGM-L), or standard formula enriched with a protein-rich MFGM fraction (MFGM-P). Primary outcome was mean weight gain per day from enrollment to age 4 months (non-inferiority margin: –3.0 g/day). Secondary (length, head circumference, tolerability, morbidity, adverse events) and exploratory (phospholipids, metabolic markers, immune markers) outcomes were also evaluated.ResultsWeight gain was non-inferior in the MFGM-L and MFGM-P groups compared with the control group. Among secondary and exploratory outcomes, few between-group differences were observed. Formula tolerance rates were high (>94%) in all groups. Adverse event and morbidity rates were similar across groups except for a higher rate of eczema in the MFGM-P group (13.9% vs control [3.5%], MFGM-L [1.4%]).ConclusionBoth MFGM-enriched formulas met the primary safety endpoint of non-inferiority in weight gain and were generally well tolerated, although a higher rate of eczema was observed in the MFGM-P group.

The nutritional composition of human milk evolves over the course of lactation, to match the changing needs of infants. This single-arm, non-inferiority study evaluated growth against the WHO standards in the first year of life, in infants consecutively fed four age-based formulas with compositions tailored to infants’ nutritional needs during the 1st, 2nd, 3rd–6th, and 7th–12th months of age. Healthy full-term formula-fed infants (n = 32) were enrolled at ≤14 days of age and exclusively fed study formulas from enrollment, to the age of four months. Powdered study formulas were provided in single-serving capsules that were reconstituted using a dedicated automated preparation system, to ensure precise, hygienic preparation. The primary outcome was the weight-for-age z-score (WAZ) at the age of four months (vs. non-inferiority margin of −0.5 SD). Mean (95% CI) z-scores for the WAZ (0.12 (−0.15, 0.39)), as well as for the length-for-age (0.05 (−0.19, 0.30)), weight-for-length (0.16 (−0.16, 0.48)), BMI-for-age (0.11 (−0.20, 0.43)), and head circumference-for-age (0.41 (0.16, 0.65)) at the age of four months, were non-inferior. Throughout the study, anthropometric z-scores tracked closely against the WHO standards (within ±1 SD). In sum, a four-stage, age-based infant formula system with nutritional compositions tailored to infants’ evolving needs, supports healthy growth consistent with WHO standards, for the first year of life.

International audience; To date, only few studies have assessed oral immunotherapy (OIT) for wheat allergy and often describe severe adverse reactions during therapy. We developed partially hydrolyzed wheat-based cereals (pHC), which were used in a multicenter, open-label, OIT pilot study, in immunoglobulin E-mediated wheat allergy children (NCT01332084). The primary objective of the study was to test whether wheat allergic patients tolerate pHC and primary end point was the presence or not of immediate adverse reactions to pHC during the 1-day initial escalation phase (stepwise increased doses of pHC), with evaluation of the maximum dose tolerated. Of the 9 patients enrolled in the trial, 4 discontinued OIT because of mild to severe reactions at the initial escalation phase. The 5 patients who passed the escalation phase consumed pHC daily for 1 to 6 months. One of these patients withdrew due to noncompliance, whereas the 4 others completed the study and successfully passed the wheat challenge test at the end of the study. About 60% of the adverse events were unrelated to the study product. Our study provides preliminary evidence that pHC is tolerated by a subset of wheat allergic patients. Further studies are warranted to test its efficacy as a potential therapeutic option for wheat allergic patients.

