Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T-Cell Responses to Mammalian Cystatins, Including the Human Self-Protein.

BACKGROUND: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. METHODS: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. RESULTS: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. CONCLUSIONS: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins.

Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T-Cell Responses to Mammalian Cystatins, Including the Human Self-Protein

Background: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. Methods: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. Results: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non–cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. Conclusion: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins. (AU)

Antecedentes: Las reacciones de hipersensibilidad de tipo inmediato y retardado a los alérgenos que están en las mascotas son comunes en las enfermedades atópicas. En este estudio, en pacientes con dermatitis atopica (DA), se analiza la reactividad cruzada con las autoproteínas y su contribución a la enfermedad. Tanto la cistatina A humana como el alérgeno felino Fel d 3 pertenecen a la familia de las cistatinas, una familia de proteínas conservadas evolutivamente. El objetivo del presente estudio fue evaluar la reactividad cruzada entre las cistatinas de mamíferos y analizar la respuestas de las células T a la cistatina en pacientes con DA sensibilizados a la caspa de las mascotas. Métodos: El ADNc que codifica la cistatina de perro se clonó a partir de piel de perro. Se analizaron sueros de 245 pacientes con sensibilización por IgE a la caspa de gato y perro para determinar la unión de IgE a cistatina felina, canina y humana expresada de forma recombinante, respectivamente. De estos 245 pacientes, 141 fueron diagnosticados de DA. Resultados: Se detectó IgE específica frente a cistatina en el 14,7% (36) de los pacientes, de los cuales 19 padecían DA. Dentro de los pacientes con DA, 9 tenían IgE medible contra las tres cistatinas. Los pacientes con DA sensibilizados frente a cistatina no difirieron de los pacientes no sensibilizados con cistatina en términos de gravedad de la enfermedad, edad o niveles totales de IgE. El análisis de citocinas de células T reveló niveles elevados de IL-4 después de la estimulación con cistatina felina y humana. Conclusión: La respuesta humoral sugiere que, además de Fel d 3, la proteína homóloga de perro también podría desempeñar un papel en la alergia. Además, la cistatina humana parece ser capaz de promover una respuesta inmune de tipo 2 en pacientes con DA sensibilizados y, por lo tanto, puede considerarse un autoalérgeno, como se ha propuesto para otras proteínas conservadas evolutivamente. (AU)

Protein and DNA-based assays as complementary tools for fish allergen detection.

BACKGROUND: Fish is one of the most important, allergenic foods worldwide. Parvalbumin is the well characterized, major allergen in fish muscle. In this study, we developed a protein- and a DNA-based method for the sensitive detection and authentication of eight commonly consumed fishes in food and compared their applicability. METHODS: Fish parvalbumins were purified. Polyclonal, anti-parvalbumin antibodies were raised in rabbits and mice. Protein extracts from food were analyzed by quantitative ELISA. Parvalbumin genes were cloned and sequenced for the design of parvalbumin gene-specific PCR-primers. DNA extracted from food was subjected to specific PCR. RESULTS: Increasing parvalbumin contents were quantified by ELISA in fresh fish, in the order of tuna < mackerel < cod < salmon/trout < redfish < carp < herring. The parvalbumin content of processed fish was up to 67% lower than in fresh fish. In spiked food samples, 1 to 15 ppm fresh fish and 30 to 170 ppm processed fish were still detectable by ELISA. The eight fishes were identified by specific PCR using 0.2 to 10 ng fish DNA. PCRs detected still 3 ppm fresh fish and 30 to 150 ppm processed fish in spiked samples. CONCLUSIONS: Both the protein- and the DNA-based method have sufficient sensitivity to protect fish-allergic consumers. The ELISA allows allergen quantification, while the PCR identifies the fish present in the food. The detection limits of both methods vary depending on different factors. Both methods need to be carefully validated for each fish and fish product when used in detection assays.

Cross-reactivity to fish and chicken meat - a new clinical syndrome.

