Characterization of the Immunogenicity and Allergenicity of Two Cow's Milk Hydrolysates--A Study in Brown Norway Rats.

Hypoallergenic infant formulas based on hydrolysed milk proteins are used in the diet for cow's milk allergic infants. For a preclinical evaluation of the immunogenicity and allergenicity of new protein ingredients for such hypoallergenic infant formulas as well as for the investigation of which characteristics of hydrolysates that contribute to allergenicity, in vivo models are valuable tools. In this study, we examine the immunogenicity and allergenicity of two hydrolysates in a Brown Norway (BN) rat model, using i.p. dosing, which allows for the use of small quantities. Intact BLG, hydrolysed BLG and a hydrolysed whey product suitable for use in extensively hydrolysed formulas were thoroughly characterized for protein chemical features and administered to BN rats by i.p. immunization with or without adjuvant. Sera were analysed for specific IgG and IgE for evaluation of sensitizing capacity, immunogenicity and antibody-binding capacity. For evaluation of eliciting capacity a skin test was performed. The study showed that the hydrolysates had no residual allergenicity, lacking the capacity to sensitize and elicit reactions in the BN rats. Dosing with or without adjuvant induced a large difference in immunogenicity. Only antibodies from rats sensitized to intact BLG with adjuvant were able to bind the hydrolysates, and the whey-based hydrolysate only showed immunogenicity when dosed with adjuvant. This study showed that hydrolysates can be evaluated by an i.p. animal model, but that the choice of in vitro tests used for evaluation of antibody responses may greatly influence the result as well as may the use of adjuvant.

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

BACKGROUND: Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion. OBJECTIVE: Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated. METHODS: Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells. RESULTS: The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes<2.0 kDa, of which approximately 50% was in aggregated complexes of Mr up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells. CONCLUSION: From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Allergen specific responses in cord and adult blood are differentially modulated in the presence of endotoxins.

BACKGROUND: Endotoxins are common contaminants in allergen preparations and affect antigen-specific cellular responses. Distinct effects of endotoxin on cells in human umbilical cord and adult blood are poorly defined. OBJECTIVES: To examine the effect of endotoxins in allergen preparations on cellular responses in human cord and peripheral blood (PB). METHODS: The endotoxin content in beta lactoglobulin (BLG), the peanut allergen Ara h 1 and the major birch pollen allergen Bet v 1 was assessed. Proliferation and cytokine response of mononuclear cells towards contaminated and lipopolysaccharide (LPS)-free allergens were evaluated at different time-points. Fractions of contaminated BLG were generated and assayed on their immuno-stimulatory capacity. The involvement of toll-like receptor (TLR) 2 and 4 was investigated by blocking antibodies and TLR-transfected human embryonic kidney cells. RESULTS: The proliferative response of cord blood (CB)-derived mononuclear cells towards allergen-preparations at day 3 was related to the level of LPS contamination. At day 7, proliferation was also detected in the absence of endotoxin. Cytokine production in CB was strongly affected by the content of endotoxin, TLR-4 dependent and not related to the allergen content. Allergen- and endotoxin-induced proliferative responses were generally significantly higher in CB than in adult blood. CONCLUSION: Endotoxins in allergen preparations confound allergen-specific cellular responses. The impact of these contaminations varies with the blood source (CB vs. PB), the type of allergen and is time- and dose-dependent.

Immunogenicity of kappa-casein and glycomacropeptide.

Glycomacropeptide (GMP), arising from the cleavage of kappa-casein by chymosin or pepsin, has been correlated with a wide variety of biological activities including immunosuppression capacity, inhibition of pathogen invasion, and induction of satiety. Due to the interest in exploiting such potential of GMP, we aimed at characterizing the immunogenic properties of GMP as an indication of its potential allergenicity. Immunogenicity of kappa-casein and GMP were investigated using 2 animal models based on different routes of immunization: 1) mice immunized intraperitoneally or subcutaneously with either kappa-casein, polymerized GMP, GMP coupled to the immunogenic carrier ovalbumin, or GMP alone; 2) mice coadministered kappa-casein or GMP and cholera toxin. The specific antibody response to GMP was evaluated as well as the antigen-specific T-cell response. The results demonstrated that immunization or feeding with kappa-casein induced GMP-specific antibodies, whereas GMP per se lacked immunogenicity independently of the mode of presentation. The size of the presented form of GMP did not influence its immunogenicity. Because the results showed that GMP did not induce a specific T-cell response, we postulate that GMP lacks the ability to stimulate antigen-specific T cells.

