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Scope: β-lactoglobulin (BLG) is a major cow milk allergen encountered by the immune system of infants fed with milk-based formulas. To determine the effect of processing on immunogenicity of BLG, this article characterized how heated and glycated BLG are recognized and internalized by APCs. Also, the effect of heat-induced structural changes as well as gastrointestinal digestion on immunogenicity of BLG is evaluated., Methods and Results: The binding and uptake of BLG from raw cow milk and heated either alone (BLG-H) or with lactose/glucose (BLG-Lac and BLG-Glu) to the receptors present on APCs are analyzed by ELISA and cell-binding assays. Heated and glycated BLG is internalized via galectin-3 (Gal-3)and scavenger receptors (CD36 and SR-AI) while binding to the receptor for advanced glycation end products (R AGE) does not cause internalization. Receptor affinity of BLG is dependent on increased hydrophobicity, β-sheet exposure and aggregation. Digested glycated BLG maintained binding to sRAGE and Gal-3 but not to CD36 and SR-AI, and is detected on the surface of APCs. This suggests a mechanism via which digested glycated BLG may trigger innate (via RAGE) and adaptive immunity (via Gal-3)., Conclusions: This study defines structural characteristics of heated and glycated BLG determining its interaction with APCs via specific receptors thus revealing enhanced immunogenicity of glycated versus heated BLG., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Scope: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated., Methods and Results: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (β-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport., Conclusions: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Background: Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood., Objective: Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection., Methods: Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively., Results: Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation., Conclusion: The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens., (Copyright © 2020. Published by Elsevier Inc.)

Lipocalins, small extracellular hydrophobic molecule carriers, can be internalized by a variety of different cells. However, to date receptors have only been identified for human lipocalins. Here, we specifically investigated uptake mechanisms for lipocalins ß-lactoglobulin and Fel d 4 in HeLa and Chinese hamster ovary (CHO) cells. We provide evidence that cell surface heparan sulphate proteoglycan is essential for internalization of these lipocalins. In HeLa cells, lipocalin uptake was inhibited by competition with soluble heparin, enzymatic digestion of cellular heparan sulphate by heparinase and inhibition of its biosynthesis by sodium chlorate. Biochemical studies by heparin affinity chromatography and colocalization studies further supported a role of heparan sulphate proteoglycan in lipocalin uptake. Finally, lipocalin uptake was blocked in CHO mutant cells defective in glycosaminoglycan biosynthesis whereas in wild-type cells it was clearly detectable. Thus, cell surface heparan sulphate proteoglycan represents a novel component absolutely participating in the cellular uptake of some lipocalins.

Scope: During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β-lactoglobulin (BLG), in their interactions with cells crucially involved in allergy., Methods and Results: BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T-cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco-2 monolayer. Uptake of glycated BLG by bone marrow-derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor-mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4 + T-cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG-specific IgE sensitized basophil cells., Conclusions: This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: β-lactoglobulin (BLG), a major allergen in cow milk (CM) can be detected in human breast-milk (BM) and is associated with exacerbation of symptoms in breastfed infants with cow milk protein allergy (CMPA). Currently, it is not known how long lactating mothers who consume dairy products need to withhold breastfeeding., Objective: To elucidate the kinetics of BLG in BM after maternal ingestion of a single dose of CM., Methods: Nineteen lactating mothers, four of whom had infants with CMPA, were instructed to avoid CM for 7 days before ingesting a single dose of CM and to continue to withhold CM thereafter throughout the study period. BLG was measured by ELISA in BM from 15 mothers of healthy infants before and at 3, 6 and 24 h, and 3 and 7 days after CM ingestion. Four pairs of mothers and CMPA infants were enrolled for BM challenge after the mothers had ingested CM., Results: After CM ingestion, the level of BLG in BM increased significantly from 0.58 ng/ml (0.58 g/L) (IQR 0.38-0.88) to a peak level of 1.23 ng/ml (IQR 1.03-2.29), p < 0.001. The BLG level on day 3 (1.15 ng/ml, IQR 0.89-1.45) and day 7 (1.08 ng/ml (IQR 0.86-1.25) after CM ingestion was significantly higher than baseline (p = 0.01 and p = 0.001, respectively). BLG was detected in all BM samples from the four mothers of CMPA infants after CM ingestion, and the level was not different from that in the mothers of the 15 healthy infants. Three of the four CMPA infants developed symptoms such as maculopapular rash and hypersecretion in the airways after BM challenge., Conclusions: BLG can be detected in BM up to 7 days after CM ingestion. Lactating mothers should suspend breastfeeding to CMPA infants more than 7 days after CM ingestion.

Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from β-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.

In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated β-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-β protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.

BLG (beta-lactoglobulin) and CBLG (cationic BLG developed by our lab) were evaluated as potential nutraceutical/drug carriers. The cationic corona conferred CBLG with superior integrity and drug retention under gastrointestinal conditions, at most 40-fold higher mucoadhesion, up to 30-fold greater transepithelial permeation and, at most 285% higher cellular uptake, compared to BLG. Furthermore, the more hydrophilic CBLG species exhibited better mucoadhesion, while the more hydrophobic one exhibited higher cellular uptake. Intriguingly, protein molecules were more cytotoxic and exhibited up to 175% higher tight junction-opening capacity than did protein nanoparticles, whereas the nanoparticles displayed up to 770% higher mucoadhesion, greater transepithelial permeation and elevated cellular uptake. Finally, all these surface properties and performances were significantly altered as CBLG bound to serum proteins. Possible mechanisms underlying these findings are discussed in detail. This research sheds some light on the development of protein-based nanoencapsulants and their performance upon oral administration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of β-lactoglobulin and anti-bovine β-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between β-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between β-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the β-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the β-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.

Background and Objectives: The digestion rate of proteins and subsequent absorption of amino acids can independently modulate protein metabolism. The objective of the present study was to examine the blood amino acid response to whey protein isolate (WPI), β-lactoglobulin-enriched WPI, hydrolysed WPI and a flavour-identical control., Methods: Eight healthy adults (four female, four male) were recruited (mean±standard error of the mean: age, 27.0±0.76 years; body mass index, 23.2±0.8 kg/cm(2)) and after an overnight fast consumed 500 ml of each drink, each containing 25g protein, in a cross-over design. Blood was taken at rest and then every 15 min for 2 h post ingestion., Results: Ingesting the β-lactoglobulin-enriched WPI drink resulted in significantly greater plasma leucine concentrations at 45-120 min and significantly greater branched-chain amino acid concentrations at 60-105 min post ingestion compared with hydrolysed WPI. No differences were observed between WPI and β-lactoglobulin-enriched WPI, and all protein drinks resulted in elevated blood amino acids compared with flavour-identical control., Conclusions: In conclusion, whole proteins resulted in a more rapid absorption of leucine and branched-chain amino acid into the blood compared with the hydrolysed molecular form of whey protein.

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

Beta-lactoglobulin (betaLG) is a protein that binds ligands like fatty acids and retinol into the hydrophobic pocket. Our purpose was to study bovine and reindeer betaLG as transporter molecules and compare their transport properties across Caco-2 cell membrane. The reindeer betaLG has more valuable binder characteristics than bovine betaLG because it has only one genetic phenotype and it seems to exhibit better immunological properties. The permeation of betaLG in Caco-2 cells was evaluated by immunoblotting, and the permeation of the model substances retinol, palmitic acid and cholesterol with and without betaLG was determined using [(3)H]-labelled ligands. Both bovine and reindeer betaLG were able to pass across a Caco-2 cell monolayer similarly. Unbound and betaLG-bound [(3)H]retinol and [(3)H]palmitic acid were equally transported across the Caco-2 cell layer, whereas [(3)H]cholesterol could not pass across Caco-2 cells with or without betaLG at any of the studied circumstances. Thus, the bovine and reindeer milk betaLG is not a suitable protein to enhance transport of ligands across the Caco-2 cell membrane, used for predicting intestinal absorption.