The gut microbiota of infants is shaped by both the mode of delivery and the type of feeding. The gut of vaginally and cesarean-delivered infants is colonized at different rates and with different bacterial species, leading to differences in the gut microbial composition, which may persist up to 6 months. In a multicenter, randomized, controlled, double-blind trial conducted in South Africa, we tested the effect of a formula supplemented with a prebiotic (a mixture of bovine milk-derived oligosaccharides [BMOS] generated from whey permeate and containing galactooligosaccharides and milk oligosaccharides such as 3′- and 6′-sialyllactose) and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446 on the bifidobacteria levels in the gut of infants born vaginally or via cesarean section in early life. Additionally, the safety of the new formulation was evaluated. A total of 430 healthy, full-term infants born to HIV-positive mothers who had elected to feed their child beginning from birth (≤3 days old) exclusively with formula were randomized into this multicenter trial of four parallel groups. A total of 421 infants who had any study formula intake were included in the full analysis set (FAS). The first two groups consisted of cesarean-delivered infants assigned to the Test formula (n = 92) (a starter infant formula [IF] containing BMOS at a total oligosaccharide concentration of 5.8 ± 1.0 g/100 g of powder formula [8 g/L in the reconstituted formula] + B. lactis [1 × 10 7 colony-forming units {cfu}/g]) or a Control IF (n = 101); the second two groups consisted of vaginally delivered infants randomized to the same Test (n = 115) or Control (n = 113) formulas from the time of enrollment to 6 months. The primary efficacy outcome was fecal bifidobacteria count at 10 days, and the primary safety outcome was daily weight gain (g/d) between 10 days and 4 months. At 10 days, fecal bifidobacteria counts were significantly higher in the Test formula than in the Control formula group among infants with cesarean birth (median [range] log: 9.41 [6.30–10.94] cfu/g versus 6.30 [6.30–10.51] cfu/g; P = 0.002) but not among those with vaginal birth (median [range] log: 10.06 [5.93–10.77] cfu/g versus 9.85 [6.15–10.79] cfu/g; P = 0.126). The lower bound of the two-sided 95% confidence interval of the difference in the mean daily weight gain between the Test and Control formula groups was more than –3 g/d in both the vaginally and cesarean-delivered infants, indicating that growth in the Test formula-fed infants was not inferior to that of Control formula-fed infants. At 10 days and 4 weeks, the fecal pH of infants fed the Test formula was significantly lower than in those fed the Control formula, irrespective of mode of delivery: for vaginal delivery: 4.93 versus 5.59; P < 0.001 (10 days) and 5.01 versus 5.71; P < 0.001 (4 weeks); for cesarean delivery: 5.14 versus 5.65, P = 0.009 (10 days) and 5.06 versus 5.75, P < 0.001 (4 weeks). At 3 months, this acidification effect only persisted among cesarean-born infants. IF supplemented with the prebiotic BMOS and probiotic B. lactis induced a strong bifidogenic effect in both delivering modes, but more explicitly correcting the low bifidobacteria level found in cesarean-born infants from birth. The supplemented IF lowered the fecal pH and improved the fecal microbiota in both normal and cesarean-delivered infants. The use of bifidobacteria as a probiotic even in infants who are immunologically at risk is safe and well tolerated.

Background The safety of an infant formula containing a new mixture of the prebiotics galacto-oligosaccharides (GOS) and fructo-oligosaccharide (FOS) and the probiotic Lactobacillus reuteri needs to be evaluated. Methods Healthy term infants in Singapore were randomly assigned (using computer-generated allocation sequences) to receive exclusively an experimental infant formula containing L. reuteri, GOS (5.50 g/L), and FOS (0.36 g/L) or a control formula containing only L. reuteri from enrollment (7–14 days of age) to 4 months of age. The primary objective of this trial was to demonstrate that weight change between birth and 4 months of age in infants fed the experimental formula was not inferior to World Health Organization (WHO) Child Growth standards. The non-inferiority margin was −0.5 standard deviations (SD). The secondary objectives were to compare changes in anthropometric measurements (weight, length, body mass index, and head circumference), digestive tolerance, stool bacterial counts, urinary D- and L- lactate concentrations, and adverse events in the two formula groups. Results The intention-to-treat (ITT) population included all randomized infants stratified by gender, (experimental group, N = 68 and control group, N = 72). The per-protocol (PP) population included 61 infants in the experimental and 62 infants in the control groups. The change in weight-for-age z-score between birth and 4 months was +0.93 (95% confidence interval [CI]: +0.63 to +1.23) SD in the experimental group and +0.92 (95% CI: +0.62 to +1.22) SD in the control group in the PP population, indicating non-inferior weight gain in both formulas groups compared with WHO standards. The ITT population had similar results. Liquid stools occurred more frequently in the experimental compared with the control group and median bifidobacteria, lactobacilli, and enterococci counts were higher in the experimental group (p

