Background: Cross-reactions between latex, chestnut, and other fruits have been demonstrated by R... more Background: Cross-reactions between latex, chestnut, and other fruits have been demonstrated by RAST, although the results show some discrepancies. Objective: To detect and characterize cross-reacting antigens in both extracts by immunoblotting. Methods: Polyclonal rabbit antisera were raised against ammoniated latex and an aqueous chestnut extract. The antigenic extracts were studied by immunoblotting using a diffusion transfer method. Cross-reactivities were measured by immunoblotting inhibition. Results: Both antisera detected a high number of antigenic proteins in both latex and chestnut extracts. Latex was a good self-inhibitor and inhibitor of chestnut with respect to both antisera. Chestnut was only a good inhibitor for the two antisera on the chestnut-blot as well as on the latex-blot when the anti-chestnut antiserum was employed. No inhibition was obtained on latex immunoblotting by chestnut using anti-latex antiserum. Conclusions: The results suggested that latex and chestnut contained common epitopes detected by antibodies raised by immunization with both antigens. The anti-latex antiserum, however, possessed antibodies that could not be inhibited by other allergens. This could explain why latex has been described as a good inhibitor of chestnut-RAST while no inhibition by chestnut was found on the latex-RAST in latex and chestnut allergic patients. Other common pollen extracts were also inhibitors of immunoblotting with the exception of the latex/anti-latex pair. This suggests that antibodies induced by immunization with latex or chestnut showed unexpected cross-reactions.
A 34-year-old Spanish woman with a lifelong history of seasonal rhinoconjunctivitis and honey int... more A 34-year-old Spanish woman with a lifelong history of seasonal rhinoconjunctivitis and honey intolerance (pyrosis and abdominal pain) developed, 3 weeks after starting ingestion of bee pollen, astenia, anorexia, abdominal pain, diarrhoea, peripheral blood hypereosinophilia and elevated serum total IgE levels. A duodenal biopsy showed eosinophilic infiltration of the mucosal layer. Other causes of hypereosinophilia were not found. Repeated parasitological stool studies, as well as a duodenal aspirate showed negative results. Symptoms, hypereosinophilia and elevated IgE levels resolved after bee pollen ingestion was stopped. This is a typical case of eosinophilic gastroenteritis by ingestion of bee pollen in a woman with intolerance to honey bee, because the patient fulfilled the usual diagnostic criteria: gastrointestinal symptoms were present, eosinophilic infiltration of the digestive tract was demonstrated by biopsy, no eosinophilic infiltration of other organs was found and the presence of parasites was excluded. Honey intolerance and/or bee pollen administration should be considered as a cause of eosinophilic gastroenteritis.
The Journal of Allergy and Clinical Immunology, Dec 1, 1984
Inhalation of wood dusts can cause immediate and/or late onset asthma, and the list of woods resp... more Inhalation of wood dusts can cause immediate and/or late onset asthma, and the list of woods responsible for such reactions is increasing. We report two patients with asthma induced by exposure to the dust of African maple wood (Triplochiton scleroxylon). Type I hypersensitivity to this dust was demonstrated by means of immediate skin test reactivity, positive passive transfer test, positive reverse enzyme immunoassay for specific IgE, and an immediate bronchial provocation test response to an African maple-dust extract. Unexposed persons did not exhibit reactivity to this wood in any of the tests listed above.
Several authors have demonstrated that renaturation is an essential step for the appropriate reco... more Several authors have demonstrated that renaturation is an essential step for the appropriate recognition of blotted proteins. The use of nonionic detergents has been described as a useful alternative to enhance the antigenicity in immunoblotting, although elution from proteins by detergents has been observed. To measure the influence of different factors on the sensitivity of specific IgE by immunoblotting, we used twenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Nonidet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing this detergent, even at high concentrations (3%), and its use gave better sensitivity than methanol. On the other hand, we detected that methanol possessed renaturing properties. A transfer method using diffusion instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method, we found that after NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effects.