Amyloid fibril forming proteins have been related to some neurodegenerative diseases and are not fully understood. In some such systems, these amyloid fibrils have been found to form radially oriented spherulite structures. The thermal dependence of formation and growth of these spherulite structures in two model protein systems, beta-lactoglobulin and insulin at low pH aqueous and high temperature conditions, have been monitored with time-lapse optical microscopy and quantified. A population-based polymerization reaction model was developed and applied to the experimental data with excellent agreement. While spherulites in the insulin solutions formed and grew at approximately 25x the rate of spherulites in the beta-lactoglobulin solutions, the temperature dependence and activation energies of both systems were found to be very similar to one another, suggesting that the underlying rate-limiting mechanisms for both formation and growth are consistent across the two systems. The similarity of both of these amyloid fibril forming protein systems provides confidence in their use as model systems for extrapolating understanding to similar systems involved in neurodegenerative diseases.

Transferrin (Tf) holds promise as a drug carrier because of overexpress transferring receptors (TfRs) on the surface of tumor cells. The purpose of this work was to conjugate Tf to PEGylated protein (Tf-PEG-protein) to improve tumor-targeted delivery of PEG-protein. After a model protein, beta-lactoglobulin B (LG), was modified by the heterobifunctional polyethyleneglycol (PEG), Tf was covalently linked to the distal end of the PEG chains on the PEG-LG (PL) conjugate. The purified Tf-PEG-LG (TPL) contained 1.4 of Tf ligand on one LG molecule. The specificity and affinity of TPL to TfR on two kinds of tumor cells (K562 and KB cells) were assessed. The results demonstrated that, TPL can bind specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. The pharmacokinetics and biodistribution studies in rodents found that TPL exhibited a significantly delayed blood clearance, the longest tumor resident time and the greatest tumor accumulation, as compared with LG and PL. Such design of the Tf conjugate would suggest a promising approach for active tumor targeting of therapeutic proteins or peptides to target cells.

The adsorption process of beta-lactoglobulin on the polyethersulfone-coated surface has been investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed simultaneous measurements of frequency and energy dissipation changes caused by the formation of a beta-lactoglobulin layer during the protein adsorption process. The results showed that the protein adsorption on the spin-coated polyethersulfone surface consisted of reversible and irreversible adsorption processes, and the irreversible adsorption was the principal factor to cause the permanent protein adsorption onto the polymer surface. The amount of irreversible adsorption of beta-lactoglobulin increased significantly, from 380.5 +/- 23.2 to 745.5 +/- 29.7 ng/cm2, when the concentration of beta-lactoglobulin solution varied from 0.1% to 2.0% (w/v). The pH also played an important role in the protein adsorption. When the pH of the solution varied from 7.0 to 3.0, the amount of irreversible adsorption of beta-lactoglobulin increased from 427.1 +/- 80.7 to 741.2 +/- 135.1 ng/cm2.

