Systemic IL-2/anti-IL-2Ab complex combined with sublingual immunotherapy suppresses experimental food allergy in mice through induction of mucosal regulatory T cells.

BACKGROUND: Therapeutic tolerance restoration has been proven to modify food allergy in patients and animal models and although sublingual immunotherapy (SLIT) has showed promise, combined therapy may be necessary to achieve a strong and long-term tolerance. AIMS: In this work, we combined SLIT with systemic administration of IL-2 associated with an anti-IL-2 monoclonal antibody (IL-2/anti-IL-2Ab complex or IL-2C) to reverse the IgE-mediated experimental allergy. MATERIALS AND METHODS: Balb/c mice were sensitized with cholera toxin and milk proteins and orally challenged with allergen to elicit hypersensitivity reactions. Then, allergic mice were treated with a sublingual administration of very low amounts of milk proteins combined with intraperitoneal injection of low doses of IL-2C. The animals were next re-exposed to allergens and mucosal as well as systemic immunological parameters were assessed in vivo and in vitro. RESULTS: The treatment reduced serum specific IgE, IL-5 secretion by spleen cells and increased IL-10 and TGF-ß in the lamina propria of buccal and duodenal mucosa. We found an augmented frequency of IL-10-secreting CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in the submaxilar lymph nodes and buccal lamina propria. Tregs were sorted, characterized and adoptively transferred to naïve mice, which were subsequently sensitized. No allergy was experienced in these mice and we encouragingly discovered a faster and more efficient tolerance induction with the combined therapy compared with SLIT. CONCLUSION: The combination of two therapeutic strategies rendered Treg-mediated tolerance more efficient compared to individual treatments and reversed the established IgE-mediated food allergy. This approach highlights the ability of IL-2C to expand Tregs, and it may represent a promising disease-modifying therapy for managing food allergy.

Immunological status of isolated lymphoid follicles after intestinal transplantation.

Intestinal transplantation (ITx) faces the challenge of grafting a high immunogenic organ, which is certainly one of the major obstacles for intestinal allograft acceptance. The allograft has to guarantee the proper functioning of the mucosal immune machinery under immunosuppressive conditions. Recently, it has been elucidated that isolated lymphoid follicles (ILFs) are an indispensable part of mucosal immunity to maintain IgA synthesis and consequently to control commensal microflora. No data about these follicular structures in the setting of ITx are available so far. Therefore, we addressed the question whether constitution, integrity and function of allograft ILFs are disturbed by immunosuppressive regimen. We compared allograft ILFs from terminal ileum of transplant patients with ILFs from nontransplant patients via flow cytometry, quantitative real-time polymerase chain reaction and immunohistochemistry. We found that host leukocytes rapidly repopulate allograft ILFs and that maintenance immunosuppressive regimen, tacrolimus and corticosteroids, does not affect their cellular integrity and function. However, allograft ILFs revealed a higher maturation state than control samples and IgA positive plasma cells were increased in number in allograft mucosa. Our results open the path for a better understanding of allograft mucosal immunity.

Hypersensitivity reactions to the Sabin vaccine in children with cow's milk allergy.

BACKGROUND: The Sabin vaccine is used world-wide, and most children with food allergies receive it without incident. However, in the 2009 vaccination campaign conducted in Argentina, four children experienced immediate-type hypersensitivity reactions following vaccination. OBJECTIVE: We aimed to review the medical history of the affected children, study their allergic condition after the episodes and analyse the presence of allergenic vaccine components. METHODS: Patients were selected based on their immediate allergic reactions following vaccination. They were assessed for allergies to cow's milk and hen's egg. The presence of cow's milk proteins in the vaccine was tested by various immunoassays involving cow's milk- or α-lactalbumin-specific polyclonal rabbit antiserum and patient sera. RESULTS: All of the patients had a history of milk allergy, and no history or current evidence of egg hypersensitivity was found. Levels of cow's milk- and Sabin vaccine-specific IgE were increased, and the result of a skin prick test with cow's milk proteins or the Sabin vaccine was positive in each patient. In addition, an ELISA using specific rabbit antiserum detected α-lactalbumin in the Sabin vaccine. When α-lactalbumin was employed as a soluble inhibitor in a competitive ELISA, binding to vaccine-coated plates by cow's milk- or α-lactalbumin-specific rabbit antiserum or by patient serum containing IgE was inhibited. CONCLUSIONS: We have demonstrated that these patients were allergic to cow's milk, and had circulating and mast cell-bound IgE antibodies specific to cow's milk proteins. We found that the Sabin vaccine contained α-lactalbumin, which may have been responsible for the reactions elicited following vaccination with the Sabin and dual viral vaccines in combination.