Hypersensitivity to cockroach antigen has been recognized as an important cause of perennial alle... more Hypersensitivity to cockroach antigen has been recognized as an important cause of perennial allergic rhinitis and asthma. To assess the frequency of cockroach hypersensitivity in our country, 150 asthmatic atopic subjects were studied using skin testing and in vitro assays for cockroach-specific IgE antibodies (Oriental and German cockroaches). Twenty-two of 61 patients who had a positive history of cockroach exposure had positive skin tests, and only 3 of 89 patients who had no history of exposure had positive skin reactions. Of 25 patients with positive skin tests, 23 showed specific IgE antibodies against oriental and German cockroaches using RAST and EIA techniques. In summary, approximately 15% of asthmatic atopics in Madrid area are sensitive to cockroaches (positive skin test + specific IgE antibodies). These results indicate that cockroach hypersensitivity should be considered in every patient with perennial asthma.
Twenty eight workers of a pharmaceutical factory were studied. The company produced morphine and ... more Twenty eight workers of a pharmaceutical factory were studied. The company produced morphine and other alkaloids extracting the active principles from shells of Papaver somniferum. Six of the exposed subjects had clinical symptoms of sensitization to this allergen and positive skin tests. A bronchial provocation test was found to be positive on 4 workers and specific IgE could be found on the 6 sensitized patients by an ELISA and a RAST test using an aqueous extract of P. somniferum. A histamine release using the same antigen was also positive in the 4 samples from sensitized patients available. An SDS-PAGE of the extract revealed a major protein band with an estimated mol wt of 52,000 d. This band had the highest IgE-binding capacity as shown by immunoblotting. All these facts suggest that P. somniferum allergy is mediated by an IgE mediated mechanism and not by a pharmacological or toxic effect of the alkaloids or polyphenols.
Objective: the ingestion of Anisakis simplex larvae may lead to the appearance of gastrointestina... more Objective: the ingestion of Anisakis simplex larvae may lead to the appearance of gastrointestinal symptoms. However, the number of reported cases of parasitization by Anisakis in Spain is lower than would be expected in a country with the second-highest fish consumption per inhabitant in the world, particularly since fish is often eaten raw or only slightly cooked. We suggest that the incidence of anisakiasis in Spain would be higher if complementary studies were used in all patients suspected of having anisakiasis. Methods: we studied 6 patients with a diagnosis of intestinal obstruction who frequently ate fish. Skin prick tests with seafood, inhalant allergen and Anisakis extracts were done. Total and specific IgE against Anisakis larvae were tested with a CAP system radioimmunoassay and immunoblot assays. Oral challenge tests with frozen larvae were also used. Results: a positive skin prick result and high levels of total and specific IgE were found in all patients. The results of immunoblot assays for IgE did not show a consistent pattern, but a group of several low (14-18 kDa) and intermediate molecular weight antigens (30-50 kDa) were found in all patients. All patients tolerated the oral challenge test well. Conclusions: in our patients with intestinal pseudo-obstruction and a history of frequent fish eating, the clinical and laboratory findings were suggestive of parasitization by Anisakis simplex larvae as the cause of the obstruction. Such complementary studies should be used whenever there is a suspicion of anisakiasis. The results of the oral provocation test show that the intake of dead larvae does not induce clinical parasitization.
A familial C1q deficiency of complement in three siblings has been established. The patients were... more A familial C1q deficiency of complement in three siblings has been established. The patients were two brothers and a sister (12, 11 and 9 years old) with clinical and pathological features of Rothmund-Thomson syndrome (Poikiloderma congenital) and mesangial proliferative glomerulonephritis with diffuse IgM deposits. Abnormality has been defined as a total lack of CH50 haemolytic activity, undetectable C1q, failure to correct the defect with functionally pure C2 to C9 complement components, normal values for C2, C3, C4 and C5 and restoration of CH50 haemolytic activity when purified human C1q was added to the assay.