Powdered infant formula is conventionally supplied in multi-serve packaging and reconstituted following multiple manual preparation steps. An advanced infant nutrition system, combining a dedicated dispenser with single-serve capsules of powdered formula, was designed to precisely deliver hygienic reconstituted formula in < 1 min. To test the performance (technical reliability and quality of reconstituted formula), a field study was conducted at three hospitals in Switzerland with a prototype system, where hospital staff used the system (n = 4) to reconstitute formula when needed by infants. Only 22 (0.9%) of the 2445 bottles prepared over the 4-mo period encountered preparation difficulties. After reconstitution, 93% of the 2445 capsules showed no solid residue, and 5% had only a few small lumps, suggesting good solubility. Additional preparations were conducted in hospitals to assess the dilution precision and microbiological quality of reconstitutes. The target for reconstituted formula weight (±3g) wa...

Cow milk proteins, particularly β-lactoglobulin (BLG) are a leading cause of food allergy in children. In vivo, gastrointestinal hydrolysis of milk proteins decreases their allergenicity, but the intestinal microbiota also contributes to their breakdown. This study investigated the effect of hydrolysis of BLG-derived tryptic-chymotryptic (TC) peptides by Bifidobacterium lactis NCC362 enzymes, on their allergenicity. B. lactis enzymes have been shown to hydrolyze both acidic and basic peptide fractions isolated from tryptic-chymotryptic hydrolysate, but not whole proteins. The IgE binding capacity of acidic TC peptides was reduced by B. lactis peptidases-driven hydrolysis. This is partly explained by the breakdown of known allergenic peptides. Moreover, the resulting peptide fragments significantly up-regulated IFN-γ and IL-10 production and down-regulated IL-4 secretion by murine splenocytes. These results indicate that B. lactis NCC362 could be a potential probiotic for preventing cow's milk allergy through hydrolysis of the allergenic portion of BLG and the release of peptides which down-regulate the allergic immune response, but further studies using living bacteria are needed to confirm these data.

Ibuprofen and antibiotics are commonly prescribed during early childhood. When given to mice at the time at which oral tolerance is induced, both treatments affect either the induction or the maintenance of oral tolerance. These results suggest that the coadministration of these and similarly acting drugs should be considered cautiously for infants at risk of allergy.

We have previously demonstrated thatLactobacillus paracaseiNCC2461 may help to prevent cow's milk allergy in mice by inducing oral tolerance to β-lactoglobulin (BLG). To investigate the mechanisms involved in this beneficial effect, we examined the possibility thatL. paracaseiinduces tolerance by hydrolyzing BLG-derived peptides and liberating peptides that stimulate interleukin-10 (IL-10) production.L. paracaseipeptidases have been shown to hydrolyze tryptic-chymotryptic peptides from BLG, releasing numerous small peptides with immunomodulating properties. We have now shown that acidic tryptic-chymotryptic peptides stimulate splenocyte proliferation and gamma interferon (IFN-γ) production in vitro. Hydrolysis of these peptides withL. paracaseipeptidases repressed the lymphocyte stimulation, up-regulated IL-10 production, and down-regulated IFN-γ and IL-4 secretion.L. paracaseiNCC2461 may therefore induce oral tolerance to BLG in vivo by degrading acidic peptides and releasing immunomodulatory peptides stimulating regulatory T cells, which function as major immunosuppressive agents by secreting IL-10.

