Sensitization to Zygophyllum fabago pollen. A clinical and immunologic study

Zygophyllum fahago is a herbaceous plant found widely in the Mediterranean area. There are no previous reports of its allergenicity. An aerobiologic and clinical survey was conducted in Murcia, southern Spain, to determine the quantity of airborne pollen and establish the possible role of this pollen as a cause of allergic symptoms. With a Hirst volumetric trap, we determined the atmospheric concentrations of this pollen in 1993, 1994,1995, and 1996. Of 1180 patients tested, 181 (15,34%) had a positive skin test. To determine its allergenicity, we divided 47 patients into three groups: in group 1, all the patients had symptoms of rhinoconjunctivitis plus asthma; in groups 2 and 3, rhinoconjunctivitis. In group 1, we performed a bronchial provocation test (BPT): in groups 2 and 3, we performed nasal provocation (NPT) and conjunctival provocation (CPT) tests, respectively, SDS-PAGE was used to characterize the antigenic fractions and RAST inhibition to determine cross-reactivity with other pollens. The pollen dispersion period is from May to September (445 grains/m³), BPT was positive in 13 of 15 patients, NPT in 14 of 16 patients, and CPT in 13 of 16 patients. RAST inhibition revealed cross-reactivity with Mercurialis, Ricinus, Olea. and Betula. SDS-PAGE identified 25 IgE antibody-binding components, five of which (60, 65, 41, 38, and 15.5/14,7 kDa) were recognized by 40% of the sera. By SDS-PAGE immunoblotting with sunflower antiprofilin rabbit serum and affinity chromatography we established that the Z. fahago extract has profilin. This study shows that this pollen becomes airborne and elicits an IgE response which triggers respiratory symptoms in allergic subjects.     

Tacrolimus metabolite cross-reactivity in different tacrolimus assays

Objectives: Tacrolimus (FK506) is an immunosuppressive drug with great clinical promise. There is a controversy regarding the role of tacrolimus metabolites in immunosuppression and toxicity, and immunoassays and immunophilin binding assays have not been adequately tested for metabolite cross-reactivity. Methods are limited to HPLC and HPLC-MS for quantifying the parent drug. Mixed lymphocyte culture assay (MLC) is the preferred functional bioassay for the measurement of parent drug and active metabolites but it is not practical for routine laboratory use. Due to differences in assay methods and reagent specificity, the concentration of tacrolimus in a given specimen may vary among different assay kit manufacturers. The objective of this study was to evaluate the degree of cross-reactivity or interference of the three first-generation tacrolimus metabolites [13-O-demethyl (M-I), 31-O-demethyl (M-II) and 15-O-demethyl (M-III)] among two different tacrolimus immunoassays (Immunoassay: PRO-Trac II FK506, Abbott IMx tacrolimus-II); and the radioreceptor assays (RRA) using minor immunophilins (14, 37, and 52 kDa immunophilins) and tacrolimus binding protein (FKBP12).

Methods: First-generation tacrolimus metabolites (M-I, M-II, and M-III) spiked in drug-free whole blood were assayed with RRA using three minor immunophilins (14, 37, and 52 kDa) and two commercial immunoassay procedures (Incstar PRO-Trac II tacrolimus, Abbott IMx tacrolimus II). The results were compared to previously published FKBP-12 RRA data and their immunosuppressive potency.

Results and conclusion: The first generation tacrolimus metabolites (M-I, M-II, and M-III) were tested using concentrations of 10 and 20 ng/mL. The significance of the metabolite interference (% of the total interference) was calculated based on the relative concentration of each metabolite present at steady-state trough concentrations in renal transplant recipients [22]. Metabolite I, which has no functional immunosuppressive activity showed minimal interference compared to M-II and M-III in all assays except the 14 kDa RRA. The Incstar PRO-Trac II tacrolimus assay showed the least M-I interference. Metabolite-II, which has a pharmacologic potency similar to the parent drug, showed a significant interference in the immunoassays and significant interference in radioreceptor assays. Metabolite III, which is pharmacologically inactive, produces 3–10% interference in the different assays if its presence in the blood is 6% of the parent drug. The total interference from these three metabolites was greater in the immunoassays than in the receptor assays. Receptor assays for tacrolimus provide results closer to the target value than do immunoassays.     

Studies on shared antigenic/allergenic components among fungi

Fungal allergens have been found to be one of the most prevalent aeroallergens in India. Knowledge of shared/unique components among different fungi is necessary for proper diagnosis and treatment of patients allergic to fungi. In the present study, crude extracts (CE) of 11 common fungi (Alternaria alternata, Aspergillus flavus, Asp. fumigatus, Asp. niger, Asp. tamarii, Asp. versicolor, Cladosporium herbarum, Curvularia lunata, Mucor hiemalis, Penicillium citrinum, and Fusarium solani) were characterized by isoelectric focusing (IEF), SDS-PAGE, and immunoblot. On IEF (pI 3–9), the number of protein bands was found to be greatest (46) in M. hiemalis extract. SDS-PAGE exhibited a varied number of bands, generally 18–40, with mol. mass ranging from 14 to 100 kDa. IgG-specific immunoprint using rabbit anti-F. solani CF antibodies demonstrated a mol. mass distribution of shared antigenic proteins of 14–100 kDa in most of the fungi. Shared allergenicity was observed in a number of allergenic proteins in fungal extracts with mol. mass ranging between 14 and 70 kDa on IgE-specific immunoblot using pooled sera of patients allergic to Fusarium. A 45-kDa protein was found to be common among these fungi on immunoblot with patients as well as with rabbit antibodies. F. solani CF extract contained more antigenic/allergenic proteins than F. solani CE. It was concluded that F. solani CF shared several antigenic/allergenic components with CE of other common fungi. This fact needs to be taken into account when fungal extracts are used in diagnosis and immunotherapy of allergic patients.     