Oil-in-water emulsions (pH 7.0 or pH 3.0) containing 30 wt% soya oil and various concentrations of lactoferrin were made in a two-stage valve homogenizer. The average droplet size (d32), the surface protein coverage (mg/m2) and composition, and the zeta-potential of the emulsions were determined. The value of d32 decreased with increasing lactoferrin concentration up to 1%, and then was almost independent of lactoferrin concentration beyond 1% at both pH 7.0 and pH 3.0. The surface protein coverage of the emulsions made at pH 7.0 increased almost linearly with increasing lactoferrin concentration from 0.3 to 3%, but increased only slightly in emulsions made at pH 3.0 at lactoferrin concentrations >1%. The surface protein coverage of the emulsions made at pH 3.0 was lower than that of the emulsions made at pH 7.0 at a given protein concentration. The emulsion droplets had a strong positive charge at both pH 7.0 and pH 3.0, indicating that stable cationic emulsion droplets could be formed by lactoferrin alone. When emulsions were formed with a mixture of lactoferrin and beta-lactoglobulin (beta-lg) (1:1 by weight), the charge of the emulsion droplets was neutralized at pH 7.0 suggesting the formation of electrostatic complexes between the two proteins. The composition of the droplet surface layer showed that both proteins were adsorbed, presumably as complexes, from the aqueous phase at pH 7.0 in equal proportions, whereas competitive adsorption occurred between lactoferrin and beta-lg at pH 3.0. At this pH, beta-lg was adsorbed in preference to lactoferrin at low protein concentrations (1%), whereas lactoferrin appeared to be adsorbed in preference to beta-lg at high protein concentrations.

Purpose: Atopy patch tests (APTs) allow the detection of delayed allergies at the skin level. The localization of beta-lactoglobulin delivered into the skin by an innovative ready-to-use APT (E-patch was investigated and the efficacy and safety of this device were assessed., Methods: The E-patch containing beta-lactoglobulin was placed for 24 h in contact with hairless rat skin mounted in a Franz diffusion cell. Transdermal passage was monitored by measurement of beta-lactoglobulin A-[methyl-(14)C] or by two-site enzyme immunoassay. An iterative skin stripping allowed measurement of the beta-lactoglobulin penetrating the first external skin layers., Results: After 24 h, 92% of beta-lactoglobulin remained on the skin. The iterative skin strippings showed a 135-fold higher concentration of beta-lactoglobulin in the stratum corneum than that found in the epidermis-dermis. Analysis of the solution in the receiver compartment by radioactivity assays or immunoassays indicates that intact protein did not cross the skin., Conclusions: The E-patch system allows native beta-lactoglobulin to concentrate in the stratum corneum, in the vicinity of immunological cells, but does not lead to its systemic delivery. Therefore, it is suggested that this delivery system creates ideal conditions for promoting a positive topical response with reduced risk of systemic anaphylactic reactions caused by the native form of the beta-lactoglobulin A.

The objective of this study was to evaluate the influence of dietary nucleotide supplementation in preterm infants during the first month of life on the intestinal permeability to lactulose, mannitol and to beta-lactoglobulin and on the development of circulating antibodies to beta-lactoglobulin and alpha-casein. Twenty-seven preterm infants were enrolled in the study; 11 of them were fed a standard low-birth weight milk formula and 16 infants were fed the same formula supplemented with nucleotides at similar levels to those found in human milk. Blood and urine samples were obtained at 1, 7 and 30 days of age. Serum beta-lactoglobulin, serum IgG antibody to alpha-casein and serum IgG antibody to beta-lactoglobulin were measured by ELISA. The lactulose/mannitol urinary excretion rate was measured by gas liquid chromatography. Neither the intestinal permeability to saccharides nor the intestinal absorption of beta-lactoglobulin were affected by the nucleotide supplementation. However, serum concentrations of IgG antibody to beta-lactoglobulin were higher in preterm neonates fed the supplemented formula than in those fed the standard formula. According to these results, dietary nucleotides might influence the maturation of the humoral immune response in preterm newborn infants.

