Meat allergy associated with galactosyl-α-(1,3)-galactose (α-Gal)-Closing diagnostic gaps by anti-α-Gal IgE immune profiling.

BACKGROUND: Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-α-(1,3)-galactose (α-Gal). It is known that α-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. α-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different α-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against α-Gal. METHODS: Three serum groups, α-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for α-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated α-Gal. RESULTS: Singleplex allergy diagnostics using the α-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against α-Gal reveals individual IgE sensitization profiles for α-Gal-containing analytes. An α-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-α-Gal) was identified, which is associated with MA. CONCLUSIONS: Detection of individual sensitization patterns with different α-Gal-containing analytes provides the basis for an individual allergy diagnosis for α-Gal-sensitized patients. Higher amounts of α-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy.

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A-gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.

Cytochemical localization of malate synthase in glyoxysomes.

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and alpha-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.

The development of microbodies and peroxisomal enzymes in greening bean leaves.

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 microm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 microm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Peanut allergens: new consolidated findings on structure, characteristics, and allergome.

Immunoglobulin E-mediated food allergy is the result of a complex pathomechanism. Factors contributing to the dysfunction of the immune system are the allergenic sources and the variable matrix effects arising from the processes involved in interaction with the gastrointestinal tract, the allergens themselves through their structural features, and the specific behavior of the individual immune system. The starting point for elucidating the pathomechanism of food allergy is the identification of allergens and the description of their structure. They are the basis for in vitro diagnostics as well as the development of immunotherapeutic drugs. With regard to Class I food allergy, peanut allergy affects by far the largest group of patients. 11 allergens have been identified in peanuts. Ara h 1, Ara h 3, and Ara h 4 belong to the cupin superfamily, Ara h 2, Ara h 6, and Ara h 7 to the prolamin superfamily; Ara h 5 (profilins) and Ara h 8 (superfamily of Bet v 1-homologous proteins) are associated with aeroallergens. Peanut lipid transfer proteins (LTP) and two peanut oleosins are listed as Ara h 9, Ara h 10, and Ara h 11 by the IUIS Allergen Nomenclature Subcommittee. Peanut agglutinin (PNA) and a third oleosin have been shown to possess allergenic properties. The effect of the above specified allergens has to be considered in the context of their matrix, which is influenced by processing factors.

Localization, release and bioavailability of pollen allergens: the influence of environmental factors.

Allergens are integral constituents of plants or animals and their normal functions and localization are being characterized. To trigger responses in humans, allergens must become bioavailable and the role of air pollutants--for example diesel-exhaust particles --in this process is causing concern. Finally, the fact that some pollen releases eicosanoid-like proinflammatory mediators may have wide implications.

A polyethylene glycol/dextran procedure for the isolation of chromatin proteins (histones and nonhistones) from wheat germ.

A new procedure is described for the isolation of both histone and non-histone chromatin proteins, based on a polyethylene glycol (PEG)/dextran two-phase partition system. Chromatin is solubilized in high salt (5 M NaC1) and mixed with PEG and dextran to separate proteins (partitioned into the upper, PEG-rich phase) from nucleic acid (DNA recovered almost exclusively in the lower, dextran-rich phase). The proteins are then absorbed onto Bio-Rex 70 by dialysis to low salt (0.05 M NaC1), followed by salt elution to recover first non-histone proteins (less than 0.55 M NaC1), then histones (2 M NaC1). Cross-contamination is not detectable in either group of proteins. The procedure is rapid, gentle, and lends itself well to scale-up. The proteins are kept in pH range 7.0--8.1, and are not exposed to the denaturing reagents characteristic of most preparative procedures for chromatin proteins. Though developed specifically for the isolation of proteins from chromatin of wheat germ, the procedure should be readily applicable to other sources as well. Wheat germ (isolated wheat embryos) appears to be an excellent source of chromatin proteins; 100 g yields 225--300 mg histones and 30--45 mg nonhistone proteins, depending on technique.

Expression of the Cucumber Hydroxypyruvate Reductase Gene Is Down-Regulated by Elevated CO2.