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine.

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1(-/-)) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1(-/-) mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.

Analysis of immune cells draining from the abdominal cavity as a novel tool to study intestinal transplant immunobiology.

During intestinal transplant (ITx) operation, intestinal lymphatics are not reconstituted. Consequently, trafficking immune cells drain freely into the abdominal cavity. Our aim was to evaluate whether leucocytes migrating from a transplanted intestine could be recovered from the abdominal draining fluid collected by a peritoneal drainage system in the early post-ITx period, and to determine potential applications of the assessment of draining cellular populations. The cell composition of the abdominal draining fluid was analysed during the first 11 post-ITx days. Using flow cytometry, immune cells from blood and draining fluid samples obtained the same day showed an almost complete lymphopenia in peripheral blood, whereas CD3(+) CD4(+) CD8(-) , CD3(+) CD4(-) CD8(+) and human leucocyte antigen D-related (HLA-DR)(+) CD19(+) lymphocytes were the main populations in the draining fluid. Non-complicated recipients evolved from a mixed leucocyte pattern including granulocytes, monocytes and lymphocytes to an exclusively lymphocytic pattern along the first post-ITx week. At days 1-2 post-Itx, analysis by short tandem repeats fingerprinting of CD3(+) CD8(+) sorted T cells from draining fluid indicated that 50% of cells were from graft origin, whereas by day 11 post-ITx this proportion decreased to fewer than 1%. Our results show for the first time that the abdominal drainage fluid contains mainly immune cells trafficking from the implanted intestine, providing the opportunity to sample lymphocytes draining from the grafted organ along the post-ITx period. Therefore, this analysis may provide information useful for understanding ITx immunobiology and eventually could also be of interest for clinical management.

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1ß and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1ß and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

Evaluation of calprotectin level in intestinal content as an early marker for graft rejection.

INTRODUCTION: The diagnosis of rejection after intestinal transplantation is still performed by endoscopic biopsy monitoring. Less invasive diagnostic procedures are desirable, although they are not available so far. Calprotectin, a stable cytosolic granulocyte protein, which previously was used as a marker of inflammatory processes, has been proposed to be a biochemical marker for rejection. The aim of the present work was to analyze the concordance between calprotectin levels in intestinal content and the presence of graft rejection after small bowel transplantation. METHODS: Calprotectin level was measured using a commercial ELISA kit on 137 samples of intestinal content randomly collected during endoscopies performed on 11 intestinal transplantation patients during 2 years' follow-up. Calprotectin determinations were correlated with histological and clinical findings. The cut-off level was determined retrospectively by receiver-operator curve analysis. RESULTS: Based on histological findings and clinical records, samples were discerned as rejection positive (37 of 137), versus negative (35 of 137) samples or those with no clinical, endoscopic, or histological findings (65 of 137 samples). A cut-off value of 65 microg of calprotectin/g of intestinal content provided the best assay parameter according to the clinical findings: a 76% sensitivity and a 47% specificity. False positive results corresponded to patients with gastrointestinal infections (13%), systemic infections (13%), ulcers (10%), or nonspecific histological alterations of the mucosa (15%). The other false positive cases corresponded to postsurgical samples (4%), or patients with concomitant gastrointestinal symptoms (2%). Most false negative results (78%) were observed during recovery from severe acute rejection episodes, among successfully treated patients. In these cases, epithelial reconstitution and no mucosal infiltration was observed. If the latter group were discarded, sensitivity rose to 93%, and specificity, to 50% with a 96% negative predictive value. Furthermore, a weak correlation was observed between calprotectin levels and the severity of rejection. CONCLUSIONS: This study confirmed the results obtained by other groups: fecal calprotectin dosage showed a good sensitivity but low specificity for the diagnosis of intestinal rejection because high calprotectin levels can also be observed in other clinical conditions. Nevertheless, it might be used as a first line for continuous evaluation of intestinal transplantation status, like other biochemical parameters that are used in kidney or liver transplantation, before considering the need for a biopsy.

Chronic inflammation as a manifestation of defects in immunoregulatory networks: implications for novel therapies based on microbial products.

