Major Cow's Milk Proteins Research Articles

Immunoglobulin G4 in eosinophilic esophagitis: Immune complex formation and correlation with disease activity.

Recent studies have shown deposition of immunoglobulin G4 (IgG4) and food proteins in the esophageal mucosa of eosinophilic esophagitis (EoE) patients. Our aims were to assess whether co-localization of IgG4 and major cow's milk proteins (CMPs) was associated with EoE disease activity and to investigate the proteins enriched in proximity to IgG4 deposits. This study included adult subjects with EoE (n = 13) and non-EoE controls (n = 5). Esophageal biopsies were immunofluorescence stained for IgG4 and CMPs. Co-localization in paired samples from active disease and remission was assessed and compared to controls. The proteome surrounding IgG4 deposits was evaluated by the novel technique, AutoSTOMP. IgG4-food protein interactions were confirmed with co-immunoprecipitation and mass spectrometry. IgG4-CMP co-localization was higher in the active EoE group compared to paired remission samples (Bos d 4, p = .02; Bos d 5, p = .002; Bos d 8, p = .002). Co-localization was also significantly higher in the active EoE group compared to non-EoE controls (Bos d 4, p = .0013; Bos d 5, p = .0007; Bos d 8, p = .0013). AutoSTOMP identified eosinophil-derived proteins (PRG 2 and 3, EPX, RNASE3) and calpain-14 in IgG4-enriched areas. Co-immunoprecipitation and mass spectrometry confirmed IgG4 binding to multiple food allergens. These findings further contribute to the understanding of the interaction of IgG4 with food antigens as it relates to EoE disease activity. These data strongly suggest the immune complex formation of IgG4 and major cow's milk proteins. These immune complexes may have a potential role in the pathophysiology of EoE by contributing to eosinophil activation and disease progression.

Natural tolerance development in cow's milk allergic children: IgE and IgG4 epitope binding.

Although most of cow's milk (CM) allergic children will outgrow their allergy, the pathomechanism of the natural development of tolerance remains poorly understood. It has been suggested that the balance between milk-specific IgE and IgG4 plays a major role. We aimed to investigate differences in IgE and IgG4 antibody binding to CM epitopes between patients with persistent CM allergy (CMA) and those that naturally became tolerant. Sera from 35 children with proven CMA (median age at inclusion of 10months) were analyzed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51months) during the study period as confirmed by a negative oral food challenge. IgE and IgG4 binding to sequential epitopes derived from five major CM proteins were measured with a peptide microarray-based immunoassay. At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5years of age compared to patients with transient CMA. Moreover, children with transient CMA had IgE and IgG4 antibodies that more often recognized the same epitopes, compared to those with persistent CMA. From baseline to the time of tolerance development, both IgE and IgG4 binding intensity decreased significantly, particularly in areas of α-s- and β-casein (P<.01, false discovery rate [FDR]<.1). Interestingly, differences between IgE and IgG4 binding intensity to CM peptides decreased when the patients became tolerant. Our results suggest that the overlap between IgE and IgG4 might be important in natural tolerance acquisition. Further studies are needed to confirm our data and can eventually lead to development of more targeted treatment of food allergy.

Composition of major proteins in cow milk differing in mean protein concentration during the first 155 days of lactation and the influence of season as well as short-term restricted feeding in early and mid-lactation

A variety of proteins contributes greatly to the unique nutritional and functional quality of dairy cow milk. Particularly, milk casein content and composition have substantial influence on the processing capabilities. In the present study, milk of 23 multiparous Holstein-Friesian cows, grouped as high- (3.49 &amp;plusmn; 0.05%; n&amp;nbsp;=&amp;nbsp;11) and low-protein (3.03 &amp;plusmn; 0.05%; n = 12) cows, was sampled approximately weekly during the first 155&amp;nbsp;days of lactation to determine the course of relative milk protein composition (&amp;alpha;-lactalbumin; &amp;beta;-lactoglobulin; &amp;alpha;-, &amp;beta;-, and &amp;kappa;-casein). Furthermore, feed restrictions by 30% of dry matter intake in early and mid-lactation as well as experimental tissue biopsies were conducted to observe their effect on milk protein composition. Milk protein composition was relatively stable and displayed similar concentration patterns throughout the experimental period between both groups. Mean relative concentrations of &amp;alpha;-, &amp;beta;-, &amp;kappa;-casein, &amp;alpha;-lactalbumin, and &amp;beta;-lactoglobulin were 34.2, 31.4, 16.0, 2.1, and 9.7% of total protein, respectively. Feed restrictions did not alter milk protein composition, whereas the season influenced &amp;alpha;- and &amp;beta;-casein as well as &amp;alpha;-lactalbumin. Further, effects were observed in both groups at times of unfamiliar stressful situations caused by taking liver or muscle biopsies. As a result, the relative concentration of &amp;beta;-casein increased. Therefore, acute stress factors may lead to a deviation in milk protein composition and should be avoided. &amp;nbsp;