We examined the effects of CO2 concentration on the white-light-stimulated expression of the cucumber (Cucumis sativus L.) Hpr gene. Hpr encodes hydroxypyruvate reductase, an enzyme important in the photorespiratory glycolate pathway, which plays an integral role in carbon allocation in C3 plants. Because CO2 is an end product of this pathway and because increased CO2 concentrations lessen the need for photorespiration, we tested whether exposure of plants to elevated CO2 would affect white-light-stimulated Hpr gene expression. Exposure of dark-adapted cucumber seedlings to elevated CO2 (2 to 3 times ambient) during a 4-h white-light irradiation significantly inhibited the accumulation of Hpr mRNA. Increasing the CO2 concentration during irradiation to 6 or 9 times ambient did not further inhibit Hpr mRNA accumulation. The depressing effect of high CO2 on Hpr mRNA accumulation was seen in both high and low light, but was more pronounced in higher light. These results suggest that maximum sensitivity to CO2 occurs in conditions near those normally encountered by the plant (high light, CO2 concentration near ambient) and support a model in which white-light-regulated Hpr expression is modulated in part by environmental CO2 concentration.

A common epitope on major allergens from non-biting midges (Chironomidae).

A synthetic peptide corresponding to sequence 91-101 of the Chironomus thummi thummi haemoglobins (Chi t I) components III and IV was used to investigate binding and cross-reactivity with polyclonal human IgE and rabbit IgG antibodies and murine IgG1 subclass monoclonal antibodies (MABs). The synthetic peptide reacted with antibodies from all three mammals. The specificity of the reaction, especially that with IgE antibodies was shown by dose dependent inhibition with native Chi t I component III. Epitope(s) reacting with these antibodies were also found in haemoglobins from 14 of the 15 chironomid species analyzed. The synthetic peptide III/IV 91-101 enabled the identification of an important antigenic/allergenic determinant of the broadly distributed insect family Chironomidae. The antigenic potency of this synthetic peptide as shown by testing with human IgE, rabbit IgG and mouse MABs, and the widespread occurrence of the epitope in an identical or homologous sequence and/or superficial location, qualifies the peptide for therapeutic applications in medicine.

Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.

Analysis of B-cell epitopes in the N-terminal region of Chi t I component III using monoclonal antibodies.

The hemoglobins of the midge Chironomus thummi thummi (Chi t I) are known to cause immediate-type hypersensitivity reactions in humans. Further knowledge of the antigenic sites of such allergens will provide new therapeutic approaches. The aim of our study was to identify and characterize linear B-cell epitopes of the hemoglobin component III of Chi t I (136 amino acid residues). Using the antigenic index algorithm of Jameson and Wolf (Jameson and Wolf (1988) Comput. Appl. Biosci. 4, 181-186), three linear binding sequences of this allergen molecule were predicted. Two mouse monoclonal antibodies (mAbs 3 and 6) raised against purified Chi t I component III were investigated by ELISA for their binding to nine synthetic peptides 19-21 residues in length, covering nearly the whole sequence of component III. MAb 6 recognized only one peptide (11-30) while mAb 3 bound to both N-terminal peptides (1-19 and 11-30), suggesting that the antibody binding site is located in the overlapping region. This assumption could be confirmed in ELISA with solid phase-bound recombinant peptides (RP) as well as in inhibition studies with free tryptic peptides indicating that identification of these linear B-cell epitopes is neither influenced by the method of peptide production nor by the kind of used immunoassay. To define the essential amino acid residues we investigated mAbs with solid phase-bound overlapping octamers. In the case of mAb 3, amino acids experimentally identified as essential for antibody binding (aa 13-17) are identical with those residues predicted as a B-cell epitope with the antigenic index of Jameson and Wolf.

Major allergen Phl p Vb in timothy grass is a novel pollen RNase.

A cDNA coding for the major group V allergen Phl p Vb was isolated from a timothy grass pollen cDNA library by immunoscreening with a specific monoclonal antibody. It was discovered for the first time that the recombinant Phl p Vb pollen allergen after expression and purification has ribonuclease activity. High homology of Phl p Vb to other group V allergens in grass pollen indicates similar function. By RNase activity gel of natural pollen extract of timothy grass and consecutive Western blot analysis of the excised proteins, the RNase active bands were shown to be group V allergens. Additionally it was demonstrated that an homologous protein to Phl p Vb in the mother plant could be induced by salicylic acid. This indicates that group Vb allergens may be involved in host-pathogen interactions because in pollen they are quickly exported RNases and in the mother plant they depend on a hormone which is related to expression of plant resistance genes.