BACKGROUND: Fish is one of the most allergenic foods. While clinical cross-reactivity among different fishes is a widely accepted feature of fish allergy, associations with other food allergies are not well understood. This study aims at analyzing the relevance of clinical cross-reactivity between fish and chicken meat in patients with allergy to chicken meat without sensitization to hen's eggs. METHODS: Patients with food allergy to fish and chicken meat (n = 29) or chicken meat only (n = 7) were recruited. IgE-reactive chicken proteins were identified (Edman, MS analysis) and quantified (ELISA). Allergens were used in IgE ELISA and skin testing. RESULTS: Chicken parvalbumin and two new allergens, aldolase and enolase, were identified at 12, 40, and 50 kDa, respectively. They were recognized by sIgE of 61%, 75%, and 83% of all patient sera which were in the majority of the cases positive for the fish homologues as well. Fish and chicken meat allergens were highly cross-reactive while high inhibition rates with fish or chicken allergens correlated with the patients' primary sensitization to fish or chicken. In cooked or roasted foods, enolase and aldolase were detectable in chicken breast while parvalbumin was detectable in chicken legs and wings. CONCLUSIONS: Fish and chicken meat are cross-reactive foods; both fish-allergic and chicken meat-allergic patients might be at risk of developing a food allergy to chicken meat or to fish, respectively. This clinical phenomenon is proposed to be termed 'fish-chicken syndrome' with cross-reactive allergens involved being parvalbumins, enolases, and aldolases.

Two galactose-α-1,3-galactose carrying peptidases from pork kidney mediate anaphylactogenic responses in delayed meat allergy.

BACKGROUND: Serum IgE antibodies directed at galactose-α-1,3-galactose (α-Gal) are associated with a novel form of delayed anaphylaxis occurring upon consumption of red meat or innards. Pork kidney is known as the most potent trigger of this syndrome, but the culprit allergens have not yet been identified. The aim of this study was the identification and characterization of pork kidney proteins mediating delayed anaphylactic reactions through specific IgE to α-Gal. METHODS: A cohort of 59 patients with specific IgE to α-Gal was screened by immunoblot for IgE-reactive proteins in pork kidney. Proteins were identified by peptide mass fingerprinting. Isolated proteins were assayed in ELISA and ELISA inhibition, basophil activation and skin prick test. RESULTS: Several IgE-binding proteins of high molecular weight (100- >200 kDa) were detected in pork kidney extracts by immunoblot using patient sera and an anti-α-Gal antibody. Two major IgE-binding proteins were identified as porcine angiotensin-I-converting enzyme (ACE I) and aminopeptidase N (AP-N). Reactivity of patient sera and anti-α-Gal antibody to both proteins was abolished by carbohydrate oxidation. The α-Gal IgE epitopes were resistant to heat denaturation. Pork kidney extract, isolated ACE I, and AP-N were able to activate patient basophils and elicit positive responses in skin prick tests. CONCLUSION: Two cell-membrane proteins carrying α-Gal epitopes were identified in pork kidney. For the first time, isolated meat proteins were shown to induce basophil activation in patients with delayed anaphylaxis to red meat providing further confirmation for the clinical relevance of these α-Gal-carrying proteins.

Development of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy.

BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.

Identification of enolases and aldolases as important fish allergens in cod, salmon and tuna: component resolved diagnosis using parvalbumin and the new allergens.