In this study, the effect ofLactobacillus paracasei(NCC 2461),Lactobacillus johnsonii(NCC 533) andBifidobacterium lactisBb12 (NCC 362) on the induction and maintenance of oral tolerance to bovine β-lactoglobulin (BLG) was investigated in mice. Germfree mice were monocolonized with one of the three strains before oral administration of whey protein to induce tolerance. Mice were then injected with BLG and sacrificed 28 or 50 days after whey protein feeding for humoral and cellular response measurement. Conventional and germfree mice were used as controls. Both humoral and cellular responses were better suppressed in conventional mice than in germfree and monoassociated mice throughout the experiment and better suppressed inL. paracasei-associated mice than in mice colonized withB. lactisorL. johnsonii.The latter two mono-associations suppressed humoral responses only partially and cellular responses not at all. This study provides evidence that probiotics modulate the oral tolerance response to BLG in mice. The mono-colonization effect is strain-dependant, the best result having been obtained withL. paracasei.

Mucosal administration of antigen is known to be appropriate for vaccine purposes as well as tolerance induction. Biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles were used to deliver both antibacterial phosphorylcholine (PC) and dietary antigen beta lactoglobulin (BLG) by mucosal route. In a first study, the protective immunity elicited by intragastric vaccination with PC encapsulated in microparticles was evaluated in a mouse model against intestinal infection by Salmonella typhimurium and pulmonary infection by Streptococcus pneumoniae. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after oral immunization with PC-loaded microparticles compared with the titers of mice immunized with free PC-thyr or blank microparticles. This antibody response correlated with a highly significant resistance to oral challenge by S. typhimurium. IgA in pulmonary secretion were not able to protect against S. pneumoniae infection. BALB/c mice were, therefore, immunized intranasally (i.n.). Immunization was followed by a rise in anti-PC IgA and IgG titers in serum and in pulmonary secretions by both free and encapsulated PC-Thyr. The survival rates were 91 and 76% in the two groups of mice, respectively. In a second study and in order to prevent allergy against milk by inducing oral tolerance, one of the major allergenic milk protein, BLG was entrapped into microparticles. Oral administration of microparticles containing BLG reduced significantly (by 10000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. These studies demonstrate the ability of microparticles to induce both mucosal immunity and oral tolerance.

The goal of the present paper was to optimize the encapsulation of beta-lactoglobulin (BLG) within poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion solvent evaporation method. The role of the pH of the external phase and the introduction of the surfactant Tween 20, in the modulation of the entrapment and release of BLG from microparticles, was studied. Reducing the solubility of BLG by decreasing the pH of the external phase to a value close to the pI of BLG resulted in a better encapsulation with, however, a larger burst release effect. By contrast, Tween 20 was shown to increase the encapsulation efficiency of BLG and reduce considerably the burst release effect. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface. In addition, Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease in the burst release. These results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.

Purpose. The goal of the present paper was to investigate the role of a surfactant, Tween 20, in the modulation of the entrapment and release of β-lactoglobulin (BLG) from poly (DL-lactide-co-glycolide) microspheres.

Poly(D,L-lactide-co-glycolide) microspheres loaded with phosphorylcholine linked to thyroglobulin (PC-Thyr) as protein carrier were prepared. The entrapment efficiency reached 80% when the initial protein:polymer ratio was 1:8. Ninety-four percent of the loaded microspheres had a diameteror = 10 microns. The encapsulation process did not alter PC-Thyr absorbance nor PC antigenic reactivity. Oral administration of these microspheres induced a specific IgA response in intestinal, pulmonary and vaginal secretions, as well as a strong specific systemic immune response in female Balb/c mice. This suggests the need to explore further the potential ability of PC-Thyr loaded microspheres to protect against infections caused by PC-bearing microorganisms which invade or colonize different mucosal sites.