Lettuce anaphylaxis: identification of a lipid transfer protein as the major allergen

BACKGROUND: Allergy to plant-derived foods is associated with birch pollinosis in central and northern Europe. Symptoms elicited are usually limited to the oropharyngeal system. By contrast, in the Mediterranean area, allergy to the same foods manifests more frequently with systemic reactions caused by nonspecific lipid transfer proteins (nsLTP), independently of an associated pollinosis. OBJECTIVE: We sought to investigate the pattern of immunoglobulin E (IgE) binding protein bands implicated in lettuce allergy, in particular the presence of an nsLTP. METHODS: Consecutive lettuce allergic patients were selected. Determination of serum-specific IgE, immunoblot, and inhibition experiments were performed in order to study the pattern of IgE binding proteins and the potential cross-reactivity to pollens. Inhibition studies with recombinant allergens were conducted to identify the lettuce allergens. The major allergen was subjected to N-terminal amino acid sequencing. RESULTS: Fourteen patients were diagnosed as being allergic to lettuce. All were sensitized to Platanus pollen. Ten of them showed specific IgE to a lettuce protein of 9-kDa. The IgE binding to this protein was completely inhibited by the cherry-LTP and peach extract. The N-terminal sequence of the 9-kDa protein showed a high degree of amino acid sequence identity to other nsLTPs. A clear partial cross-reactivity was observed between lettuce-LTP and Platanus-pollen extract. CONCLUSIONS: An LTP has been demonstrated to be a major allergen in patients suffering from lettuce allergy.     

Clustering of allergenic pollen on the basis of skin responses of atopic patients by matrix analysis

The responses of 148 atopic patients to some 43 different extracts of allergenic pollen were tested by prick tests. The measure of dissimilarity was introduced and calculated for all pairs of allergens. The investigated allergens were clustered into groups, according to their unbiased greatest similarity, by a matrix-structuring method. Results indicate that subgroups of allergens can be distinguished even within groups of closely related pollen allergens that were believed to be fully cross-reactive. A few cases are demonstrated for various varieties of olives, pecans, date palms, and turf grasses and for some wild chenopods and amaranths. The usefulness of the suggested solution for allergy research and for clinical practice is discussed.     

Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.     

Sensitization to Dermatophagoides siboney, Blomia tropicalis, and other domestic mites in asthmatic patients

Mite species adapted to warm, humid climates are commonly found in house dust in the tropics. In Cuba, Dermatophagoides pteronyssinus, D. siboney , and Blomia tropicalis are the most common and abundant mite species in house dust. To investigate the pattern of Sensitization of Cuban asthmatic patients to common mite species, we skin-prick-tested (SPT) 148 patients with a clinical history of asthma and possible mite allergy, and determined specific IgE antibodies against mite allergens ( D. pteronyssinus, D. farinae, D. siboney, B. tropicalis, Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae , and Glycyphagus domesticus ). The prevalence of positive SPT was high to D. siboney (88%), D. pteronyssinus (87%), A. siro (85%), B. tropicalis (85%), and D. farinae (83%). The largest skin reactions were obtained with D. siboney and B. tropicalis extracts. The skin test response to the D. siboney extract correlated to those of D. farinae, D. pteronyssinus, B. tropicalis , and A. siro. The highest IgE levels were found to Dermatophagoides species and B. tropicalis. IgE to D. siboney and B. tropicalis were found in 97% and 96% of the patients, respectively. The prevalence of specific IgE to the other mites studied varied from 46 to 65%. D. siboney and B. tropicalis are important sensitizers among asthmatic patients in Cuba.     

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase

BACKGROUND: Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens. METHODS: Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients' sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients. RESULTS: Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients' sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients' sera. CONCLUSIONS: Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust.     

Recombinant fish parvalbumins: Candidates for diagnosis and treatment of fish allergy

Fish and fish products represent one of the most important causes of IgE-mediated food hypersensitivity. In sensitized individuals contact with and consumption of fish can lead to severe health problems, ranging from urticaria and dermatitis to angiedema, diarrhoea, asthma and, at worst, systemic anaphylactic reactions and death. Parvalbumin, a small calcium-binding protein present in the muscles of vertebrates, was identified as the major fish allergen. We describe the isolation and characterization of cDNA clones coding for carp parvalbumin by IgE immunoscreening of a carp muscle expression library. These clones will be the basis for the production of recombinant carp parvalbumin, a useful tool for in vitro and in vivo diagnosis of fish allergy.