BACKGROUND: The majority of fish-allergic patients are sensitized to parvalbumin, known to be the cause of important IgE cross-reactivity among fish species. Little is known about the importance of fish allergens other than parvalbumin. OBJECTIVE: The aim of this study was to characterize hitherto undefined fish allergens in three commonly consumed fish species, cod, salmon and tuna, and to evaluate their importance for in vitro IgE-diagnosis in addition to parvalbumin and fish gelatin. METHODS: Sixty-two patients were diagnosed by clinical history, skin prick tests and specific IgE to fish extracts. Two new fish allergens from cod, salmon and tuna were identified by microsequencing. These proteins were characterized by immunoblot, ELISA and mediator release assay. Purified parvalbumin, enolase, aldolase and fish gelatin were used for quantification of specific IgE in ELISA. RESULTS: Parvalbumin and two other allergens of 50 and 40 kDa were detected in IgE-immunoblots of cod, salmon and tuna extracts by most patient sera. The 50 and 40 kDa proteins were identified as beta-enolase and fructose-bisphosphate aldolase A respectively. Both purified enzymes showed allergenic activity in the mediator release assay. Indeed, 72.6% of the patients were sensitized to parvalbumin, 20% of these had specific IgE to salmon parvalbumin only. IgE to enolases were found in 62.9% (0.5-95.0 kUA /L), to aldolases in 50.0% (0.4-26.0 kUA /L) and to fish gelatin in 19.3% (0.4-20.0 kUA /L) of the patients. Inter-species cross-reactivity, even though limited, was found for enolases and aldolases by IgE-inhibition ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: Fish enolase and aldolase have been identified as important new fish allergens. In fish allergy diagnosis, IgE to enolase and aldolase are especially relevant when IgE to parvalbumin are absent.

Evaluation of two new recombinant guinea-pig lipocalins, Cav p 2 and Cav p 3, in the diagnosis of guinea-pig allergy.

BACKGROUND: Guinea-pigs, classical laboratory animals now often kept as pets, regularly elicit important allergic reactions. Two guinea-pig allergens have been described previously to some extent; however, biomolecular and immunological data are lacking. OBJECTIVE: To identify major guinea-pig allergens, produce them as recombinant proteins and define their allergenic potential and clinical importance in allergic patients. METHODS: Protein extracts were prepared from various guinea-pig tissues and allergens were purified by ion exchange chromatography. The N-termini of two immunoglobulin E (IgE)-reactive proteins were determined and degenerate primers were designed for cDNA amplification by RT-PCR. Allergenic properties of recombinant proteins were assayed by immunoblotting, ELISA and mediator release assays. Specific IgE were quantified in the sera of 26 guinea-pig-allergic patients. RESULTS: Major IgE-reactive proteins were detected in submaxillary and harderian gland extracts. Two proteins were identified and the cDNAs were cloned. The 17 kDa protein expressed in the harderian gland corresponds to the previously described Cav p 2. The 19 kDa protein, Cav p 3, is a new allergen expressed in the submaxillary gland. Recombinant Cav p 2 and Cav p 3 were recognized by IgE antibodies from 65% and 54% of guinea-pig-allergic patients, respectively. Both proteins demonstrated equivalent allergenic activity in the mediator release assays. CONCLUSION AND CLINICAL RELEVANCE: Two major guinea-pig allergens, Cav p 2 and Cav p 3, have been characterized and produced as recombinant proteins. Combined to guinea-pig serum albumin, the new allergens proved to be valuable in the component-resolved diagnosis of guinea-pig allergy.

Important variations in parvalbumin content in common fish species: a factor possibly contributing to variable allergenicity.

BACKGROUND: Although 95% of fish-allergic patients are sensitized to the major fish allergen parvalbumin, clinical reactions to different fish species vary considerably in symptoms, intensity and frequency in allergic subjects. This study aimed at the quantification of parvalbumin levels in salmon, trout, cod, carp, mackerel, herring, redfish and tuna. METHODS: Fish muscle extracts were separated by SDS-PAGE and parvalbumin content was estimated by densitometric band quantification. Individual antisera were raised in BALB/c mice against parvalbumins purified from seven fish species. Parvalbumin content was quantified in fish (raw/processed) and skin prick test (SPT) solutions by ELISA using the corresponding anti-serum for detection and the purified parvalbumins as standards. RESULTS: Using SDS-PAGE scanning, parvalbumin contents were <0.5 mg per gram tissue for mackerel, 0.5-2 mg for salmon and trout, and >2 mg for cod, carp, redfish and herring. Using ELISA, parvalbumin content ranged from <0.05 mg for tuna, 0.3-0.7 mg for mackerel, 1-2.5 mg for salmon, trout and cod to >2.5 mg per gram raw muscle for carp, herring and redfish. The parvalbumin content of processed samples (cooked/commercial) was 20-60% lower. Allergen content in SPT samples ranged from 20 to 70 µg parvalbumin/ml of extract. No parvalbumin was found in tuna SPT solution. CONCLUSION: The parvalbumin content of most commonly consumed fish species varies considerably. Differences range from severalfold to one hundredfold. This has to be taken into account when designing food challenge tests and advising fish-allergic patients.