Early recovery from cow's milk allergy is associated with decreasing IgE and increasing IgG4 binding to cow's milk epitopes

The dynamics and balance of allergen-specific IgE, IgG4, and IgA binding might contribute to the development of tolerance in patients with cow's milk allergy (CMA). Profiling of antibody binding to cow's milk (CM) protein epitopes might help in predicting the natural history of allergy. We sought to investigate differences in IgE, IgG4, and IgA binding to CM epitopes over time between patients with early recovery or with persisting CMA. We studied serum samples at the time of diagnosis (mean age, 7 months), 1 year later, and at follow-up (mean age, 8.6 years) from 11 patients with persisting IgE-mediated CMA at age 8 to 9 years and 12 patients who recovered by age 3 years. We measured the binding of IgE, IgG4, and IgA antibodies to sequential epitopes derived from 5 major CM proteins with a peptide microarray-based immunoassay. We analyzed the data with a novel image-processing method together with machine learning prediction. IgE epitope-binding patterns were stable over time in patients with persisting CMA, whereas binding decreased in patients who recovered early. Binding patterns of IgE and IgG4 overlapped. Among patients who recovered early, the signal of IgG4 binding increased and that of IgE decreased over time. IgE and IgG4 binding to a panel of alpha(s1)-, alpha(s2)-, beta-, and kappa-casein regions predicted outcome with significant accuracy. Attaining tolerance to CM is associated with decreased epitope binding by IgE and a concurrent increase in corresponding epitope binding by IgG4.

B-cell epitopes as a screening instrument for persistent cow's milk allergy.

Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE-binding epitopes of all 6 major cow's milk proteins (alpha(s1)-, alpha(s2)-, beta-, and kappa-casein; alpha-lactalbumin; and beta-lactoglobulin) and had some evidence suggesting that IgE antibodies from patients with persistent cow's milk allergy (CMA) recognize different epitopes on cow's milk proteins than do those from patients who were likely to outgrow their allergy. In this study we sought to assess whether recognition of IgE antibodies of certain epitopes of cow's milk proteins would clearly separate the patients with life-long CMA from those who will become clinically tolerant to cow's milk. According to the known IgE-binding regions of cow's milk proteins, 25 decapeptides of alpha(s1)-casein, alpha(s2)-casein, kappa-casein, alpha-lactalbumin, and beta-lactoglobulin, comprising the core epitopes, were synthesized on a cellulose-derivatized membrane. Sera from 10 patients with persistent CMA and 10 patients who subsequently outgrew their milk allergy were used to investigate the differences in epitope recognition. Five IgE-binding epitopes (2 on alpha(s1)-casein, 1 on alpha(s2)-casein, and 2 on kappa-casein) were not recognized by any of the patients with transient CMA but showed binding by the majority of the patients with persistent allergy. The presence of IgE antibodies against at least 1 of 3 epitopes (amino acid [AA] 123-132 on alpha(s1)-casein, AA 171-180 on alpha(s2)-casein, and AA 155-164 on kappa-casein) identified all patients with persistent CMA. The presence of IgE antibodies to distinct allergenic epitopes of cow's milk proteins can be used as a marker of persistent CMA. Prospective studies are needed to investigate the usefulness of these informative epitopes in predicting life-long CMA in young children.

