Ricardo Palacios

Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen

BACKGROUND Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America. OBJECTIVE To clone olive allergens and to characterize immunologically the purified recombinant allergens. METHODS Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced. Recombinant proteins were produced in Escherichia coli by the use of two different expression systems. Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive. RESULTS Significant sequence polymorphism was found in both allergens. The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column. One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1. The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin. Both recombinant allergens shared B cell epitopes with the corresponding natural allergens. CONCLUSION IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes.

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Sensitization to Sunflower Pollen: Only an Occupational Allergy?

Sunflower (Helianthus annuus) pollen sensitization has been reported as an occupational allergy. In this report, the sensitization of the general population living in sunflower-growing areas to Helianthus pollen was studied. Both RAST results in 32 adults with summer symptoms previously diagnosed as allergic to Artemisia pollen, and cross-reactivity studies between H. annuus and other Compositae suggested that H. annuus pollen was the main allergen involved in the hypersensitivity reaction of those patients. Good correlation was found between RAST and SPT to Helianthus and between RAST and conjunctival provocation test to Helianthus. Bronchial challenge tests performed on 8 of the 32 patients confirmed the clinical implication of Helianthus pollen in suspected subjects. Five workers, handling sunflower pollen, who suffered from related symptoms were subjected to the same study, showing lesser wheal areas and lesser specific IgE levels than a non-worker group. Thirteen patients with RAST values > or = class 2 showed 2 IgE-binding fractions at 34.0 and 42.8 kD in 65% of sera and 3 IgE-binding fractions at pI 4.9, 9.6 and 10.2 in 54% of sera. By means of micropreparative high-resolution chromatography, it was possible to purify a 34-kD major allergen. Analysis performed by RAST inhibition with sera from atopic patients and ELISA inhibition with experimental anti-Helianthus rabbit sera demonstrated a cross-reactivity between Helianthus and other Compositae, but low affinity of specific anti-Helianthus antibodies for heterologous antigens. Taking into account the above-mentioned data, and the high prevalence of Helianthus pollen in the atmosphere during harvesting (in spite of its entomophilous character), Helianthus pollen should be considered as an allergenic source to be investigated in the general population living in sunflower-growing regions suffering from seasonal summer allergy.

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin)

BACKGROUND Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Pollen allergy in peach-allergic patients : Sensitization and cross-reactivity to taxonomically unrelated pollens

BACKGROUND Fruit allergy has been attributed to cross-reactive IgE to pollens and has been associated with a particular pollen sensitization. OBJECTIVE The aim of the study was to evaluate sensitization to several taxonomically unrelated pollens in peach- and pollen-allergic patients and to study cross-reactivity between them. METHODS One hundred sixty-five patients were evaluated: 70 peach- allergic patients together with 95 pollen-allergic patients (control group). Pollen skin tests in duplicate were performed to 5 grasses, 8 trees, and 7 weeds. Cross-reactivity between peach and taxonomically diverse pollens was determined by radioallergosorbent inhibition and Western blot inhibition tests. Experiments were also carried out after preadsorption of the sera with purified natural profilin. RESULTS The skin test results revealed that peach-allergic patients frequently reacted to most pollens-grasses, weeds, and trees-even when some of these are not found in our geographic area. There was a statistically significant increase in sensitization frequency to most trees and weeds, with a statistically higher occurrence of asthma (odds ratio 2.98, 95% confidence interval 1.46-6.09). Inhibition test results provided evidence that taxonomically unrelated grasses, weeds, and trees produced various and substantial degrees of inhibition in specific IgE to peach and that the peach extract elicited strong inhibitions to those pollens. Profilin was found to be a relevant cross-reactive antigen in these patients. CONCLUSION The results of this study provide evidence that peach allergy is linked to sensitization to several taxonomically unrelated pollens. This is attributable to the ubiquitous nature of the IgE binding determinants-such as profilins-between peach and taxonomically unrelated pollens.