High intakes of milk, but not meat, increase s-insulin and insulin resistance in 8-year-old boys.

OBJECTIVE: Our objective was to examine if a high animal protein intake from milk or meat increased s-insulin and insulin resistance in healthy, prepubertal children. A high animal protein intake results in higher serum branched chain amino acids (BCAA; leucine, isoleucine and valine) concentrations, which are suggested to stimulate insulin secretion. Furthermore, milk possesses some postprandial insulinotrophic effect that is not related to its carbohydrate content. DESIGN: A total of 24 8-y-old boys were asked to take 53 g protein as milk or meat daily. At baseline and after 7 days, diet was registered, and insulin, glucose, and amino acids were determined. Insulin resistance and beta cell function were calculated with the homeostasis model assessment. RESULTS: Protein intake increased by 61 and 54% in the milk- and meat-group, respectively. In the milk-group, fasting s-insulin concentrations doubled, which caused the insulin resistance to increase similarly. In the meat-group, there was no increase in insulin and insulin resistance. As the BCAAs increased similarly in both groups, stimulation of insulin secretion through BCAAs is not supported. CONCLUSIONS: Our results indicate that a short-term high milk, but not meat, intake increased insulin secretion and resistance. The long-term consequences of this are unknown. The effect of high protein intakes from different sources on glucose-insulin metabolism needs further studying.

Caseinomacropeptide self-association is dependent on whether the peptide is free or restricted in kappa-casein.

There is a general agreement that the experimentally determined molecular weight (MW) of caseinomacropeptide (CMP) is greater than the theoretical MW. Some studies suggest that this is due to a pH-dependent aggregation of monomeric CMP. How this aggregation is influenced by pH is not understood. This study was carried out to study the nature of CMP aggregates and to clarify which conditions affect aggregation of CMP. The apparent MW of CMP at different pH values was determined using size-exclusion chromatography. Caseinomacropeptide was further characterized by immunochemical analysis, sodium dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of kappa-casein (CN), and 2 commercial CMP products. Both commercial products and CMP resulting from chymosin-hydrolysis of kappa-CN (at pH 6.6) had elution volumes with a MW corresponding to 35 kDA at pH 8.0 and 3.4. Caseinomacropeptide prepared from pepsin-hydrolysis of kappa-CN (at pH 2.5) eluted as multiple peaks with apparent MW of 35, 18, and 9 kDa, again independently of pH. Hydrolysis of kappa-CN with chymosin or pepsin at different pH values (pH 2.5, 3.4, and 6.6) produced differently sized aggregates of CMP, largely depending on the pH of the hydrolysis. These results indicate that, whereas CMP molecules are irreversibly associated, CMP in kappa-CN may associate reversibly in a pH-dependent manner. We suggest that interactions between para-kappa-CN parts of the kappa-CN molecules may be a requisite for the pH-dependent dissociation/association.

Localization and functional significance of a polymorphic determinant in the third component of human complement.

A polymorphic epitope in the third component of human complement was studied. This allotypic system is distinct from the electrophoretically determined C3 S/F polymorphism and is defined by the recognition of one allotype by a monoclonal antibody. Allotypic protein variants, C3F+ (reactive with this antibody) and C3S- (non-reactive with the antibody), were purified. Deglycosylation studies and N-terminal sequencing of CNBr fragments, reactive with the antibody, revealed that the polymorphic epitope was present in a beta chain fragment of mol. wt 20,000. In the intact C3 molecule, this fragment is situated with N-terminus at residue No. 202, using the numbering of the cDNA derived amino acid sequence of human prepro C3. Addition of Fab fragments from the alloselective antibody preferentially inhibited the activity of C3F+ in a haemolytic assay which is selective for the C3 activity in the alternative complement pathway.

Human complement component C2: production and characterization of polyclonal and monoclonal antibodies against C2.

Rabbit antibodies to human complement component C2 were produced by immunization of rabbits with precipitates from line immunoelectrophoresis, and the antibodies were used to monitor a classical chromatographic purification of C2 and for affinity purification of C2. Twelve monoclonal antibodies with specificity for human complement component C2 were produced by fusion of myeloma cells with spleen cells from mice immunized with the affinity purified C2. The specificity of the monoclonal antibodies was confirmed by their reaction with antigen-antibody precipitates where C2 was the antigen, and by their specific reaction with C2 after separation in SDS-PAGE followed by immunoblotting. The affinity of the monoclonal antibodies varied as demonstrated by the titration curves in ELISA. The antibodies will be of importance for immunospecific purification of human C2 and C2 fragments, for specific depletion of C2 from human serum, and for quantification of C2 for clinical purposes.