Isolation of the specific mRNA coding for a CALLA-like protein and partial characterization of its cell-free translational products.

In a cell-free translation directed mRNA from the human lymphoblastoid cell line KM3, common ALL-Antigen (CALLA)-like proteins were detected immunologically. For immunoprecipitation a combination of three monoclonal antibodies and a rabbit heteroantiserum with anti-CALLA specificity were used. Employing a cocktail of two anti-CALLA monoclonal antibodies CALLA directing mRNA could be purified by specific polysomal immunoadsorption. The CALLA-like protein defined in the cell-free products of total mRNA and mRNA derived from polysomes showed a Mr of 95,000 daltons. Additional CALLA-like proteins could be identified in the cell-free translational products when mRNA from polysomes precipitated by the anti-CALLA heteroantiserum was used. The calculated Mr of these proteins were 80,000 97,000 and 135,000 daltons.

Interaction between Aeroallergens and Airborne Particulate Matter.

The in situ interaction between pollen and airborne particulate matter (APM) as well as the effect of extracts of APM on grass pollen (Dactylis glomerata) was studied. The samples were processed for structural analysis using scanning and transmission electron microscopy as well as for determination of protein content and release using immunoblot techniques. The results indicate a direct in situ interaction between pollen surfaces and APM. This effect is prominent in industrialized regions with high emission of organic pollutants. It is also found to occur near roads with heavy traffic. There is morphological evidence for preactivation of pollen by organic extracts of APM. Aqueous extracts, however, directly induce the release of allergens with altered antigenicity. It is concluded that the generation and release of allergenic aerosols in a humidified air is initiated and mediated by substances adsorbed to APM.

Identification of inflammatory cell phenotypes in human oral carcinomas by means of monoclonal antibodies.

Monoclonal antibodies reacting with human T cell sub-populations, Langerhans cells and macrophages were used to examine the quantitative distribution of immune-competent cells in normal oral mucosa and invasive oral carcinomas. Both immunofluorescent and immunoperoxidase procedures were applied. In normal oral epithelia, the dominant immune-reactive cell was the Langerhans cell, positive for OKT 6 and expressing HLA-DR gene products (OKIa1+). Many intra-epithelial non-epithelial cells (non-keratinocytes), belonged to the lymphocyte system carrying the suppressor/cytotoxic phenotype (OKT 8+). This lymphocyte sub-population was also the most prominent cell type in the normal mucosal stroma. The quantitative evaluation of immune-competent cells in squamous cell carcinomas revealed elevated numbers of all the inflammatory cell sub-populations investigated (suppressor/cytotoxic lymphocytes, helper/inducer lymphocytes, Langerhans cells, macrophages) compared with the normal oral mucosa. There was a striking increase in suppressor/cytotoxic lymphocytes (OKT 8+) and in cells of the macrophage system, including Langerhans cells (OKIa1+, OKM 1+, OKT 6+). In the stroma distant to the tumour complexes, many helper/inducer lymphocytes (OKT 4+) were also observed.

Sensitivity to aspirin: a new serological diagnostic method.

Certain adverse reactions to aspirin (ASA), nonsteroidal anti-inflammatory drugs (NSAIDs) and pyrazolone derivatives resemble IgE-mediated hypersensitivity. However, convincing evidence of antigen-antibody interactions or inhibition of the cyclooxygenase pathway of arachidonic acid metabolism, stimulating the production of leukotrienes (LTs) and decreasing the production of prostaglandins (PGs), has not been presented. In this study, two types of specific IgE antibodies have been found in six serum samples from eight ASA-sensitive patients with salicyloyl and O-methylsalicyloyl disks using the Radio Allergo Sorbent Test (RAST), whereas no positive result could be found with acetylsalicyloyl disks. Further investigation on the specificity of these IgE antibodies and the chemical structure of their epitopes were performed by cross-inhibition studies. The results are in favor of an IgE-dependent mechanism involved in ASA sensitivity, and suggest that determination of specific IgE antibodies would be a safe diagnostic method for ASA sensitivity in vitro.

Successful technique for the selective production of monoclonal antibodies against a major allergenic component in timothy pollen extract.

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.