Our objective was to examine the permeability of the gut to protein macromolecules and sugar probes and their possible association in celiac disease patients. We studied the permeability to human alpha-lactalbumin, beta-lactoglobulin, mannitol, and lactulose on 46 occasions in 33 celiac disease patients in various phases of the disease; in addition, mannitol and lactulose permeability was studied in 18 healthy controls. Lactalbumin absorption was detected in 19 of 42 patients tested, more often in celiac disease patients with villous atrophy than in those with normal jejunal biopsy (p = 0.01). Higher absorption of lactalbumin was found in patients with subtotal villous atrophy than in those with normal biopsy (p = 0.02). beta-lactoglobulin was found in four of 42 patients tested. Less mannitol was absorbed by patients with either subtotal or partial villous atrophy than by those with normal histology (p = 0.001 and 0.006, respectively). Lactulose recovery was higher in newly diagnosed patients and patients with subtotal villous atrophy than in controls (p = 0.007 and 0.03, respectively). The lactulose/mannitol ratio was higher in newly diagnosed patients and patients with villous atrophy than in controls (p = 0.002 and 0.002, respectively). The correlation between permeability to lactalbumin and mannitol and lactulose was poor. We conclude that permeability to proteins and sugar molecules is abnormal in celiac disease patients with mucosal damage and that they probably reflect different mechanisms of penetration.

Background & Aims: Intestinal inflammation resulting in disruption of the mucosal barrier function has been proposed as a cause of increased incidence of allergic diseases. This study was designed to evaluate whether intestinal inflammation is able to change the immune responsiveness to sensitization and antigen challenge responses., Methods: Guinea pigs orally sensitized to cow's milk proteins were either treated or not treated with trinitrobenzenesulfonic acid (TNBS) to induce intestinal inflammation and compared with control animals (not sensitized). Systemic immune and local responsiveness to antigen challenge were assessed by measuring antibody serum titers, colonic fluid secretion, mucosal histamine level, and mucus depletion. Intestinal permeability was evaluated from 51Cr-ethylenediaminetetraacetic acid (EDTA) recovery and beta-lactoglobulin serum level., Results: Immunoglobulin E titers were higher in TNBS-treated animals than in non-TNBS-treated sensitized animals. Antigen challenge in TNBS-treated animals induced a fourfold increase of colonic secretion and greater histamine and mucus depletion than in non-TNBS-treated animals. Permeability to 51Cr-EDTA increased 5 days after TNBS treatment but was unchanged after antigen challenge. In contrast to controls, beta-lactoglobulin was not detected in the sera of challenged sensitized and TNBS-treated animals., Conclusions: Intestinal inflammation increasing gut permeability enhances the sensitization process. Therefore, local anaphylactic reactions are exacerbated after antigen challenge.

We investigated gut permeability to human alpha-lactalbumin (ALA) and bovine beta-lactoglobulin (BLG) in 20 infants from birth to 8 months or until weaning, before which they were on a strictly cow's-milk-free diet. We measured the proteins with a sensitive, solid-phase, double-sandwich immunofluorometric assay. Median (range) levels of serum ALA on days 3-4 after birth, and at 1 and 2 months of age were 31 (12-225), 6 (0-55), and 2 (0-16) micrograms/l serum per g ALA given per kg body weight, respectively. At 3, 5, and 8 months of age, only trace amounts of ALA were found. One week after weaning, serum BLG was found in 5/13 infants (38%) and at 2 weeks in 3/14 infants (21%), with median concentrations of 7 and 4 micrograms/l serum per g BLG given per kg body weight, respectively. No ALA could be detected in any of these samples. In absorption of ALA, the four infants who had allergic symptoms did not differ from those without symptoms. Thus, systemic absorption of ALA and BLG does occur in infants. Absorption of ALA is greatest after birth, when 3 x 10(-4) (median) of the given antigens are absorbed, but absorption decreases rapidly. The gut may often be transiently permeable to BLG when cow's-milk-based formula is started.