Monosensitivity to pangasius and tilapia caused by allergens other than parvalbumin.

Fish allergy is one of the most common food allergies in populations where fish is a major part of the diet. Most fish-allergic patients react to the panallergen parvalbumin present in multiple fish species. Our aim was to investigate the clinical case of a patient with oral allergy syndrome to pangasius and Nile tilapia but tolerance of other fish and seafood. The temporal relationship between fish consumption and allergic symptoms, the positive skin prick tests, and the basophil activation test results for both fish species strongly supported the diagnosis of an immunoglobulin (Ig) E-mediated allergy. This was confirmed by the detection of specific IgE to 18-kDa and 45-kDa proteins in immunoblot analysis. Notably, the patient was not sensitized to parvalbumin, as shown by enzyme-linked immunosorbent assay using purified allergens. Cross-reactivity between fish species can result from sensitization to allergens other than parvalbumin. This case report emphasizes the applications of flow cytometry-assisted analysis in the diagnosis of food allergy.

Occupational inhalant allergy to pork followed by food allergy to pork and chicken: sensitization to hemoglobin and serum albumin.

BACKGROUND: Animal-derived proteins are implicated in primary food allergies, but also in inhalant allergies with secondary food allergy symptoms. The objective of this study was to define the allergen(s) implicated in a case of food allergy to chicken meat, which developed in a person previously sensitized to pork after occupational exposure. METHODS: A 42-year-old female with a history of occupational inhalant allergy to pork reported rhinitis, asthma, dysphonia and conjunctivitis 30 min after ingestion of chicken. Skin tests were positive to chicken meat. Protein extracts were prepared from chicken meat. Allergens were characterized by IgE immunoblotting, N-terminal sequencing and ELISA. RESULTS: The patient showed specific IgE binding to chicken meat proteins at 12, 14, 26, 55 and 65 kDa. N-terminal amino acid sequencing identified the 12- and 14-kDa proteins as the alpha- and beta-chain of hemoglobin. ELISA and immunoblot showed specific IgE binding to hemoglobin purified from chicken blood. IgE antibodies to chicken serum albumin were detected by ELISA. Inhibition studies with chicken and porcine hemoglobin as well as with serum albumins demonstrated cross-reactive IgE antibodies. CONCLUSIONS: We report a case of confirmed occupational inhalant allergy due to pork followed by food allergy to pork and 3 years later by food allergy to chicken. Porcine and chicken hemoglobin were found to be cross-reactive allergens. Cross-reactivity between porcine and chicken serum albumin was possibly linked to a prior sensitization to cat serum albumin.

Clinical and immunological aspects of HLA class I deficiency.

