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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: Sera from 35 children with proven CMA (median age at inclusion of 10 months) were analyzed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51 months) 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. RESULTS: At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5 years 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 ß-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. CONCLUSIONS: 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.

Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family.

BACKGROUND: IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. OBJECTIVE: We attempted to identify pistachio allergens and to clone the encoding genes. METHODS: Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. RESULTS: Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. CONCLUSION: Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.

[Activity of the enzymes of ethanol and acetaldehyde metabolism in rats with varying initial sensitivity to alcohol]. / Aktivnost' fermentov metabolizma étanola i atsetal'degida u krys s razlichnoi iznachal'noi chuvstvitel'nost'iu k alkogoliu.

The experiments were carried out on male rats with different sensitivity to alcohol. To assess sensitivity to ethanol effects, we have used ethanol-induced sleep time and variations in rectal temperature of alcohol-intoxicated animals. Activity of alcohol dehydrogenase, microsomal ethanol-oxidizing system, catalase and aldehyde dehydrogenase in the liver as well as ethanol and acetaldehyde levels in the blood were determined after alcohol intoxication (3.5 g/kg, i.p., 8 days). The development of alcohol tolerance was accompanied by induction of the microsomal ethanol-oxidizing system in long-sleeping rats and in short-sleeping rats as well as by an increase in ethanol and acetaldehyde levels in the blood.

[Metabolic adaptation to alcohol in rats with different preference of ethanol over water]. / Metabolicheskaia adaptatsiia k alkogoliu u krys, razlichaiushchikhsia po predpochteniiu étanola vode.

Recent studies have shown that the phenomenon of ethanol preference by animals and of alcohol consumption by humans may be related to the intensity of its metabolism in the body and depend on the activities of the ethanol and aldehyde metabolizing systems which are potential regulators of the acetaldehyde level in the cell. The special features of adaptative reactions of this system (alcohol dehydrogenase, microsomal ethanol oxidizing system, catalase, aldehyde dehydrogenase) were examined in rats, differing by the preference to water or ethanol (5%, 10%, 15%) under condition of a long contact with alcohol.

[Effects of catalase activators and inhibitors on ethanol pharmacokinetic characteristics and ethanol and aldehyde-metabolizing enzyme activities in the rat liver and brain].

The effects of catalase regulators (aminotriazole, lead acetate, taurine, di-2-ethylhexylphthalate) on the preference for ethanol, its pharmacokinetics, and activities of rat liver and brain ethanol and acetaldehyde-metabolizing enzymes were studied. Lead acetate (100 mg/kg, i.p., 7 days), aminotriazole (1 g/kg, i.p., 7 days), and taurine (650 mg/kg, i.g., 14 days) decreased ethanol consumption under conditions of free choice (10% ethanol water), whereas di-2-ethylhexylphthalate (300 mg/kg, i.g., 7 days) did not exert any effect on this parameter. Taurine, lead acetate and di-2-ethylhexylphthalate significantly activated liver ADH, MEOS and catalase peroxidase activity. Aminotriazole also activated ADH and MEOS, but inhibited liver catalase. The activities of liver and brain A1DH as well as catalase were insignificantly changed by this treatment. The 7-day administration of lead acetate, di-2-ethylhexylphthalate and aminotriazole administrations significantly influenced the ethanol (2 g/kg., i.p.) pharmacokinetic parameters: the area under the pharmacokinetic curve and the elimination half-life time were significantly reduced, whereas the elimination constant and clearance were increased. This unequivocally indicates accelerated ethanol elimination. The 14-day ingestion of taurine insignificantly changed the parameters of ethanol pharmacokinetics in rats.

Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy.

