IgE antibodies to hydrolysates of cow milk proteins in children with cow milk allergy

Milk substitutes such as protein hydrolysates are largely used in children with cow milk allergy. The clinical benefit of these preparations is still a matter of debate. In the present study, IgE directed against protein hydrolysates was detected in 6/13 in children with cow milk allergy.     

Ontogeny of antibody response to Brucella abortus in turkeys

No differences were detected between large White Nicholas and small White Hybrid turkeys in antibody (AB) titers in birds vaccinated with Brucella abortus. Maximal AB titers occurred by 6 weeks of age and remained high throughout life. B. abortus elicits an AB response when injected at day of hatch while bursectomy at day of hatch and 2 days of age significantly depressed this response. There was an apparent immunological maturation process occurring to B. abortus in the bursa of Fabricius which contained for approximately 6 weeks.     

Immunoglobulin A antibody response to respiratory syncytial virus structural proteins in colostrum and milk.

Immunoglobulin A (IgA) antibody response to respiratory syncytial virus (RSV) structural proteins in colostrum and milk was investigated by a radioimmunoprecipitation assay. By using [35S]methionine-labeled RSV-infected HEp-2 cells and antiserum to human IgA as the capture antibody, IgA antibody responses to large glycoprotein, fusion protein, nucleoprotein, phosphoprotein, and matrix protein were demonstrated in colostrum and milk. The IgA antibody response was mainly directed against fusion protein, whereas IgA activity against matrix protein was more variable and was not comparable to the antibody responses to other structural proteins. Maternal mammary IgA response after RSV infection in the infant was monitored in four cases, and the appearance of anti-RSV IgA activity against several RSV structural proteins was observed in convalescent-stage milk samples of two mothers in whom RSV infection was demonstrated.     

Contact urticaria to cow milk

A patient with contact urticaria to cow milk is presented. Contact sensitivity was documented to whole cow milk, skim milk, commercial condensed milk, and milk heated to 80 degrees C for 30 min. Contact sensitivity was also documented to casein and lactalbumin. Unlike a previous report our patient's sensitivity to heated milk precluded its use in the diet. Thus, heating milk does not necessarily reduce allergenicity to its component proteins.     

Ontogeny of mouse caudal proteins identified by a monoclonal antibody

A monoclonal antibody (mAb) D2G4 directed to human spermatozoa recognized antigens on the acrosomal region of both human and mouse spermatozoa and reacted with two proteins of molecular weights 45 kd and 26 kd. Immunohistochemical staining with this antibody indicated that only the epithelial cells of the cauda epididymis were stained and not the sections of testis, caput or corpus epididymis. These observations suggest that antigens recognized by D2G4 were acquired by the spermatozoa during their passage through the cauda epididymis and appear to have a role in the maturation of spermatozoa. The ontogeny of these antigens in mice was studied during sexual maturation. These antigens could be detected in cauda epididymis from day 50 onwards by immunohistochemistry. The highest concentration of these antigens was observed in the cauda epididymis of 80-day-old mice. SDS-PAGE analysis indicated that the 26 kd protein recognized by D2G4 was visible from day 50 onwards, confirming the immunohistochemical observations. The plasma testosterone levels showed a significant increase from days 40 to 60 followed by a decrease. The fact that these epididymal proteins appear during sexual maturation and at the time of the testosterone surge indicates that they are androgen dependent.     

The postvaccination antibody response to influenza virus proteins

The radio-immunoblot (RIB) assay was used to examine the antibody response to proteins of the vaccine strains induced after influenza vaccination. Vaccination stimulated an antibody response to the surface glycoproteins (HA and NA) and to the internal antigens (NP and M) of the three vaccine strains. Antibodies were detected to both the monomeric form of the haemagglutinin (HA) and its two subunits HA1 and HA2. In addition, antibody to the monomeric form of NA was detected. A wide range of response patterns was observed to the viral proteins. All three major antibody classes (IgG, IgA and IgM) were induced after vaccination and in the majority of volunteers the antibody reactivity increased one week after vaccination. IgM antibodies had a wider reactivity pattern, recognising proteins and subunits which were not fully processed or slightly degraded. The varied antibody response induced after influenza vaccination reflects the differing infection histories of the volunteers with influenza. We show some of the practical limitations of studying the antibody response to influenza vaccination.     