Based on a unifying theory presented here, it is predicted that the immune defects resulting in chronic inflammation rather than effective immune responses could be rectified by the therapeutic use of agents prepared from micro-organisms. With appropriate molecular patterns, these should be able to induce protective immunoregulatory networks or to reprogramme defective ones. In contrast to acute inflammation, chronic inflammation appears to have no beneficial role, but is a state of sustained immune reactivity in the presence or progression of a disease process. This results in an escalating cycle of tissue damage followed by unproductive tissue repair, breaks in self-tolerance, malignant transformation or deleterious changes in tissue morphology and function. Such inappropriate immune reactivity is an underlying characteristic, either in initiation or maintenance, of a diverse range of disease states including chronic infection, autoimmunity, allergy, cancer, vascular disease and metabolic alterations. Evidence is presented that the inappropriate immune reactivity is due, at least to some extent, to failures in the establishment of immunoregulatory networks as a result of hygiene-related factors. Such networks are the result of activation of antigen-presenting cells, principally dendritic cells, by molecular patterns of micro-organisms encountered sequentially during life and establishing the 'biography' of the immune system.

Duodenal intraepithelial lymphocytes of children with cow milk allergy preferentially bind the glycan-binding protein galectin-3.

A breakdown in intestinal homeostasis results in inflammatory bowel diseases including coeliac disease and allergy. Galectins, evolutionarily conserved beta-galactoside-binding proteins, can modulate immune-epithelial cell interactions by influencing immune cell fate and cytokine secretion. In this study we investigated the glycosylation signature, as well as the regulated expression of galectin-1 and -3 in human duodenal samples of allergic and non-allergic children. Whereas galectin-1 was predominantly localized in the epithelial compartment (epithelial cells and intraepithelial lymphocytes) and the underlying lamina propria (T cells, macrophages and plasma cells), galectin-3 was mainly expressed by crypt epithelial cells and macrophages in the lamina propria. Remarkably, expression of these galectins was not significantly altered in allergic versus non-allergic patients. Investigation of the glycophenotype of the duodenal inflammatory microenvironment revealed substantial alpha2-6-linked sialic acid bound to galactose in lamina propria plasma cells, macrophages and intraepithelial lymphocytes and significant levels of asialo core 1 O-glycans in CD68+ macrophages and enterocytes. Galectin-1 preferentially bound to neutrophils, plasma cells and enterocytes, while galectin-3 binding sites were mainly distributed on macrophages and intraepithelial lymphocytes. Notably, galectin-3, but not galectin-1 binding, was substantially increased in intraepithelial gut lymphocytes of allergic patients compared to non-allergic subjects, suggesting a potential role of galectin-3-glycan interactions in shaping epithelial-immune cell connections during allergic inflammatory processes.

Immunochemical characterization of Glycine max L. Merr. var Raiden, as a possible hypoallergenic substitute for cow's milk-allergic patients.

BACKGROUND: Cows' milk allergy (CMA) is the most common cause of food allergy in infancy. The only proven treatment is the complete elimination of cows' milk proteins (CMPs) from the diet by means of hypoallergenic formulas. Soybean-based formulae are widely used although intolerance to soy has been reported to occur in 15-40% of infants with CMA. OBJECTIVE: The aim of this work was to analyse the in vitro reactivity of the soybean cultivar Raiden, which naturally lacks glycinin A(4)A(5)B(3), to evaluate whether this genotype could be a safe CMP substitute for CMA patients. METHODS: The reactivity of conventional soybean (CS) and Raiden soybean (RS) genotypes and also recombinant glycinin A(4)A(5)B(3) and alphabeta-conglycinin with casein-specific monoclonal antibodies and CMP-specific polyclonal serum was evaluated by immunoblotting and ELISA. A sequential competitive ELISA with the polyclonal antiserum and different soluble inhibitors was performed. In addition, an indirect ELISA with sera of atopic children with CMA was carried out to analyse the IgE-binding capacity of the different soybean components. RESULTS: We have shown that CS contains four components that cross-react with CMP, while RS has only one. The remaining cross-reactive component in RS was identified as alpha-subunit beta-conglycinin. By means of inhibitory ELISA, we demonstrated that CS, RS and the alpha-subunit beta-conglycinin extracts inhibited the binding of CMP-specific antibodies to the CMP-coated solid phase. Finally, we showed that CS, RS and the recombinant proteins were recognized by human CMP-specific IgE antibodies. CONCLUSION: This work shows that although Raiden has fewer cross-reactive components than conventional soybean, it still has a residual cross-reactive component: the alpha-subunit beta-conglycinin. This reactivity might make this genotype unsuitable to treat CMA and also explains adverse reactions to soybean in CMA infants.