BACKGROUND: Approximately two-thirds of egg-allergic infants become tolerant within the first 5 years of life. OBJECTIVE: We sought (1) to compare the recognition of sequential (linear) and conformational binding sites of ovomucoid, ovalbumin and ovotransferrin, by IgE antibodies of children with persistent and transient egg allergy, (2) to identify immunodominant IgE-and IgG-binding epitopes of ovomucoid, and (3) to compare epitope-specificity of IgE antibodies between patients with differing natural histories of egg allergy. METHODS: Using immunodot-blots or ImmunoCAPs, IgE-antibodies against conformational (native) and sequential (reduced and alkylated) egg proteins were determined at the time of clinical reactivity in patients who retained their allergy and in those who developed clinical tolerance. IgE- and IgG-binding epitopes were mapped for ovomucoid using overlapping decapeptides on SPOTs membranes. Recognition of the major IgE-binding epitopes were compared between patients with differing natural histories of egg allergy. RESULTS: The patients with long-lasting egg allergy had a higher concentrations of IgE antibodies against sequential and native ovomucoid and ovalbumin than the children who subsequently gained tolerance (P < 0.01). Four major IgE-binding epitopes were identified in ovomucoid at amino acid 1-10, 9-20, 47-56, and 113-124. IgE antibodies of all seven patients with persistent egg allergy recognized these epitopes whereas none of the 11 children who outgrew their egg allergy did so. CONCLUSIONS: Patients with persistent egg allergy develop IgE antibodies against more sequential and conformational epitopes of ovomucoid and ovalbumin. The presence of serum IgE antibodies to specific sequential epitopes of ovomucoid may be used as a screening instrument for persistent egg allergy.

Humoral and cellular responses to cow milk proteins in patients with milk-induced IgE-mediated and non-IgE-mediated disorders.

BACKGROUND: Cow milk allergy (CMA) is one of the most common food allergies in childhood. Patients with CMA present with a wide range of immunoglobulin (Ig)E- and non-IgE-mediated clinical syndromes. Limited information is known about the specific humoral and cellular responses to cow milk proteins in these various forms of CMA. OBJECTIVE: The aim of the study was to determine IgE, IgA, IgG1 and IgG4 antibody levels and lymphocyte proliferative responses to the major cow milk allergens in patients with IgE- and non-IgE-mediated CMA. METHODS: One hundred and forty cow milk allergic patients, 6 months to 22 years of age, were included in the study. One hundred and thirteen patients had IgE-mediated CMA, 11 had milk protein-induced enterocolitis syndrome and 16 had allergic eosinophilic gastroenteritis. Twenty-one patients without food allergy, 8 months to 18 years of age, served as controls. Serum IgE, IgA, IgG1 and IgG4 antibodies to alpha-, beta-, and kappa-casein, alpha-lactalbumin and beta-lactoglobulin were measured using enzyme-linked immunosorbent assays. For a subset of these patients, we performed lymphocyte proliferation assays to the various milk allergens. RESULTS: Patients with IgE-mediated CMA had higher specific IgE concentrations to casein compared with whey proteins (P < 0.001). In this group of patients, there was a positive correlation between IgE levels and levels of the other isotypes for all four milk proteins (P < 0.001). In general, the caseins were the more allergenic and antigenic proteins in all groups of patients. Patients with enterocolitis syndrome produced less milk protein-specific IgG4 (P < 0.05) and had a trend for higher IgA antibody levels when compared to the control group. Lymphocyte proliferative responses in all groups with CMA were significantly higher than controls (P < 0.05), although this response was similar in patients with IgE- and non-IgE-mediated CMA. CONCLUSION: There is a distinct pattern of humoral antibody response in the different forms of CMA. Patients with IgE-mediated CMA have an elevated polyisotypic response to cow milk protein. The relative lack of specific IgG4 production in patients with enterocolitis syndrome may be involved in the pathogenesis of the disease. In general, caseins appear to be the predominant allergen in patients with CMA.

[Adrenal glucocorticoid function in rats with various ethanol-induced sleeping time following ethanol administration].