Human antibody response to human cytomegalovirus-specific DNA-binding proteins

Human antibody responses to human cytomegalovirus (HCMV) specific DNA-binding proteins were studied in serum samples by the Western blot technique. The molecular weights of six DNA-binding proteins found in HCMV-infected cells, ranged from 52kD to 18kD. The sera obtained from patients with acute HCMV infections reacted well with the six HCMV specific DNA-binding proteins. The strongest reactivity was observed with the 52kD and 35kD proteins. The sera from healthy HCMV seropositive donors reacted only with the 52kD DNA-binding protein as visualized in Western blots, but 2 out of 8 sera failed to react with any HCMV specific DNA-binding proteins.     

Kinetics of antibody response to Ehrlichia canis immunoreactive proteins

Immunoreactive proteins of Ehrlichia canis and Ehrlichia chaffeensis that have been characterized include a family of 28-kDa major outer membrane proteins (p28) and two large antigenically divergent surface glycoprotein orthologs. We previously demonstrated that recombinant E. canis p28 and the 140- and 200-kDa glycoproteins gp140 and gp200, respectively, react strongly with serum antibodies from suspect canine ehrlichiosis cases that were positive for E. canis by immunofluorescent antibody test and in various phases of acute or chronic infection (J. Clin. Microbiol. 39:315-322, 2001). The kinetics of the antibody response to these potentially important vaccine and immunodiagnostic candidates is not known. Acute-phase serum antibody responses to whole-cell E. canis lysates and recombinant p28, gp140, and gp200 were monitored for 6 weeks in dogs experimentally infected with E. canis. Irrespective of the inoculation route, a T-helper 1-type response was elicited to E. canis antigens consisting of immunoglobulin G2 antibodies exclusively in both acute and convalescent phases in most dogs. Analysis of immuoreactive antigens for peak intensity and relative quantity identified major immunoreactive E. canis antigens recognized early in the infection as the 19-, 37-, 75-, and 140-kDa proteins. Later in infection, additional major immunoreactive E. canis proteins were identified, including the 28-, 47-, and 95-kDa proteins and the recently identified 200-kDa glycoprotein. All dogs had developed antibody against the recombinant gp140, gp200, and p28 in the convalescent phase. Immunoreactivity and antibody response kinetics suggest that major immunoreactive proteins identified are immunodominant, but early recognition suggests increased dominance by some antigens.     

Cow's milk allergy and eczema: patterns of the antibody response to cow's milk in allergic skin disease

The pattern of the humoral-immune response to cow's milk was examined in children with eczema and in children who had acute urticarial and/or angioedematous reactions to cow's milk. Each patient group was compared to a group of age-matched controls. Whereas the eczematous patients had significantly elevated IgG, IgA and IgE milk-antibodies, patients, allergic to cow's milk, had elevated IgE milk-antibodies. These differences in the pattern of antibody response to cow's milk suggests that these two patient groups constitute separate allergic populations, and that different pathogenic mechanisms may be operative in these two skin diseases associated with elevated levels of IgE antibodies to cow's milk.     

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.     

IgE antibody response to vertebrate meat proteins including tropomyosin.

BACKGROUND: Although meat is a main source of proteins in western diets, little information is available regarding allergy to vertebrate meats or the allergens implicated in these reactions. OBJECTIVE: To evaluate the in vitro IgE antibody response to different vertebrate meats in suspected meat-allergic subjects, as well as the possible role of tropomyosin in meat allergy and to analyze the cross-reactivity between vertebrate meats and the effect of heating on the IgE-binding to meat proteins. METHODS: Fifty-seven sera from suspected meat-allergic subjects were tested by grid blot to extracts of beef, lamb, pork, venison, chicken, and turkey and to four mammalian tropomyosins of different origins. RESULTS: Meat-allergic subjects have IgE antibodies to proteins in different mammalian meats (43/57 subjects); cross-reactivity with avian meat was limited: less than 50% (19/43) of meat positive sera reacted to chicken. In contrast, most of the poultry-positive sera also reacted to different mammalian meats. In general, there was stronger IgE reactivity to raw meats in comparison to cooked meats; an exception was six cases in which IgE reactivity to cooked poultry was stronger. Weak IgE reactivity to tropomyosin was detected in only 2/57 sera tested. CONCLUSIONS: Suspected meat-allergic subjects have serum IgE directed to meat proteins. In vitro cross-reactivity among mammalian meats appears to be important, while cross-reactivity to poultry is limited indicating mammalian-specific proteins. Although cooking in general denatures meat proteins rendering them less allergenic, in some cases the process of cooking may result in the formation of new allergenic moieties. The muscle protein tropomyosin is not an important vertebrate meat allergen.     