An Observational Study on Outgrowing Food Allergy during Non-Birch Pollen-Specific, Subcutaneous Immunotherapy

Background: Birch pollen-specific immunotherapy (SIT) decreases allergy to foods containing birch pollen-homologous allergens. Cross-reactivity was also observed between plane tree pollen and some vegetable foods. Objective: The aim of this study was to evaluate the outgrowing of food allergy by patients suffering from vegetable food allergy associated with plane tree pollinosis (rhinoconjunctivitis and/or asthma) during plane tree pollen SIT. Methods: An observational and prospective study was conducted in 16 adult patients suffering from vegetable food allergy (hazelnut, walnut, lettuce, peach and cherry) and from plane tree pollinosis receiving plane tree pollen SIT for 1 year. Open oral challenges with the implicated food were performed before and after SIT. Blood samples were drawn for measurement of pollen- and food-specific IgE and IgG4 before and after treatment. Results: Plane tree SIT resulted in a significant decrease in food allergy, since the mean food quantity provoking objective symptoms increased from 2.19 to 13.74 g (p < 0.05), and 6 of the 11 patients tolerated the highest level (25 g) of the challenged food after plane tree SIT. Laboratory data also showed a decrease in IgE levels and an increase in IgG4 levels after immunotherapy. Conclusion: SIT with plane tree pollen has a positive impact on food allergy in plane tree pollen-allergic subjects.

Sequence polymorphism and structural analysis of timothy grass pollen profilin allergen (Phl p 11)

Three cDNA clones encoding timothy grass pollen profilin (Phl p 11) were newly isolated. Comparison of the sequences of four cDNA clones, including a previously isolated clone, showed a low level of polymorphism. Isoelectrofocusing of highly purified timothy grass profilin indicated the existence of at least five isoforms. One recombinant profilin showed similar immunological properties to natural timothy grass profilin. Tertiary structure of Phleum pratense profilin was obtained by homology-based molecular modeling.

Occupational allergy caused by carnation (Dianthus caryophyllus)

BACKGROUND Occupational respiratory symptoms caused by decorative flowers are seldom reported in the literature. In our area a large portion of the population works in carnation (Dianthus caryophyllus) winter quarters, and many workers have symptoms of rhinitis and asthma related to exposition. OBJECTIVE The purposes of this study were to investigate whether the symptoms induced by carnation were IgE-mediated and to study the possible allergens involved. METHODS A total of 16 subjects employed in indoor carnation cultivation with symptoms during exposition time were studied along with 15 patients with allergic asthma who were not exposed to carnations and 15 healthy carnation workers used as control subjects. Skin prick tests with carnation extract and RASTs were performed. Protein bands were isolated by SDS-PAGE, and afterwards immunoblotting was performed to characterize the extract. Specific nasal provocation and nonspecific bronchial provocation tests were performed for all the asthmatic patients. Diurnal variation in peak expiratory flow was also measured. RESULTS Skin prick test responses with carnation extract were positive in 15 of the 16 patients and negative in all control subjects. Nasal provocation test responses with carnation extract were positive in 13 of 16 patients. A significant correlation was seen between RAST and nasal provocation results (P <.01). Immunoblotting of sera from 13 patients showed 2 major IgE-binding fractions of 34 and 35 kd in most of the patients, which could constitute the major allergen. Methacholine PD20 showed a variable degree of nonspecific bronchial hyperresponsiveness in all asthmatic subjects. CONCLUSION Data demonstrate the involvement of carnation in occupational allergy, mediated by an IgE-dependent mechanism.

Allergen immunotherapy decreases LPS-induced NF-κB activation in neutrophils from allergic patients

Allergen‐specific immunotherapy (IT) is widely used to treat allergic diseases. The molecular mechanisms have not been clarified yet completely. The present work was undertaken to analyze the effect of IT in the activation of NF‐κB.

Rhinoconjunctivitis and asthma caused by vine pollen: A case report

BACKGROUND The vine (Vitis vinifera) is a cultivated plant that is found in some European and American countries. Its pollen gathers in small quantities during a short pollination period in the months of May and June. Allergy to vine pollen has not been previously documented. OBJECTIVE We sought to describe a case report of allergy to vine pollen documented on the basis of anamnesis, cutaneous, provocation, and specific IgE determination tests. METHODS An allergenic extract was obtained from collected V vinifera pollens by aqueous standard procedures. Pollen counts and pollination periods of this and other common pollens in the area where the patient became symptomatic were studied. Cutaneous tests and the presence of specific IgE to the pollen extracts were performed by prick, CAP, and RAST techniques. Bronchial and conjunctival tests with the involved pollen extracts were also carried out to identify the sensitizing allergens. Five healthy subjects and 5 pollinic patients were used as control subjects and underwent the same tests. RESULTS Skin prick test responses with vine pollen at different concentrations were positive for the studied patient and negative for the control subjects. Patient serum revealed a total IgE titer of 334 IU/mL and a specific IgE value of 1.3 PRU/mL (RAST class 2) to vine pollen. Bronchial and conjunctival provocation test responses were also positive when the patient was challenged with V vinifera extract. CONCLUSION Exposure to the pollen of the vineyard plants (V vinifera) can induce immunologic sensitization and rhinoconjunctivitis/asthma.