The absorption of alpha-lactalbumin (ALA) and bovine beta-lactoglobulin (BLG) was investigated in 23 healthy preterm infants with gestational ages of 32 to 36 wk. The concentrations of ALA and BLG in serum after a milk feeding were measured at intervals during the first 8 mo of life. We used a time-resolved fluoroimmunoassay to measure the proteins. Measurable amounts of ALA were found on d 7 after birth, and at 1, 2, 3, 5, and 8 mo in 23 of 23, 13 of 18, 13 of 18, six of 17, eight of 16, and five of 13, respectively, of the infants tested; median serum levels of ALA at the respective ages were 120 (range, 19-2598), 16 (range, 0-177), 5 (range, 0-40), 0 (range 0-3), 0.8 (range 0-38), and 0 (range, 0-22) micrograms/L serum/g ALA given/kg body wt, respectively. The rate of decline in ALA absorption was comparable among the infants. Tests for BLG were begun after the introduction of cow's milk. At 2, 3, 5, and 8 mo of age BLG was detected in two of 7, two of 9, eight of 10, and two of 12, respectively, of the infants tested, where median levels in positive cases were 13, 17, 15, and 3 micrograms/L serum/g BLG given/kg, respectively. The amounts of absorbed ALA and BLG were 10(-5) to 10(-3) of the oral dose. Serum levels of ALA or BLG did not depend on the gestational age of the infant. Few of the infants had any detectable absorption of either protein shortly after weaning.(ABSTRACT TRUNCATED AT 250 WORDS)

The absorption of beta-lactoglobulin (BLG) by cultured explants of the normal adult duodenal mucosa has been studied in vitro using immunocytochemical techniques. Membrane filters (1 mm2) were soaked in medium containing BLG and placed on the mucosal surface. Explants were cultured for < or = 60 min, and histological sections were immunostained for BLG. Increasing the BLG concentration in the filters and/or the exposure time increased the number and immunoreactivity of the immunopositive epithelial cells present, suggesting increased BLG absorption. Increased BLG penetration of the duodenal mucosa in untreated coeliac disease was also shown using these techniques. In some studies horseradish peroxidase (HRP) was used as an alternative probe and pin-pointed in epithelial cells by ultracytochemical techniques, confirming vesicular uptake of macromolecules. Colocalisation experiments using BLG and HRP and immunofluorocytochemical techniques suggested linkage between the mechanisms of absorption of both proteins into epithelial cells. Short-term organ culture and protein challenge followed by immunolocalisation has been shown to be of value in studying the absorption of BLG and HRP by the upper small intestine and possibly of other protein macromolecules as well.

We studied absorption of the potentially allergenic protein beta-lactoglobulin during acute rotavirus diarrhea in infants and assessed the relationship of this macromolecular absorption with intestinal sugar permeability. After oral rehydration, 38 patients with acute gastroenteritis were given orally a 100-ml solution containing 4 g (11.7 mmol/L) of lactulose and 0.8 g (4.4 mmol/L) of mannitol, and their recovery rate as shown in urine passed during the subsequent 5 h was measured. A blood sample was taken 2 h after a milk feed for ELISA measurement of beta-lactoglobulin in circulating immune complexes. Twelve nondiarrhea patients were studied after an overnight fast as controls. Immune complexes containing beta-lactoglobulin were found in the serum of all, but the levels [median (range)] were significantly higher in patients with rotavirus diarrhea [686 (36-4352)] than in nondiarrhea patients [165 (0-2594)]; p = 0.007. The mean (95% confidence interval) lactulose/mannitol urinary recovery ratios were increased in patients with acute diarrhea [0.19 (0.10, 0.30)] compared to nondiarrhea patients [0.01 (0.005, 0.02)]; p = 0.0001. Thus, a significant correlation between beta-lactoglobulin absorption and sugar permeability was found; Spearman's rank correlation coefficient = 0.42, p = 0.004. This correlation was not, however, direct but was due to an inverse relationship between urinary recovery of mannitol and serum beta-lactoglobulin immune complexes. These results indicate that rotavirus gastroenteritis is associated with enhanced beta-lactoglobulin absorption and elevated lactulose/mannitol permeability test results, but these represent different phenomena.