A simple alternative pathway for hemolytic assay of human complement component C3 using methylamine-treated plasma.

A quantitative, alternative pathway (AP) hemolytic assay for human complement component C3 has been developed. This AP-C3 assay is inexpensive, rapid, simple, reproducible and insensitive to C3 degradation products. The AP-C3 assay uses rabbit erythrocytes as complement activator and methylamine-treated plasma, depleted of C3 and C4, as complement source. Rabbit erythrocyte sensitivity varies little from batch to batch, and remains unaltered for at least one month in Alsever's solution. Methylamine plasma may be stored at -20 degrees C for 3 months. AP-C3 lysis of 5-25% of erythrocytes is complete in 20 min and does not change subsequently. The AP-C3 assay is optimally stable at 2 mM Mg2+, 5 mM EGTA and at 5 X 10(7) erythrocytes/ml, yet insensitive to at least 20% deviation in these concentrations. The AP-C3 assay is specific to functional C3 and well suited for determination in plasma samples and during C3 preparation. Adding excessive amounts of C3c or plasma components other than C3 does not change the hemolytic response. The level of native C3 in plasma from 14 donors relative to a reference plasma pool ranged from 0.78 to 1.23. The standard deviation of relative C3 determinations did not exceed 2%.

Conjugation to preadsorbed preactivated proteins and efficient generation of anti peptide antibodies.

A solid phase conjugation method is described based on the preadsorption of proteins to aluminium hydroxide adjuvant followed by activation of the adsorbed carrier proteins with iodoacetic acid N-hydroxysuccinimidester or other conjugation reagents. Cysteine-containing peptides were coupled to the iodoacetic acid-activated carrier-adjuvant particles through their SH groups. No dialysis is required since the reaction product is isolated at each step of the procedure by a simple centrifugation and can easily be extensively washed between individual manipulations. The method generates peptide-carrier-adjuvant particles with sterically defined presentation of the peptides at the surface of the particles. When used for immunization of mice and rabbits the conjugates elicited high-titered specific anti-peptide sera, which reacted well with the parent protein in ELISA. The strongest reactions were with the denatured form of the parent protein. On immunoblots antisera to the N- and C-terminus of calreticulin recognized the same M, 52,000 protein.

Serum N-Terminal Propeptide of Collagen Type I is Associated with the Number of Bone Metastases in Breast and Prostate Cancer and Correlates to Other Bone Related Markers.

BACKGROUND: A number of biomarkers have been proven potentially useful for their ability to indicate bone metastases (BM) in cancer patients. The aim of this study was to investigate the relative utility of a newly developed N-terminal propeptide of collagen type I (PINP) human serum assay for the detection of BM in cancer patients. This assay has a corresponding rat PINP assay which in the future might help in translational science between rodent and human trials. METHODS: Participants were 161 prostate, lung and breast cancer patients stratified by number of BM (Soloway score). PINP was assessed and correlated to number of BM. Additionally, the PINP marker was correlated to bone resorption of young (ALPHA CTX-I)- and aged bone (BETA CTX-I); number of osteoclasts (Tartrate-resistant acid phosphatase 5b, TRACP5B) and osteoclast activity (CTX-I/ TRACP5B). RESULTS: PINP was significantly elevated in breast- and prostate cancer patients +BM, compared to -BM (P < 0.001), however not in lung cancer patients. A strong linear association was seen between PINP and the number of BMs. Significant elevation of PINP was observed at Soloway scores 1-4 (<0 BM) compared with score 0 (0 BM) (P < 0.001). The correlation between bone resorption of young bone or aged bone and bone formation was highly significant in patients +BM and -BM (P < 0.0001). CONCLUSIONS: Data suggest that the present PINP potentially could determine skeletal involvement in patients with breast or prostate cancer. Correlations suggested that coupling between bone resorption and bone formation was maintained in breast- and prostate cancer patients.

Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP)--assessment of corresponding epitopes.

OBJECTIVES: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. METHODS: Monoclonal antibodies were raised against corresponding rat and human PINP sequences and competitive assays were developed for each species. They were evaluated in relevant pre-clinical or clinical studies. RESULTS: The antibody characterizations indicated that PINP indeed was recognized. Technical robust assays were obtained. Rat PINP and tALP showed similar patterns in the gold standard osteoporosis rat ovariectomized (OVX) model. No liver contribution was observed in the liver fibrosis rat bile duct ligation model (BDL). In an osteoporosis study, the human serum PINP levels were significantly decreased after ibandronate treatment compared to placebo. CONCLUSIONS: The two corresponding PINP assays were specific and these bone turnover markers may improve translational science for the evaluation for bone-related diseases.