Background: Oral administration of an antigen has been shown to suppress the specific immune response to this antigen. This approach, called oral tolerance, has been demonstrated with intact proteins in animal models for prevention of allergy and autoimmune diseases. Objective: The purpose of this study was to determine whether oral tolerance can be induced with protein peptides. Partially hydrolyzed and extensively hydrolyzed cow's milk formulas were compared for their capacity to induce tolerance to cow's milk proteins. Methods: Five-week-old Sprague-Dawley rats were fed cow's milk formulas ad libitum from day 1 to day 19. All animals were immunized with β-lactoglobulin and ovalbumin on day 5 and bled on day 19. Sera were analyzed for specific IgE and IgG antibodies by ELISA and for functional IgE response by in vitro mast cell mediator (tritiated serotonin) release. In vivo modulation of intestinal mast cells was analyzed by the specific release of the rat mast cell protease II, and T-cell response was determined by tritiated thymidine incorporation into lymph node lymphocytes. Results: Oral administration of a partially hydrolyzed cow's milk formula suppresses specific serum IgE and IgG anti-β-lactoglobulin antibodies, as well as mediator release from rat mast cells and T-lymphocyte response. This suppression was shown to be antigen-specific and dose-dependent. An extensively hydrolyzed formula was unable to achieve the induction of such an oral tolerance. Conclusion: These results support the view that partially hydrolyzed proteins are able to induce specific oral tolerance, whereas extensively hydrolyzed proteins are not. (J Allergy Clin Immunol 1997;100:266-73.)

Background: Oral tolerance against food proteins has been achieved in different animal models with use of native or moderately hydrolyzed proteins as inducers. However, native proteins remain highly allergenic, although it has been demonstrated that protein hydrolyzates and resulting peptides can lose their allergenicity. Objective: This study was designed to evaluate the ability of β-lactoglobulin hydrolyzate and peptides to induce oral tolerance to native β-lactoglobulin and to identify tolerogenic β-lactoglobulin peptides with low allergenicity. Methods: β-Lactoglobulin was hydrolyzed by trypsin and fractionated by ion exchange chromatography. Peptide enrichment of fractions was evaluated. Balb/c mice were fed β-lactoglobulin hydrolyzate or fractions by single gavage at day 1. Five days later animals were challenged intraperitoneally with native β-lactoglobulin. At day 27 delayed-type hypersensitivity was performed. Twenty-four hours later mice were bled, and intestinal contents and spleens were collected. Oral tolerance was measured by titrating specific IgE in sera and intestinal samples. Specific T-cell responses were analyzed by splenocyte proliferation. Antigenicity of hydrolyzate and fractions was evaluated by specific ELISA inhibition. Results: Mice fed either β-lactoglobulin hydrolyzate or 2 fractions of the hydrolyzate were tolerized against β-lactoglobulin. Specific serum and intestinal IgE were suppressed. Delayed-type hypersensitivity and proliferative responses were inhibited. One tolerogenic fraction was found to be 50 times less antigenic than the total β-lactoglobulin hydrolyzate was. Conclusion: These findings support the strategy of inducing oral tolerance in "at-risk" patients by means of tolerogenic cow's milk peptides or hydrolyzate. (J Allergy Clin Immunol 2000;105:514-21.)

Oral administration of antigen is known to be appropriate for some vaccine purposes as well as oral tolerance induction. In the present study, oral administration of β-lactoglobulin (BLG) loaded poly( d , l -lactide-co-glycolide) ( d , l -PLG) microspheres induced tolerance was evaluated. A single feeding of 5 μg of encapsulated BLG tolerised BALB/c mice to subsequent BLG parenteral challenge, suppressing the specific humoral, intestinal and cellular responses. The tolerogenic efficient dose was then reduced 10,000 times, compared to oral administration of soluble BLG. This suggests that loading food proteins into d , l -PLG microspheres might be a potential tool for inducing oral tolerance with allergens.

This study was designed to confirm the tolerogenic properties of beta-lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed beta-lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non-specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP-fed, versus saline-fed mice, by measuring specific seric and intestinal antibody responses, delayed-type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three-week-old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti-BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP-fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) versus an increase of IL-10 and transforming growth factor-beta (TGF-beta) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows' milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.