We present eight patients allergic to latex and fruit (chestnut and banana), seven of whom are wo... more We present eight patients allergic to latex and fruit (chestnut and banana), seven of whom are women, and aged 17 to 42 years (mean 25 years). Four had family and five personal atopic histories. The total IgE varied from 41 to 520 Ku/L (mean 263). The symptoms followed ingestion of fruit (anaphylaxis) in four patients and contact with rubber (contact urticaria and anaphylaxis) in the other four. Skin prick test (SPT) with latex and radioallergosorbent test to latex were positive in all the patients. Histamine release (HR) to latex was carried out on six patients and was positive in three. In the six patients with symptoms after having eaten chestnuts the SPT was positive and specific IgE was detected in five of them. Histamine release to chestnuts was positive in three of the six patients tested and one of them (-SPT and + IgE) tolerated the fruit. Two out of five patients with symptomatic banana allergy had negative SPT with banana while the test was positive in one patient who tolerated this fruit, this being the only case with specific IgE to banana. Histamine release with banana was only positive in one case. The important correlation between SPT, RAST, and HR results to latex and chestnut together with the total inhibition of the chestnut RAST with a serum pool by preincubation with latex suggests cross-reactivity among these allergens.
Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful... more Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97-1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 microg/ml mite extract. Analysis of the data obtained with 1.6 microg/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91-1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non-allergic children produced a cut-off of 8% activated basophils (AUC = 1.0) with 16 microg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 microg/ml extract (AUC = 97%, 95% CI = 0.92-1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.
Molecular and Biochemical Parasitology, Aug 1, 2008
Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnos... more Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnosis usually leads to the dietary recommendation of avoiding fish and seafood consumption. The protein Ani s 4 is a clinically relevant allergen due to its heat and pepsin resistant properties and its importance in the anaphylaxis process. The attempt of cloning Ani s 4 has led to the identification and characterisation of a new isoform that differs only in one amino acid with the previously published. This isoform was produced as an His tagged recombinant protein and its allergenic properties were tested by IgE immunoblot and by a flow cytometry basophil activation test. The results were compared to the allergenic properties of the isoform previously described. Both isoforms of Ani s 4 showed different capacities to bind IgE from sensitised patients and different potencies in the basophil activation test.
Background: Cross-reactions between latex, chestnut, and other fruits have been demonstrated by R... more Background: Cross-reactions between latex, chestnut, and other fruits have been demonstrated by RAST, although the results show some discrepancies. Objective: To detect and characterize cross-reacting antigens in both extracts by immunoblotting. Methods: Polyclonal rabbit antisera were raised against ammoniated latex and an aqueous chestnut extract. The antigenic extracts were studied by immunoblotting using a diffusion transfer method. Cross-reactivities were measured by immunoblotting inhibition. Results: Both antisera detected a high number of antigenic proteins in both latex and chestnut extracts. Latex was a good self-inhibitor and inhibitor of chestnut with respect to both antisera. Chestnut was only a good inhibitor for the two antisera on the chestnut-blot as well as on the latex-blot when the anti-chestnut antiserum was employed. No inhibition was obtained on latex immunoblotting by chestnut using anti-latex antiserum. Conclusions: The results suggested that latex and chestnut contained common epitopes detected by antibodies raised by immunization with both antigens. The anti-latex antiserum, however, possessed antibodies that could not be inhibited by other allergens. This could explain why latex has been described as a good inhibitor of chestnut-RAST while no inhibition by chestnut was found on the latex-RAST in latex and chestnut allergic patients. Other common pollen extracts were also inhibitors of immunoblotting with the exception of the latex/anti-latex pair. This suggests that antibodies induced by immunization with latex or chestnut showed unexpected cross-reactions.