Serological prevalence of anti-latex IgE antibodies in unselected blood donors in Argentina.

The prevalence of specific IgE to natural rubber latex proteins in the general population remains uncertain. The purpose of this study was to determine the prevalence of sera containing specific IgE antibodies to latex proteins using immunoenzymatic methods. A population of 500 unselected adult voluntary blood donors was the source of the sera used in this study. Two different immunoenzymatic methods (EAST and CARLA) were used to analyze the presence of specific IgE antibodies. Confirmation assay was carried out by inhibition ELISA and immunoblotting. Sera from healthy nonatopic individuals were also used as control. Two hundred and twenty five sera showed higher than normal total IgE levels. Of those, three presented latex specific IgE antibodies, which could be inhibited in a dose-response manner with the natural rubber latex and glove extracts. Several latex allergens were recognized by the IgE antibodies from these positive sera. This low seroprevalence (0.66%) indicates that latex hypersensitivity is not an important problem in the general population. We believe that prevention of latex exposure is only necessary in high risk groups of patients.

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk.

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods.

Evaluation of the residual antigenicity and allergenicity of cow's milk substitutes by in vitro tests.

BACKGROUND: This study aimed the to investigate presence of residual allergenic cow's milk proteins (CMP) in some milk substitutes employed in the treatment of cow's milk allergy (CMA). These allergens may interfere with the treatment, and elicit allergic reactions in sensitized individuals. METHODS: The protein composition of the different extracts was evaluated by Lowry's method and tricine SDS-PAGE. Different immunoenzymatic methods were used (ELISA, EAST and immunoblotting) to quantify total serum IgE and specific serum IgE, as well as to detect the presence of antigenic and allergenic components. RESULTS: The results showed a higher protein content in mammalian milks (cow, sheep, mare, goat, and human) than in hydrolyzed substitutes (partially or extensively hydrolyzed casein or whey proteins). Residual native, processed, or contaminant polypeptides have been identified in the moderate hydrolysates, whereas extensive hydrolysates did not show the presence of residual components by immunoblotting. However, specific antibodies with capacity to bind to peptides have been detected by EAST and ELISA, suggesting that extensive hydrolysates contain residual peptides that preserve immunoreactive epitopes. We were unable to demonstrate either residual antigenicity or allergenicity in an amino-acid-based formula. CONCLUSIONS: Immunoenzymatic methods were used to detect the presence of cross-reactive components in mammalian milks. Residual allergenic components from cow's milk could be identified in both the moderate and extensive hydrolysates analyzed. This information may be relevant to the treatment of CMA.

Identification of allergenic proteins in condoms by immunoenzymatic methods.

BACKGROUND: A large increase of allergy to latex proteins has been observed lately probably as a result of a great use of latex-containing goods. At present these untoward reactions have led to consideration of this problem as a health and occupational hazard. It is therefore, necessary to identify the allergens contained in latex-manufactured products and to develop effective diagnostic tools to detect sensitized individuals. OBJECTIVE: The objective of this study is to identify antigenic and allergenic components in latex condoms by using chemical, immunochemical, and immunoenzymatic methods. METHODS: The protein content of extracts obtained from several brands of condoms was determined and characterized by using a modified Lowry method, a quantitative ELISA assay and SDS-PAGE. The allergenic behavior of these proteins was studied by IgE immunoblotting, EAST and ELISA techniques, using sera from subjects allergic to latex products, particularly to latex condoms. RESULTS: Wide variations in the protein content (38 to 740 microg/g product) and composition were observed. The SDS-PAGE protein profiles showed components ranging from 7 to 94 kD of relative molecular weights; most of them were also detected in natural rubber latex. The most prominent bands were revealed in the 14 and 30 kD zones. A strong band of 69 kD in the SDS-PAGE profiles would correspond to a neoantigen, since it was not observed in natural latex. The immunoblotting analysis employing sera from 5 patients allergic to latex condoms showed the presence of 4 components with IgE binding capacity (14, 30, 69, and 94 kD). The EAST and ELISA methods showed the presence of allergens in all the condom brands studied. CONCLUSIONS: The presence of allergenic proteins in several condom brands was demonstrated by different immunoenzymatic methods.