The purpose of this study was to investigate the in-vitro interaction between [3H]ampicillin-loaded polyisohexylcyanoacrylate nanoparticles and murine macrophages (peritoneal and J774) infected with Salmonella typhimurium. The multiplicity of infection was ten bacteria to each macrophage and the mean (+/- S.D.) diameter of the nanoparticles was 220 (+/- 20 nm), corresponding to an ampicillin concentration of 2 g/L. The uptake of nanoparticle-bound [3H]ampicillin by non-infected J774 and peritoneal macrophages was six- and 24-fold greater respectively than that of free [3H]ampicillin. For infected cells, uptake by J774 and peritoneal macrophages was nine- and 20-fold greater respectively. However, there was no difference between nanoparticle-bound ampicillin and free ampicillin in terms of bactericidal activity against intracellular S. typhimurium. This unexpected observation might be accounted for by bacterium-induced inhibition of phagosome-lyosome fusion within the macrophages, thereby preventing contact between the bacteria in the phagosomes and the nanoparticles in the secondary lysosomes.

Objective: To assess growth of infants fed starter formula supplemented with milk fat globule membranes (MFGM).

370 Effect of Partially Hydrolyzed and Extensively Hydrolyzed Milk Formulas on the Oral Tolerance Development in Infants at Risk of Allergy

Objective: To assess growth of infants fed starter formula supplemented with either prebiotics or symbiotic. In addition, gut microbiota was studied. Methods: Healthy, full term newborns (n= 240) were enrolled before the 14th day of their life and randomly assigned between 3 exclusively formula fed groups: starter IF, starter IF + prebiotics (CMOS+GOS), starter IF + prebiotics (CMOS+GOS) and probiotics (BL999 + LPR). Growth was assessed by body weight gain during 4 months. Gut microbiota profile was examined by measuring fecal counts in Bifidobacteria, Lactobacilli, Enterobacteria, Clostridium at 2 months of age. Incidence of morbidity was appraised. Results: Non inferiority in growth was established between the two experimental groups and the control at the end of the 4 months intervention period. 90% confidence intervals for the effects of prebiotics versus control and symbiotic versus control were (-1.24, 3.17) and (-2.35, 2.02) respectively. Higher levels of fecal Bifidobacteria were observed in the experimental groups; this difference being significant between the symbiotic and control groups (8.8 +/-;1.7 versus 9.87+/-;1.2 p< 0.05). Furthermore, Clostridium levels were lower in both experimental vs control groups. No statistically significant differences of adverse events were detected between groups, except for a higher incidence of colics in the prebiotic and, to a lesser degree, in the symbiotic group vs control. Conclusion: Growth of newborns receiving IF containing prebiotics or symbiotic was non inferior to the growth observed in controls and within the limits defined by WHO growth reference curves. A bifidogenic effect due to symbiotic formulation was demonstrated.

When healthy volunteers were given a daily dose of 3 x 10(8) life-dehydrated Saccharomyces cerevisiae cells for 5 days, the volunteers excreted 10(5) living yeast cells per g of feces at first, but the yeast cells disappeared within 5 days of the end of treatment. In gnotobiotic mice, S. cerevisiae administered alone colonized the intestinal tract but did not interfere with previous or subsequent colonization by a variety of potentially enteropathogenic microorganisms. When these microorganisms were present, the intestinal counts of S. cerevisiae were greatly reduced.

The ecological impact of roxithromycin given orally at 300 mg/day on the intestinal floras in six human volunteers was studied. The resulting fecal concentrations of active roxithromycin were in the range of 100 to 200 micrograms/g of feces. Consecutive modifications in the composition of the fecal floras were limited to a decrease in counts of total members of the family Enterobacteriaceae. The rest of the intestinal floras, including the predominant anaerobic floras, changed little. No overgrowth of Pseudomonas aeruginosa, staphylococci, fungi, or highly erythromycin-resistant strains of the family Enterobacteriaceae was observed. The strains of Enterobacteriaceae and of anaerobes isolated during treatment were not markedly more resistant to roxithromycin than those isolated before treatment started. Changes in intestinal resistance to colonization by exogenous microorganisms in gnotobiotic mice inoculated with human fecal flora were studied and were also found to be minimal. The impact of oral roxithromycin on the intestinal microbiota appears to be weaker than that previously observed with oral erythromycin, perhaps because the concentrations of roxithromycin in the feces were lower than those previously found for erythromycin.