Cellular and Humoral Immunity against Cow's Milk Proteins in Type 1 Diabetes

Cow's milk β-casein has been proposed as a candidate trigger of autoimmunity associated with type 1 diabetes. In this study, cellular and humoral immunity against β-casein was compared to that against other major cow's milk proteins in patients with recent onset type 1 diabetes and control subjects. T cell responses were found against α-casein, β-casein, β-lactoglobulin and bovine serum albumin in both patients with type 1 diabetes (stimulation index: 0.2–22.8, n=23) and control subjects (stimulation index: 0.1–18.2, n=22), with no significant differences between groups. Twelve (52%) patients and nine (41%) control subjects had stimulation indices >3 to at least one protein, including 9 (39%) patients and 4 (18%) control subjects against β-casein, all but one of these also having elevated responses to α-casein. The highest responses (stimulation index >9) were against α- and β-casein in some patients and control subjects who had the HLA DR3 allele. Antibody levels against α-casein, β-casein and β-lactoglobulin were low in both patients (n=59) and control subjects (n=52). Nevertheless, significantly higher IgG binding to both α-casein in ELISA (P=0.02) and β-casein using ELISA (P=0.02) and RIA (P=0.04) was observed in patients aged <15 years compared to control subjects of similar age. No relationship was found between cellular and humoral immunity against individual antigens. These data show that immune responses to cow's milk are not limited to patients with diabetes and not solely against β-casein.

Characterization of antigens and allergens in hypo-allergenic infant formulae

The antigenicity and allergenicity of so-called hypo-allergenic infant formulae is mainly determined by the degree of hydrolysis and ultrafiltration. Five different formulae were investigated by means of immunoblotting and RAST in order to characterize the antigens and allergens regarding their molecular weights, molecular origin and their ability to bind human IgG and IgE antibodies: A non hydrolysed infant formula (I-F), a mixture of the major cow's milk proteins (PM), a whey-based infant formula (W-H), a whey-based and ultra-filtrated infant formula (U-H), a casein/whey-based infant formula (CW-H). By immunoblotting we demonstrated that all tested formulae still contain antigens with molecular weights from 3 to 67 kD. But when compared with I-F and PM the antigen content of the hydrolysed formulae was considerably lower. The lowest antigen content could be demonstrated in U-H, which contains casein fragments (3-6 kD) and beta-lactoglobulin and its fragments (6-18 kD). W-H and CW-H contain bovine serum albumin, beta-lactoglobulin, casein and their fragments (3-67 kD). All hydrolysed formulae tested showed a reduced IgE-binding capacity. Three out of 12 cow's milk allergic children possessed IgE binding to U-H or W-H, and 5 of them IgE against CW-H. Conclusion. The enzymatic hydrolysis plus ultra-filtration seems to be the most efficient method to reduce the antigen content of so-called hypo-allergenic infant formulae.

217 EVALUATION OF AN IGE-ELISA SPECIFIC FOR BOVINE α-CASEINS

ELISA microtiter plates ( Greiner ) were coated with highly purified bovine α-caseins and incubated with diluted sera. Specific IgE was detected by successive incubation with three antibodies, the last one conjugated with horseradish peroxidase. Twelve patients ( age 1/2 - 3 years ) with positive provocation and skin prick test to bovine caseins were compared with 12 children ( age 1/2 -12 years ) with negative tests. The results were compared to CAP-RAST ( Code f78, Pharmacia ) data. Additionally IgE-immunoblots were made as a qualitative criterion and to distinguish between immunreactions to the different caseins and the other major cow milk proteins.67% of the cow milk allergic patients had positive RAST ( class > 1 ) and 92% were positive ( OD > 0.1 ) in the ELISA. In the immunoblot 83% showed distinct reactions to α-caseins. None of the control group gave positive reactions. Only one child's serum showed a positive reaction to α-caseins in the immunoblot.In conclusion, compared with the clinical allergy of the children studied our ELISA proved to have a higher sensitivity than the RAST; specitivity of both tests equalled 100%.

Food-processing approaches to altering allergenic potential of milk-based formula

All the major cow milk proteins in their native states are potential allergens in infants with milk allergy. Heat treatment can reduce the antigenicity of whey proteins considerably, but it has virtually no effect on the antigenicity of casein. Infants allergic to milk still react to heat-denatured whey proteins. Therefore heat denaturation alone cannot produce a formula with low allergenicity. Enzyme hydrolysis reduces the antigenicity and allergenicity of protein. Partial hydrolysis produces hydrolysate consisting mainly of large peptides, whereas extensive hydrolysis produces hydrolysate containing a mixture of large and small peptides and free amino acids. Enzyme hydrolysis often produces bitter peptides and destroys the physical functionality of protein. When hydrolysate formula is made, casein or whey protein hydrolysate is ultrafiltered to remove large residual peptides. Certain amino acids are fortified to provide a balanced amino acid profile. The final formulation must comply with the recommendations of the Codex Alimentarius Commission (FAO/WHO) and the U.S. Infant Formula Act to provide adequate infant nutrition. Protein hydrolysate-based formula can be improved by further reducing the residual allergenicity, increasing the small peptide content, and improving the taste.