A 34-year-old Spanish woman with a lifelong history of seasonal rhinoconjunctivitis and honey int... more A 34-year-old Spanish woman with a lifelong history of seasonal rhinoconjunctivitis and honey intolerance (pyrosis and abdominal pain) developed, 3 weeks after starting ingestion of bee pollen, astenia, anorexia, abdominal pain, diarrhoea, peripheral blood hypereosinophilia and elevated serum total IgE levels. A duodenal biopsy showed eosinophilic infiltration of the mucosal layer. Other causes of hypereosinophilia were not found. Repeated parasitological stool studies, as well as a duodenal aspirate showed negative results. Symptoms, hypereosinophilia and elevated IgE levels resolved after bee pollen ingestion was stopped. This is a typical case of eosinophilic gastroenteritis by ingestion of bee pollen in a woman with intolerance to honey bee, because the patient fulfilled the usual diagnostic criteria: gastrointestinal symptoms were present, eosinophilic infiltration of the digestive tract was demonstrated by biopsy, no eosinophilic infiltration of other organs was found and the presence of parasites was excluded. Honey intolerance and/or bee pollen administration should be considered as a cause of eosinophilic gastroenteritis.
The Journal of Allergy and Clinical Immunology, Dec 1, 1984
Inhalation of wood dusts can cause immediate and/or late onset asthma, and the list of woods resp... more Inhalation of wood dusts can cause immediate and/or late onset asthma, and the list of woods responsible for such reactions is increasing. We report two patients with asthma induced by exposure to the dust of African maple wood (Triplochiton scleroxylon). Type I hypersensitivity to this dust was demonstrated by means of immediate skin test reactivity, positive passive transfer test, positive reverse enzyme immunoassay for specific IgE, and an immediate bronchial provocation test response to an African maple-dust extract. Unexposed persons did not exhibit reactivity to this wood in any of the tests listed above.
Several authors have demonstrated that renaturation is an essential step for the appropriate reco... more Several authors have demonstrated that renaturation is an essential step for the appropriate recognition of blotted proteins. The use of nonionic detergents has been described as a useful alternative to enhance the antigenicity in immunoblotting, although elution from proteins by detergents has been observed. To measure the influence of different factors on the sensitivity of specific IgE by immunoblotting, we used twenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Nonidet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing this detergent, even at high concentrations (3%), and its use gave better sensitivity than methanol. On the other hand, we detected that methanol possessed renaturing properties. A transfer method using diffusion instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method, we found that after NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effects.
Hypersensitivity to cockroach antigen has been recognized as an important cause of perennial alle... more Hypersensitivity to cockroach antigen has been recognized as an important cause of perennial allergic rhinitis and asthma. To assess the frequency of cockroach hypersensitivity in our country, 150 asthmatic atopic subjects were studied using skin testing and in vitro assays for cockroach-specific IgE antibodies (Oriental and German cockroaches). Twenty-two of 61 patients who had a positive history of cockroach exposure had positive skin tests, and only 3 of 89 patients who had no history of exposure had positive skin reactions. Of 25 patients with positive skin tests, 23 showed specific IgE antibodies against oriental and German cockroaches using RAST and EIA techniques. In summary, approximately 15% of asthmatic atopics in Madrid area are sensitive to cockroaches (positive skin test + specific IgE antibodies). These results indicate that cockroach hypersensitivity should be considered in every patient with perennial asthma.
Twenty eight workers of a pharmaceutical factory were studied. The company produced morphine and ... more Twenty eight workers of a pharmaceutical factory were studied. The company produced morphine and other alkaloids extracting the active principles from shells of Papaver somniferum. Six of the exposed subjects had clinical symptoms of sensitization to this allergen and positive skin tests. A bronchial provocation test was found to be positive on 4 workers and specific IgE could be found on the 6 sensitized patients by an ELISA and a RAST test using an aqueous extract of P. somniferum. A histamine release using the same antigen was also positive in the 4 samples from sensitized patients available. An SDS-PAGE of the extract revealed a major protein band with an estimated mol wt of 52,000 d. This band had the highest IgE-binding capacity as shown by immunoblotting. All these facts suggest that P. somniferum allergy is mediated by an IgE mediated mechanism and not by a pharmacological or toxic effect of the alkaloids or polyphenols.