The effect of single and chronic ethanol (Eth) administration (25 % solution, 3.5 g/kg) on functional activity of the hypophyseal-adrenal system in rats with different sensitivity to the hypnotic action of ethanol (short-sleep - SS; non-sleep--NS, long-sleep--LS, intermediate group--IG), was studied. It has been shown that, after a single Eth administration, the concentration of corticosterone (K) in LS rat plasma was 1.5-fold higher than that in the NS animals although it did not differ from the K level in SS and Ig those. After repeated ethanol load, the corticosterone contents in the NS rat blood plasma was 3.5-fold and 4.9-fold lower compared to the control and LS groups, respectively. The data obtained indicate that the SS and LS animals had initially different basal blood plasma glucocorticoid level. The SS animals showed a decreased blood plasma K, whereas the LS ones--an increased one. The features of the glucocorticoid status are suggested to be a factor determining the sensitivity of rats to the ethanol hypnotic effect.

Identification of IgE- and IgG-binding epitopes on alpha(s1)-casein: differences in patients with persistent and transient cow's milk allergy.

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children less than 2 years of age, but about 80% become clinically tolerant within the first 3 years of life. Casein is one of the major allergens responsible for CMA and seems to play an important role in persistent allergy. Previous studies on egg allergy suggested that linear epitopes are associated with long-lasting food allergy. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on alpha(s1)-casein and to determine whether the patterns of epitope recognition are associated with the natural history of CMA. METHODS: According to the known amino acid (AA) sequence, 96 overlapping decapeptides representing the entire length of alpha(s1)-casein were synthesized on a cellulose-derived membrane. Sera from 24 children with milk allergy were used to identify IgE- and IgG-binding epitopes. RESULTS: Six major and 3 minor IgE-binding, as well as 5 major and 1 minor IgG-binding, regions on alpha(s1)-casein were identified. Two IgE-binding regions (AA 69-78 and AA 173-194) were recognized by the majority of patients over 9 years of age with persistent allergy (67% and 100%, respectively) but by none of the children less than 3 years of age who are likely to outgrow CMA. No differences in IgG binding between the groups were observed. CONCLUSION: There appears to be a difference in epitope recognition between patients with different natural histories of CMA. Screening for IgE antibodies to these epitopes may be useful in identifying children who will have persistent milk hypersensitivity.

Isolation of a virus from chimpanzee liver cell cultures inoculated with sera containing the agent of non-A, non-B hepatitis.

A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources. Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated. Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.

Identification of IgE and IgG binding epitopes on beta- and kappa-casein in cow's milk allergic patients.

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children aged less than 2 years of age. Although beta- and kappa-casein are considered among the major allergens responsible for CMA, no data are available on their allergenic epitopes in humans. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on beta- and kappa-casein and to determine whether the pattern of epitope recognition is associated with the natural history of CMA. METHODS: Overlapping decapeptides representing the entire length of beta- and kappa-casein, respectively, were synthesized on a cellulose-derivatized membrane. Sera from 15 milk-allergic children, 4-18 years of age, with high levels of specific IgE antibodies to cow's milk were used to identify IgE- and IgG-binding epitopes. In addition, IgE epitopes were screened with pooled or individual sera from younger patients aged less than 3 years and who had low levels of specific serum IgE, who are likely to outgrow CMA. RESULTS: Six major and three minor IgE-binding epitopes, as well as eight major and one minor IgG binding regions, were identified on beta-casein. Eight major IgE-binding epitopes, as well as two major and two minor IgG-binding epitopes, were detected on kappa-casein. Three of the IgE binding regions on beta-casein and six on kappa-casein were recognized by the majority of patients in the older age group, but not by the younger patients. CONCLUSION: Information regarding the immunodominant epitopes in beta- and kappa-casein may be important for understanding the pathophysiology and natural history of CMA. Differences in epitope recognition may be useful in identifying children who will have persistent milk hypersensitivity.

IgE and IgG binding epitopes on alpha-lactalbumin and beta-lactoglobulin in cow's milk allergy.