Amino acid sequence of endothiapepsin. Complete primary structure of the aspartic protease from Endothia parasitica.

The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined. The enzyme consists of 330 residues. The sequence determination was performed exclusively at the protein level. The homology of this fungal milk-clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25-30% identity. The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology.

Detection of potentially allergenic material in 12 hydrolyzed milk formulas.

Hypoallergenic milk formulas are used as an alternative diet for infants who have allergies to cow's milk when breast-feeding is not possible. These products are based on proteins, which have been heat-treated and hydrolyzed to a different degree in order to cleave antibody-binding structures. Even extensively hydrolyzed products have occasionally been observed to elicit allergic reactions in sensitized infants, however. Therefore, the parameters of relevance to allergenic potential require more investigation. The objective of the present study was to investigate 12 different hydrolyzed milk formulas for their contents of potentially allergenic protein material, i.e. material that may induce allergenicity or elicit allergic responses in already sensitized individuals. Analytical methods applied were gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE, immunoblotting, dot-immunobinding, and ELISA. Care was taken to assure that all protein fractions were investigated, including supernatants and precipitates following centrifugation of the milk formulas. By gel filtration, protein material with apparent molecular masses of 7 to >30 kDa was detected. Analysis by SDS-PAGE of formula precipitates showed that proteins with a molecular mass above 20 kDa were present even in some of the extensively hydrolyzed formulas. Residual antigenic beta-lactoglobulin was found by ELISA in all products. By immunoblotting and dot-immunobinding with antibodies against total whey, caseins, or Kunitz soybean trypsin inhibitor, we observed antigenic material mainly in partially hydrolyzed products. We concluded that SDS-PAGE of formula supernatants and precipitates gave the most differentiated profile of hydrolyzed formulas and that this method is well suited for screening potential allergenicity.

Identification of immunogenic maize proteins in a casein hydrolysate formula.

Cow's milk-based formulas used for infants with cow's milk allergy are based on hydrolyzed proteins. The formulas that are successful in preventing allergic responses are extensively hydrolyzed. Nevertheless, reactions to such formulas are occasionally reported, and protein material of higher molecular weight than expected has been detected by binding immunoglobulin E (IgE) from patients' sera. This paper presents the identification of high-molecular-weight material in the extensively hydrolyzed casein formula, Nutramigen. The material was concentrated by simple centrifugation. The proteins in the pellet were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein-containing bands were analyzed by protein sequencing after electroblotting. The proteins were identified as maize zeins, which are water-insoluble proteins of apparent M(r) 20,000 and 23,000, presumably originating from the maize starch in Nutramigen. Rabbits immunized with this formula developed antibodies against zeins but not against milk proteins. The maize zeins are probably identical to the recently reported components in Nutramigen (1), detected by binding of IgE from milk allergic patients, but not correlated to clinical allergic reactivity. The clinical relevance of maize proteins in Nutramigen remains to be established.

Combined immunostaining and Coomassie Brilliant Blue staining of polyvinylidene difluoride membranes without organic solvent.

A method for staining proteins on polyvinylidene difluoride membranes without using organic solvent is described. The method uses preblocking of the membrane with either Tween 20 or polyethylene glycol followed by staining with 0.01% Coomassie Brilliant Blue. No destaining of the membrane is needed afterwards. Preblocking with polyethylene glycol is compatible with microsequencing while Tween 20 leads to very low initial yields. Preblocking with Tween 20 has the additional advantage of allowing immunostaining followed by Coomassie Brilliant Blue staining for total protein on the same membrane.

The solubilities of denatured proteins in different organic solvents.

The solubilities of heat-denatured and reduced, S-carboxymethylated proteins have been investigated in various organic solvents. Polar, protic solvents (formic acid, trifluoroacetic acid, 3-mercaptopropionic acid) were found to be good solvents for the denatured proteins (20-40 mg ml-1), and the solubilities of the reduced, S-carboxymethylated proteins were generally higher than those of the heat-denatured forms. Most other organic solvents were less effective in solubilizing the denatured proteins. Apolar solvents did not solubilise denatured proteins, but low solubilizing powers were observed for polar, aprotic solvents. Heat-denaturation was observed to result in the formation of large intermolecular aggregates, which, for ovalbumin and lysozyme, were formed by intermolecular S-S bonds, but for bovine serum albumin involved intermolecular isopeptide bonds.