Ontogeny of the erythrocyte-dependent IgM antibody responses to cell membrane antigens

Our previous experiments characterized the T-cell independent type 2 B-cell responses to cell membrane antigens that are controlled by two donor cell types with different antigen-presenting(AP) activities. We here report about the ontogeny of this novel type of responses with special reference to the mutual relation of the development among two AP activities and their acceptor functions. The responses of mice to H-2^d antigens on allogeneic cells and hapten (fluorescein isothiocyanate) antigens on syngeneic cells were examined in parallel. The positive AP activity displayed by red blood cells(RBC) for induction of antihapten responses was fully developed in the fetus, although H-2^d antigens on the RBC for induction of anti-H-2^d responses was immature in mice under 7 days old. In contrast, the negative AP activity displayed by spleen cells(B cells) for inhibition of the RBC-dependent anti-hapten and anti-H-2^d responses was first developed in mice about 3 weeks old. The B cell functions accepting the positive and negative AP activities were also matured by that time. The possible significance of these findings in the physiology and pathology of the unique responses was discussed.     

Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus

Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n = 4) that were persistently infected with EAV, and from horses (n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.     

Ontogeny of the mucosal immune response

Conclusions In the 8-week period from 11 weeks to 19–20 weeks gestation human fetal intestine develops organised Peyer's patches and mucosal T cells in the lamina propria and epithelium (Table 2). T cell numbers are, however, low compared to post-natal intestine, and the Peyer's patches only contain primary B cell follicles. There is little information on changes which occur between 20 weeks gestation and birth. However, there is little further development of human gut-associated lymphoid tissue until the gut is exposed to food and bacterial antigens. Thus, there are no plasma cells in newborn human intestine. It would, therefore, appear that the mucosal immune system is functionally mature by 20 weeks gestation, but remains quiescent until birth.These studies also emphasise that one should be extremely careful in extrapolating from rodent data, where prenatal intestinal lymphoid Tissue is virtually absent, to the situation in man.     

Ontogeny of the allergic inflammatory response

The ability to produce allergic responses begins early in fetal life along with the development of other elements of the immune system. Among the most interesting questions related to the development of allergic disease are whether the fetus in utero commonly is exposed to sufficient allergen to induce IgE production and how much the mother's immune responses affect the developing fetal immune system. After birth, it seems that many factors, including the frequency and severity of infections and the timing and intensity of allergen and animal exposures, continue to influence immune development.     

Prospective estimation of IgG, IgG subclass and IgE antibodies to dietary proteins in infants with cow milk allergy

Prospectively, serum levels of IgE, specific IgE antibodies (AB) to whole cow milk protein (CMP), bovine se-albumin, bovine immunoglobulin, bovine lactoferrin, bovine lactalbumin and beta-lactoglobulin (BLG), IgG and IgG subclass antibodies to ovalbumin (OA) and BLG, and IgG4 RAST to CMP (bovine whey) were measured in 39 infants with cow milk protein allergy (CMPA) at birth (cord blood), at time of diagnosis and before and after milk challenge at the age of 12 months. Immunological measurements were also undertaken in 33 control infants without CMPA at birth, at 6 months and at 18 months. At no time, were differences found between the levels of IgG and IgG subclass AB to OA and BLG in control versus infants with CMPA. In the 39 infants with CMPA no correlation was found between the levels of IgE, IgG and IgG subclass AB in cord blood and subsequent levels of these values, irrespective of the type of CMPA (IgE-mediated (CMA) or non-IgE-mediated (CMI)), and irrespective of whether remission had occurred. In cord blood 25/33 (76%) of the infants with CMPA had specific IgE-AB to one or more of the bovine milk proteins indicating a prenatal intrauterine sensitization to cow milk protein. At 6 months the frequency of specific IgE-AB to bovine milk proteins was significantly (p less than 0.05) higher in infants with CMA versus CMI, and at 12 months total serum-IgE and the increase of these specific IGE-AB and RAST to CMP were significantly higher (p less than 0.05) in infants with persistent CMA. From 6 to 12 months withholding milk resulted in a significant fall in specific IgE-AB to CMP, and IgG, IgG1 and IgG4 anti-BLG followed by an increase after milk challenge. Decreasing levels of IgG anti-OA from birth to 6 months reflect passive maternal transfer of IgG through the placenta, and increasing levels of IgG anti-BLG, already from birth to 6 months, may represent an early exposure to CMP in all infants. Significantly higher levels (p less than 0.05) of IgG anti-OA AB, IgG1 and IgG4 anti-BLG AB were found in infants with persistent CMA, indicating a close relation between the synthesis of IgE and IgG and between IgE and IgG subclasses (IgG1 and IgG4) in symptomatic cow milk-allergic individuals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human antibody response to outer membrane proteins of Campylobacter jejuni during infection.