BACKGROUND: Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE and IgG binding epitopes for alpha(s1)-casein, a major cow's milk allergen, and found an association between recognition of certain epitopes and clinical symptoms of cow's milk allergy (CMA). Since alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) are suspected to be significant allergens in cow's milk, we sought to determine the structure of sequential epitopes recognized by IgE antibodies to these proteins. We further sought to assess the pattern of epitope recognition in association with the clinical outcome of CMA. METHODS: According to the known amino acid sequence of ALA and BLG, 57 and 77 overlapping decapeptides (offset by two amino acids), respectively, were synthesized on a cellulose derivatized membrane. Sera from 11 patients 4-18 years of age with persistent CMA (IgE to cow's milk >100 kU(A)/l) were used to identify IgE binding epitopes. In addition, 8 patients < 3 years of age and likely to outgrow their milk allergy (IgE to cow's milk < 30 kU(A)/l) were used to investigate the differences in epitope recognition between patients with 'persistent' and those with 'transient' CMA. Seven patients 4-18 years of age were used for assessing the IgG binding regions. RESULTS: In patients with persistent allergy, four IgE binding and three IgG binding regions were identified on ALA, and seven IgE and six IgG binding epitopes were detected on BLG. The younger patients that are likely to outgrow their allergy recognized only three of these IgE binding epitopes on BLG and none on ALA. CONCLUSIONS: The presence of IgE antibodies to multiple linear allergenic epitopes may be a marker of persistent CMA. The usefulness of IgE binding to distinct epitopes on whey proteins in defining the patients that would have a lifelong CMA needs to be investigated in further studies.

[Effect of acetaldehyde on ethanol- and aldehyde-metabolising systems of the liver and brain of rats]. / Vliianie atsetal'degida na étanol- i al'degidmetaboliziruiushchie sistemy pecheni i mozga krys.

Lately the mechanism of craving for alcohol has been related to the local level of brain acetaldehyde occurring in ethanol consumption and depending on the activities of the brain and liver ethanol and acetaldehyde-metabolizing systems. In this connection, we studied the effect of chronic acetaldehyde intoxication on the activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), the microsomal ethanol oxidizing system (MEOS) and liver and brain catalase as well as ethanol and acetaldehyde levels in the blood. The results showed that the chronic acetaldehyde intoxication did not alter significantly the activities of liver ADH, MEOS and catalase as well as liver and brain ALDH. In parallel with this, the systemic acetaldehyde administration led to shortened time of ethanol narcosis and activation of catalase in the cerebellum and left hemisphere, which may indicate involvement of this enzyme into metabolic tolerance development.

Role of conformational and linear epitopes in the achievement of tolerance in cow's milk allergy.

BACKGROUND: Cow's milk (CM) is one of the leading causes of food allergy in children. However, approximately 85% of milk-allergic children become clinically tolerant to CM within the first 3 years of life. The mechanisms involved in the achievement of tolerance remain unknown. OBJECTIVE: To study whether IgE antibodies from children with persistent cow's milk allergy (CMA) differ from children who become clinically tolerant in their ability to recognize linear and conformational epitopes of alpha(s1)- and beta-casein. METHODS: Thirty-six milk-allergic children were included in the study: 11 of the children became clinically tolerant, and 25 had persistent CMA. Blood was obtained from all patients during the time they showed clinical reactions to milk challenge. Six non-milk-allergic children served as controls. Specific IgE antibodies against linear (denatured) as well as conformational (native) milk proteins were determined by probing dot-blots with patients' sera. In addition, selected decapeptides from alpha(s1)- and beta-casein, previously found to be suggestive of persistent CMA, were synthesized on a cellulose-derivatized membrane and probed with individual sera from 10 patients who outgrew CMA and from 10 patients with persistent CMA. RESULTS: Analysis of immunodot-blots showed that, in comparison to tolerant patients, milk-allergic children with persistent symptoms had a significantly higher ratio of specific IgE antibodies to linearized than to native alpha- and beta-casein (P < 0.005 and P < 0.02, respectively). Comparing the selected decapeptides, six of the 10 patients with persistent allergy recognized the peptide corresponding to amino acids 69-78 from alpha(s1)-casein while none of the patients who outgrew CMA had IgE binding to this epitope. CONCLUSION: Patients with persistent milk allergy possess higher detectable levels of IgE antibodies to linear epitopes from alpha(s1)- and beta-casein than children who have achieved tolerance. Specific IgE binding to particular linear epitopes in alpha(s1)-casein may be a predictive factor for persistence of CMA.