Objective: the ingestion of Anisakis simplex larvae may lead to the appearance of gastrointestina... more Objective: the ingestion of Anisakis simplex larvae may lead to the appearance of gastrointestinal symptoms. However, the number of reported cases of parasitization by Anisakis in Spain is lower than would be expected in a country with the second-highest fish consumption per inhabitant in the world, particularly since fish is often eaten raw or only slightly cooked. We suggest that the incidence of anisakiasis in Spain would be higher if complementary studies were used in all patients suspected of having anisakiasis. Methods: we studied 6 patients with a diagnosis of intestinal obstruction who frequently ate fish. Skin prick tests with seafood, inhalant allergen and Anisakis extracts were done. Total and specific IgE against Anisakis larvae were tested with a CAP system radioimmunoassay and immunoblot assays. Oral challenge tests with frozen larvae were also used. Results: a positive skin prick result and high levels of total and specific IgE were found in all patients. The results of immunoblot assays for IgE did not show a consistent pattern, but a group of several low (14-18 kDa) and intermediate molecular weight antigens (30-50 kDa) were found in all patients. All patients tolerated the oral challenge test well. Conclusions: in our patients with intestinal pseudo-obstruction and a history of frequent fish eating, the clinical and laboratory findings were suggestive of parasitization by Anisakis simplex larvae as the cause of the obstruction. Such complementary studies should be used whenever there is a suspicion of anisakiasis. The results of the oral provocation test show that the intake of dead larvae does not induce clinical parasitization.
A familial C1q deficiency of complement in three siblings has been established. The patients were... more A familial C1q deficiency of complement in three siblings has been established. The patients were two brothers and a sister (12, 11 and 9 years old) with clinical and pathological features of Rothmund-Thomson syndrome (Poikiloderma congenital) and mesangial proliferative glomerulonephritis with diffuse IgM deposits. Abnormality has been defined as a total lack of CH50 haemolytic activity, undetectable C1q, failure to correct the defect with functionally pure C2 to C9 complement components, normal values for C2, C3, C4 and C5 and restoration of CH50 haemolytic activity when purified human C1q was added to the assay.
We present eight patients allergic to latex and fruit (chestnut and banana), seven of whom are wo... more We present eight patients allergic to latex and fruit (chestnut and banana), seven of whom are women, and aged 17 to 42 years (mean 25 years). Four had family and five personal atopic histories. The total IgE varied from 41 to 520 Ku/L (mean 263). The symptoms followed ingestion of fruit (anaphylaxis) in four patients and contact with rubber (contact urticaria and anaphylaxis) in the other four. Skin prick test (SPT) with latex and radioallergosorbent test to latex were positive in all the patients. Histamine release (HR) to latex was carried out on six patients and was positive in three. In the six patients with symptoms after having eaten chestnuts the SPT was positive and specific IgE was detected in five of them. Histamine release to chestnuts was positive in three of the six patients tested and one of them (-SPT and + IgE) tolerated the fruit. Two out of five patients with symptomatic banana allergy had negative SPT with banana while the test was positive in one patient who tolerated this fruit, this being the only case with specific IgE to banana. Histamine release with banana was only positive in one case. The important correlation between SPT, RAST, and HR results to latex and chestnut together with the total inhibition of the chestnut RAST with a serum pool by preincubation with latex suggests cross-reactivity among these allergens.
Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful... more Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97-1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 microg/ml mite extract. Analysis of the data obtained with 1.6 microg/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91-1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non-allergic children produced a cut-off of 8% activated basophils (AUC = 1.0) with 16 microg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 microg/ml extract (AUC = 97%, 95% CI = 0.92-1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.
Molecular and Biochemical Parasitology, Aug 1, 2008
Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnos... more Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnosis usually leads to the dietary recommendation of avoiding fish and seafood consumption. The protein Ani s 4 is a clinically relevant allergen due to its heat and pepsin resistant properties and its importance in the anaphylaxis process. The attempt of cloning Ani s 4 has led to the identification and characterisation of a new isoform that differs only in one amino acid with the previously published. This isoform was produced as an His tagged recombinant protein and its allergenic properties were tested by IgE immunoblot and by a flow cytometry basophil activation test. The results were compared to the allergenic properties of the isoform previously described. Both isoforms of Ani s 4 showed different capacities to bind IgE from sensitised patients and different potencies in the basophil activation test.
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