Two techniques were used to isolate outer membrane proteins from Campylobacter jejuni, EDTA-lysozyme extraction and sodium-N-lauroylsarcosinate (Sarkosyl) solubilization. The protein profiles of the two preparations were similar, with a few additional bands in the EDTA-lysozyme preparations. The major outer membrane protein was 43,000 (43K) daltons, and there were 8 to 10 minor bands ranging from 92K to 14K daltons. There was no difference in the protein profile of a strain causing an infection (strain 17) and the resulting stool isolate (strain 17J). Sera collected before the infection and during the acute and convalescent stages were used with Western blotting and immunoautoradiographic techniques to determine the antigenicity of outer membrane proteins. A number of antigenic proteins were detected before the infection by their reaction with preinfection serum (61K, 51K, 43K, 40K, 34K, and 31K daltons), and three additional bands appeared during the infection when acute and convalescent sera were used (92K, 56K, and 19K daltons). Furthermore, an area of the gel at less than 14.4K daltons that did not stain with Coomassie brilliant blue became visible in the immune blots when the convalescent serum was used.     

Gamma-interferon production in cow milk allergy

The aim of this study was to develop an assay to assist in the diagnosis of delayed onset of adverse responses to cow milk in children, by measurement of gamma-interferon (GIFN) produced in vitro in response to β-lactoglobulin-stimulated blood mononuclear cells. Diagnostic procedures identified 75 children with immediate reactions who had high total IeE and IgE-isotype responses to cow milk, 17 children who developed reactions after 24 h and had low total IgE and low IgE-isotype response to cow milk and 59 milk-tolerant children. GIFN production was less in children with immediate reactions compared to those with late reactions ( P ≤0.009) or milk-tolerant children ( P =0.022). The results of this study suggest enhanced T-cell reactivity may be involved in the immuno-pathogenesis of non-immediate cow milk allergy, but GIFN production was not a clinically useful diagnostic test.     

Photochemical linking of primary aromatic amines to carrier proteins to elicit antibody response against the amine haptens

Two chemical methods, diazocoupling and reaction with isocyanates, are commonly used to conjugate primary aromatic amines with carrier proteins in order to elicit antibody responses against the aromatic amine haptenic group. Limitations of these conjugation techniques include the requirement for specific functional groups on the carrier protein which generally limits the degree of haptenic substitution obtainable, the many possible side reactions yielding hapten-hapten and carrier-carrier conjugates which waste valuable materials and lower desired hapten-carrier conjugate yields, and, in some cases, conjugation conditions which may denature the carrier protein (e.g., alkaline coupling conditions). We report here a photolabeling approach for conjugating primary aromatic amines to carrier proteins which avoids some of the problems of other conjugation methods and which was used to elicit antibodies against the primary aromatic amine hapten. The method described here is of general application for coupling primary aromatic amines to the carrier proteins and circumvents many of the problems inherent in the isocyanate or diazocoupling methods. 3-Azido-N-ethylcarbazole (ANEC), the azido analog of 3-amino-N-ethylcarbazole, was conjugated to bovine serum albumin (BSA), human transferrin (TR), thyroglobulin (TH), poly-(lysine X tyrosine), and poly-(lysine X phenylalanine) using standard photolabeling procedures. After photolysis, the conjugated proteins or polypeptides were separated from the unbound products of ANEC photolysis on a Sephadex G-10 column. The conjugated proteins were extracted with isobutanol which demonstrated that approximately 20% of the ANEC was covalently coupled to the protein carriers and that the larger portion of the aromatic haptens was non-covalently and hydrophobically bound to the carriers. The ANEC-protein conjugates used for immunization demonstrated a total covalently and non-covalently bound ANEC epitope density of 90 per BSA, 107 per TR and 800 per TH molecule. Rabbits were immunized with the three conjugated proteins and the production of antibody specific for the 3-amino-N-ethylcarbazole hapten was demonstrated by enzyme-linked immunosorbent assay and by inhibition studies using hapten-carrier conjugates of free hapten. The results demonstrate that antibodies against aromatic amine haptens may be raised by immunizing animals with hapten-carrier protein conjugates produced by photolabeling. Since the coupling conditions are very mild and the functional group requirements are so general (requiring only the presence of C-H, N-H, C = O, C = S, or S-H bonds) most carrier proteins should be suitable for use in this method.(ABSTRACT TRUNCATED AT 400 WORDS)