Detection of allergens in Aedes albifasciatus mosquito (Diptera: Culicidae) extracts by immunological methods.

Aedes albifasciatus (Diptera: Culicidae) is the most common mosquito species in Argentina and it has been demonstrated to be the vector for some pathogens. The objective of this study was to describe the allergen composition of this mosquito species endemic to Argentina using SDS-PAGE and immunoblotting methods. Sera from mosquito-bite allergic subjects were employed. The protein extracts, obtained from thoraxes containing salivary glands, showed a protein pattern with components of apparent molecular weights ranging from 14 to over 94 kDa. Some of the components could bind IgE in the 16, 20, 30, 36 and 50-67 kDa-zones, whereas the 14 kDa fraction detectable by SDS-PAGE did not behave as an allergen with any positive serum. This protein extract was used to develop in vitro assays to detect the presence of serum-specific IgE against proteins from A. albifasciatus (RAST and ELISA). Thirty-five sera from patients showing local reactions after mosquito bites were tested. The 21 positive sera were from subjects with clinical histories of atopic signs. Through immunoblotting, these sera revealed IgE reactivity against several fractions, mainly in the 16, 20, 30, 36 and 50-67 kDa zones. Comparing the serum IgE reactivity pattern against A. albifasciatus and Aedes aegypti, we observed that the main difference was found in the 14 kDa region where a strong reactivity was seen. The immunoblotting inhibition results indicate that there might be species-unique and species-shared antigens between A. albifasciatus and A. aegypti.

Serological investigation of latex allergy in Argentina.

Atopy to latex has been reported in different high-risk groups of subjects in whom it is mainly caused by proteins from natural latex that are responsible for eliciting a variety of clinical symptoms, some of them systemic. Thus, identification of subjects sensitized to latex proteins is of great health importance, because it would become possible to advise them to avoid contact with latex-containing products. Protein extracts from ammoniated latex were prepared by incubation with PBS buffer either with or without detergents, followed by ultracentrifugation. Three immunoenzymic methods were developed (EAST, ELISA, and immunoblotting) to detect the presence of specific IgE antibodies against latex proteins in sera of patients from different groups at risk. The protein content of the latex extracts ranged from 5.3 to 8.8 mg/mL. The prevalence of specific IgE against latex proteins was 9/28 (32.1%) in children with multiple surgeries, 17/98 (17.3%) in health care workers, and 23/123 (18.6%) in outpatients assisted at the Allergy Department. None of the sera belonging to the healthy control group showed the presence of specific IgE. Therefore, this protein extract from latex could be used to detect specific IgE antibodies in serum by immunoenzymic methods. Serologic results for latex-specific IgE found are in accordance with those reported in the literature of other countries.

Prevalencia de hipersensibilidad al látec en pacientes con mielomeningocele / Prevalence of latex hypersensibility in patients with myelomeningocele

Se estudió la presencia de IgE específica para proteínas del látex en el suero de 27 pacientes con mielomeningocele. La IgE se determinó por RAST y ELISA y su reactividad se analizó por inmunoblottig frente a un extracto proteico de látex natural amoniacado. Se detectaron 8 sueros con IgE específica para látex por todos los métodos ensayados. Todos ellos mostraron IgE elevada y revelaron una proteína de 14 kD. Estos resultados indican una incidencia del 29 por ciento de hipersensibilidad en este grupo, la que es comparable con la informada internacionalmente

Prevalencia de hipersensibilidad al látec en pacientes con mielomeningocele / Prevalence of latex hypersensibility in patients with myelomeningocele

Se estudió la presencia de IgE específica para proteínas del látex en el suero de 27 pacientes con mielomeningocele. La IgE se determinó por RAST y ELISA y su reactividad se analizó por inmunoblottig frente a un extracto proteico de látex natural amoniacado. Se detectaron 8 sueros con IgE específica para látex por todos los métodos ensayados. Todos ellos mostraron IgE elevada y revelaron una proteína de 14 kD. Estos resultados indican una incidencia del 29 por ciento de hipersensibilidad en este grupo, la que es comparable con la informada internacionalmente

Identification of casein as the major allergenic and antigenic protein of cow's milk.

The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., alpha- and beta-casein, beta-lactoglobulin, and alpha-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to beta-lactoglobulin, and 5/80 showed reactivity to alpha-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with beta-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.