High concentrations of ciprofloxacin have been shown to persist in the faeces of volunteers for several days after a week of oral treatment with this drug, which was also found to have a prolonged effect on aerobic Gram-negative intestinal bacteria. To determine whether a shorter course of ciprofloxacin would have the same prolonged effect, we treated ten healthy adult volunteers with a single oral dose of 750 mg ciprofloxacin and found that this was not followed by any significant changes in the counts of anaerobes or streptococci, but that there was a mean decrease of 2.5 log10 cfu/ml in the counts of faecal Enterobacteriaceae, which lasted for a full week. We attributed this to the persistence of high faecal ciprofloxacin concentrations for several days in all the volunteers. We did not observe any significant increase in the MICs of ciprofloxacin for faecal Enterobacteriaceae, or any faecal overgrowth of staphylococci, fungi, Pseudomonas aeruginosa or Clostridium difficile.

Intestinal endogenous members of the family Enterobacteriaceae were eliminated in 12 human volunteers treated with 400 or 800 mg of oral norfloxacin per day for 5 days. No clones resistant to quinolone derivatives were isolated. Counts of aerotolerant streptococci were affected to various degrees, depending on their susceptibility to norfloxacin. During treatment, counts of anaerobes remained above 9.8 log10 CFU/g of feces. A total of 932 anaerobic isolates from the predominant flora (over 10(9) CFU/g) in fecal samples obtained before or during norfloxacin treatment were classified by a simple morphological and physiological scheme. The composition of this flora was fairly stable from one sample to another before treatment and was not substantially modified by norfloxacin. Intestinal resistance to colonization by exogenous microorganisms was studied in gnotobiotic mice associated with a human fecal flora. The composition of the fecal flora of the human donor and the fecal concentrations of norfloxacin in the volunteers were reproduced in the intestine of the mice. Resistance to colonization by exogenous microorganisms was reduced by norfloxacin for only 2 of 14 (14%) of the strains tested. These results suggest that norfloxacin is a good candidate for selective antimicrobial modulation of the intestinal tract in humans.

Intestinal members of the family Enterobacteriaceae were eliminated in five human volunteers treated with oral ofloxacin for 5 days. No emergence of resistant Enterobacteriaceae was observed. Counts of group D streptococci were significantly reduced. Colonization by Candida sp. was observed in all five volunteers during ofloxacin treatment. The anaerobic flora was fairly stable from one sample to another before treatment and was not substantially modified by ofloxacin.

The milk model protein, beta lactoglobulin (BLG), was encapsulated into microspheres prepared by a multiple emulsion/solvent evaporation method. The effect of the pH of the outer aqueous phase on protein encapsulation and release as well as on microsphere morphology has been investigated. At all tested pH values, the encapsulation efficiency was shown to decrease with increasing the initial amount of BLG. This was correlated with the reduced stability of the primary emulsion as the initial BLG increased. In addition, reducing the solubility of BLG in the external aqueous phase by decreasing the pH to the isoelectric point of BLG (pI 5.2) resulted in an improved protein encapsulation. Moreover, it was shown that combining pH modification and optimal stability of the first emulsion yielded microspheres with a high encapsulation efficiency. However, release kinetic studies revealed that a significant burst release was observed with microspheres loaded with large amounts of BLG, especially when prepared in a medium at pH 5.2. This burst effect was attributed to morphology changes in the microsphere surface which was characterized by the presence of pores or channels able to accelerate the release of BLG. These pores were assumed to result from the presence of large amounts of protein molecules on the microsphere surface, that aggregate during microsphere formation at pH 5.2. Indeed, single adsorption experiments have shown that BLG had a higher affinity for the particle surface when the pH was close to the pI. Thus, reducing the solubility of a protein in the external aqueous phase allows the product of microspheres with a better encapsulation efficiency, although this benefit is provided by a strong adsorption of the protein on microsphere surface.

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.