Effects of cooking methods on peanut allergenicity.

BACKGROUND: Allergy to peanut is a significant health problem. Interestingly, the prevalence of peanut allergy in China is much lower than that in the United States, despite a high rate of peanut consumption in China. In China, peanuts are commonly fried or boiled, whereas in the United States peanuts are typically dry roasted. OBJECTIVE: The aim of this study was to examine whether the method of preparing peanuts could be a factor in the disparity of allergy prevalence between the 2 countries. METHODS: Two varieties of peanuts grown in the United States were roasted, boiled, or fried. Proteins were analyzed by using SDS-PAGE and immunoblotting. Allergenicity was compared by using immunolabeling with sera from 8 patients with peanut allergy. RESULTS: The protein fractions of both varieties of peanuts were altered to a similar degree by frying or boiling. Compared with roasted peanuts, the relative amount of Ara h 1 was reduced in the fried and boiled preparations, resulting in a significant reduction of IgE-binding intensity. In addition, there was significantly less IgE binding to Ara h 2 and Ara h 3 in fried and boiled peanuts compared with that in roasted peanuts, even though the protein amounts were similar in all 3 preparations. CONCLUSION: The methods of frying or boiling peanuts, as practiced in China, appear to reduce the allergenicity of peanuts compared with the method of dry roasting practiced widely in the United States. Roasting uses higher temperatures that apparently increase the allergenic property of peanut proteins and may help explain the difference in prevalence of peanut allergy observed in the 2 countries.

A murine model of peanut anaphylaxis: T- and B-cell responses to a major peanut allergen mimic human responses.

BACKGROUND: Peanut allergy affects 0.6% of the US population. At the present time, allergen avoidance is the only therapeutic option. Animal models of food-induced anaphylaxis would facilitate attempts to design novel immunotherapeutic strategies for the treatment of peanut allergy. OBJECTIVE: The purpose of this study was to develop a murine model of IgE-mediated peanut hypersensitivity that closely mimics human peanut allergy. METHODS: C3H/HeJ mice sensitized orally with freshly ground whole peanut and cholera toxin as adjuvant were challenged orally 3 and 5 weeks later with crude peanut extract. Anaphylactic reactions were determined. T- and B-cell responses to Ara h 1 and Ara h 2, the major peanut allergens, were characterized by evaluating splenocyte proliferative responses and IgE antibody concentrations. Furthermore, IgE antibodies in the sera of patients with peanut allergy and mice were compared for antibody binding to Ara h 2 isoforms and allergenic epitopes. RESULTS: Peanut-specific IgE was induced by oral peanut sensitization, and hypersensitivity reactions were provoked by feeding peanut to sensitized mice. The symptoms were similar to those seen in human subjects. Ara h 1- and Ara h 2-specific antibodies were present in the sera of mice with peanut allergy. Furthermore, these Ara h 2-specific IgE antibodies bound the same Ara h 2 isoforms and major allergenic epitopes as antibodies in the sera of human subjects with peanut allergy. Splenocytes from mice with peanut allergy exhibited proliferative responses to Ara h 1 and Ara h 2. CONCLUSION: This murine model of peanut allergy mimics the clinical and immunologic characteristics of peanut allergy in human subjects and should be a useful tool for developing immunotherapeutic approaches for the treatment of peanut allergy.