Human leukocyte antigen (HLA) class I deficiency is a rare disease with remarkable clinical and biological heterogeneity. The spectrum of possible manifestations extends from the complete absence of symptoms to life-threatening disease conditions. It is usually diagnosed when HLA class I serological typing is unsuccessful; flow cytometric studies then reveal a severe reduction in the cell surface expression of HLA class I molecules (90-99% reduction compared to normal cells). In most cases to date, this low expression is due to a homozygous inactivating mutation in one of the two subunits of the transporter associated with antigen processing (TAP), critically involved in the peptide loading of HLA class I molecules. Although asymptomatic cases have been described, TAP deficiencies are usually characterized by chronic bacterial infections of the upper and lower airways, evolving to bronchiectasis, and in half of the cases, also skin ulcers with features of a chronic granulomatous inflammation. Despite the defect in HLA class-I-mediated presentation of viral antigens to cytotoxic T cells, the patients do not suffer from severe viral infections, presumably because of other efficient antiviral defence mechanisms such as antibodies, non-HLA-class-I-restricted cytotoxic effector cells and CD8+ T-cell responses to TAP-independent antigens. Treatment is at present exclusively symptomatic, and should particularly focus on the prevention of bronchiectasis, which requires early detection.

IgE antibodies of fish allergic patients cross-react with frog parvalbumin.

BACKGROUND: The major allergens in fish are parvalbumins. Important immunoglobulin (Ig)E cross-recognition of parvalbumins from different fish species has been shown. Recently frog parvalbumin alpha has been found to be responsible for a case of IgE-mediated anaphylaxis triggered by the ingestion of frog meat. The aim of this study was to investigate whether IgE antibodies of fish allergic persons cross-react with frog parvalbumin and to appreciate its clinical relevance. METHODS: The sera of 15 fish allergic patients and one fish and frog allergic patient were tested by IgE-immunoblotting against frog muscle extract. Sera were tested against recombinant parvalbumin alpha and beta from Rana esculenta. Skin prick tests were performed in selected patients with recombinant frog parvalbumin. Ca(2+) depletion experiments and inhibition studies with purified cod and frog recombinant parvalbumin were done to characterize the cross-reactive pattern. RESULTS: Fourteen of the sera tested had IgE antibodies recognizing low molecular weight components in frog muscle extract. Calcium depletion experiments or inhibition of patient sera with purified cod parvalbumin led to a significant or complete decrease in IgE binding. When tested against recombinant parvalbumins, three of 13 sera reacted with alpha parvalbumin and 11 of 12 reacted with beta parvalbumin from R. esculenta. Skin prick tests performed with recombinant frog parvalbumin were positive in fish allergic patients. Inhibition studies showed that a fish and frog allergic patient was primarily sensitized to fish parvalbumin. CONCLUSION: Cod parvalbumin, a major cross-reactive allergen among different fish species, shares IgE binding epitopes with frog parvalbumin. This in vitro cross-reactivity seems to be also clinically relevant. Parvalbumins probably represent a new family of cross-reactive allergens.

Severe IgE-mediated anaphylaxis following consumption of fried frog legs: definition of alpha-parvalbumin as the allergen in cause.

BACKGROUND: IgE-mediated allergic reactions to bullfrog and edible frog have been reported. The implicated allergens have not been defined so far. The frog material and the patient's serum from a case of severe food-induced anaphylaxis were used to define the implicated allergen at the protein and DNA level. METHODS: Immunoblotting techniques and N-terminal protein microsequencing were used to define the allergen recognized by the patient's serum. Back translation from the identified protein sequence was used to design degenerated primers to amplify the allergen's cDNA by polymerase chain reaction (PCR). We defined the nucleotide sequence of the allergen from the frog of Indonesian origin that was consumed by the patient, and the homologous cDNA from Rana esculenta. RESULTS: Protein microsequencing revealed that the implicated frog allergen belonged to the parvalbumin family. cDNAs coding for alpha- and beta-parvalbumin of R. esculenta and Rana species were cloned. Recombinant proteins were expressed in Escherichia coli. The patient's serum IgE antibodies recognized parvalbumin prepared from frog muscle and recombinant alpha-parvalbumin from R. species but not from R. esculenta. Recombinant beta-parvalbumin was not recognized by the IgE antibodies. CONCLUSION: This work defines at the protein and DNA levels alpha-parvalbumin as the allergen implicated in a case of IgE-mediated anaphylaxis to frog muscle. It also shows that a protein belonging to the parvalbumin family is implicated in type I allergies outside the fish species.