The nature of the adsorbed protein on a non-biological biomaterial surface is known to influence cell adhesion. This work deals with the contribution of adherent platelets towards the redistribution of preadsorbed proteins. A redistribution was only observed with spread platelets on adsorbed fibrinogen and fibronectin, but not with adsorbed beta-lactoglobulin. The labelled protein images showed a dark outer ring with an inner region having fluorescent areas, with greater intensity than regions not covered by platelets. The fluorescent patterns observed may be a result of the binding of adsorbed fluorescent protein to the glycoprotein IIb/IIIa receptors of platelets, followed by their redistribution within the plasma membrane.

We evaluated the clinical response to oligoallergenic dietary treatment and the intestinal absorption of a protein antigen, cow milk beta-lactoglobulin (BLG) in 24 patients with chronic urticaria/angioedema syndrome 13 of whom also suffered from joint symptoms. Sixteen patients (77% of those with arthralgia) responded to diet (RD) with marked reduction of symptoms; the others did not respond (NR). Ten (all but one RD with arthralgia) had increased permeability to BLG after oral administration of cow milk. Four with high titers of IgG to BLG showed the highest absorption of BLG and the groups with arthralgia showed higher BLG levels than those without arthralgia. In all cases, specific IgE to cow milk was absent. These data suggest that the symptoms of a subgroup of patients with chronic urticaria, and especially patients with joint complaints that subside with diet, are related to excess intestinal permeability. The measurement of gut permeability to food proteins may be useful to define those who may benefit from dietary restriction.

Intestinal absorption of antigenic (intact) and degraded beta-lactoglobulin and horseradish peroxidase was studied in rabbits experimentally infected at weaning with the rabbit-specific Escherichia coli strain RDEC-1 (015:NM). Transepithelial fluxes of both proteins were measured at four stages after infection: early, peak, late, and recovery, on segments of ileum and Peyer's patches mounted in Ussing chambers. During this period of observation, absorption of antigenic beta-lactoglobulin and intact horseradish peroxidase decreased significantly in the ileum and Peyer's patches of age-matched controls. No significant age-related decrease was observed in the controls for the transport of these proteins in degraded form. RDEC-1 infection caused, first, a rise in degraded protein fluxes across Peyer's patches only, during the early phase of the disease. Second, during the peak phase of infection, absorption of antigenic beta-lactoglobulin by the ileum rose to 74.0 ng/hr/cm2 (90% confidence interval: 63.1, 86.8) vs 15.8 (5.8, 42.8) in the controls (P = 0.07), and absorption of intact horseradish peroxidase rose to 21.8 (16.5, 26.7) ng/hr/cm2 vs 4.4 (0.7, 26.6) in control ileum, (P = 0.09). These rises, which were similar in Peyer's patches, delayed the physiologic decrease in protein absorption that occurs with age. These results indicate that increased antigen absorption is observed during bacterial diarrhea and that it might interfere with the immune responses of the host.

Intestinal closure to cow's milk beta-lactoglobulin occurs within 6 days of birth in the guinea pig. Passive intestinal permeability to lactulose persists through the suckling period. The uptake of small water-soluble markers does not reflect macromolecular absorption, and has no place in the measurement of immunologic protein handling by the gut.

Results from experiments in this laboratory using 59Fe suggest that bovine lactoferrin (Lf) has no effect on iron absorption in rats. A study was therefore carried out in newborn infants to measure the effects of Lf on iron retention. Bovine Lf was labeled with the stable isotope 58Fe and fed to 7-day-old infants in a standard milk formula. Iron retention was estimated by measuring the unabsorbed 58Fe excreted in the feces during the following 3 days using neutron activation analysis. The results were compared with those obtained from a group of infants fed a similar level of iron as ferric chloride, labeled with 58Fe, together with 30 mg ascorbic acid. There was a very wide variation in percent iron retention amongst the infants but no overall difference between the Lf and ferric chloride groups. This confirms the previous findings in rats that Lf does not influence the availability of nonheme iron.