Design and evaluation of a hypoallergenic peptide-based vaccine for Salsola kali allergy.

BACKGROUND: The Salsola kali (S. kali) pollen is one of the most important causes of allergic rhinitis in the deserts and semi-desert areas. Immunotherapy with allergen extracts remains the only available treatment addressing the underlying mechanism of allergy. However, given the low efficacy of this method, it is necessary to find more effective and alternative therapeutic interventions using molecular biology and bioinformatics tools. In this study, a hypoallergenic vaccine was designed on the basis of B-cell epitope approach for S. kali immunotherapy. METHODS: Using the Immune Epitope Database (IEDB), a 35-mer peptide was selected and chemically conjugated to a keyhole limpet hemocyanin (KLH) molecule. Specific IgG and IgE from immunized BALB/c mice sera against the vaccine (Sal k 1-KLH), S. kali extract and the recombinant protein, rSal k 1, were measured using ELISA. Also, inhibition of IgE by mouse IgG was evaluated using an inhibitory ELISA. Finally, the IgE reactivity and T-cell reactivity of the designed vaccine were evaluated by dot blot assay and MTT assay. RESULTS: Vaccination with the vaccine produced high levels of protective IgG in mice, which inhibited the binding of patients IgE to recombinant proteins. The result showed that the designed vaccine, unlike the recombinant protein and extract, did not induce T-cell lymphocytes response and also exhibited decreased IgE reactivity. CONCLUSION: The designed vaccine can be considered as a promising candidate for therapeutic allergen-specific immunotherapy.

A Hypoallergenic Polygalacturonase Isoform from Olive Pollen Is Implicated in Pollen-Pollen Cross-Reactivity.

BACKGROUND: Cross-reactivity reactions between allergenic polygalacturonases (PGs) from different biological sources, especially foods and pollens from the Oleaceae family, have been described using Salsola kali PG (Sal k 6). No PG from olive pollen has been characterized to date, hampering further knowledge about cross-reactions through PGs. OBJECTIVES: The aim of this work was to determine the potential allergenicity of the PG from olive pollen and clarify its role in cross-reactivity. METHODS: A cDNA-encoding olive pollen PG sequence was subcloned into the pET41b vector and used to transform BL21(DE3) Escherichia coli cells to produce a His-tag fusion recombinant protein. The allergenic properties of olive pollen PG were determined by immunoblotting and ELISA in comparison to Sal k 6. The cross-reactivity potential of the protein with other pollen sources was analyzed by inhibition immunoassays. RESULTS: The existence of other isoforms of Ole e 14 with different allergenicity was confirmed by proteomics and a meta-analysis of the recently reported olive genome. Sal k 6 showed a higher IgE recognition than Ole e 14 regardless of patient sensitization, suggesting the existence of more allergenic Ole e 14 isoforms in olive pollen. IgG and IgE inhibition assays supported the existence of cross-reactions between them and with other PGs from Oleaceae and Poaceae plant families. CONCLUSIONS: A new allergen from olive pollen, Ole e 14, has been identified, produced as a recombinant isoform, and structurally and immunologically characterized. Its role in cross-reactivity has been confirmed and, due to its smaller IgE binding capacity, it could have an important role for therapeutic purposes.

Extensive Cross-reactivity Between Salsola kali and Salsola imbricata.

BACKGROUND: There are no studies on cross-reactivity between Salsola kali and Salsola imbricata pollens. The main goals of the present study were to compare the degree of the cross-reactivity between S kali and S imbricata and to compare the various allergenic components shared by S kali and S imbricata. METHODS: erum samples were obtained from rhinitis patients with or without asthma living in Kuwait and presenting with a positive skin test result to S kali. SDS-PAGE/IgE Western blot and ELISA inhibition assay were performed. RESULTS: The study population comprised 37 patients. The most frequent IgE proteins against S imbricata weighed around 12, 15, 18, 37, and 50+55 kDa. 2D electrophoresis revealed a correlation between S kali and S imbricata at 40, 60, and 75 kDa, with similar isoelectric points. ELISA inhibition revealed an Ag50 value of 1.7 µg/mL for S kali and 500.5 µg/mL for S imbricata when the solid phase was S kali and an Ag50 value of 1.4 µg/mL for S kali and 3.0 µg/mL for S imbricata when the solid phase was S imbricata. CONCLUSIONS: ELISA inhibition revealed strong cross-reactivity between S kali and S imbricata. This finding might be clinically relevant for the efficacy of allergen-specific immunotherapy. We report, for the first time, the allergenic profile of S imbricata and potentially allergenic proteins for S kali and S imbricata.

Tolerability and immunological effect of short up-dosing immunotherapy with 2 standardized native allergen extracts derived from the pollen of Salsola kali and Cupressus arizonica

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A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis.

BACKGROUND: The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Allergens of weed pollen: an overview on recombinant and natural molecules.

Weeds represent a botanically unrelated group of plants that usually lack commercial or aesthetical value. Pollen of allergenic weeds are able to trigger type I reactions in allergic patients and can be found in the plant families of Asteraceae, Amaranthaceae, Plantaginaceae, Urticaceae, and Euphorbiaceae. To date, 34 weed pollen allergens are listed in the IUIS allergen nomenclature database, which were physicochemically and immunologically characterized to varying degrees. Relevant allergens of weeds belong to the pectate lyase family, defensin-like family, Ole e 1-like family, non-specific lipid transfer protein 1 family and the pan-allergens profilin and polcalcins. This review provides an overview on weed pollen allergens primarily focusing on the molecular level. In particular, the characteristics and properties of purified recombinant allergens and hypoallergenic derivatives are described and their potential use in diagnosis and therapy of weed pollen allergy is discussed.

Identification of methionine synthase (Sal k 3), as a novel allergen of Salsola kali pollen.

Salsola kali pollen is a common cause of pollinosis during summer and early fall in desert and semi-desert regions. The aim of this study was the identification and characterization of Sal k 3, a new allergen from S. kali pollen. S. kali pollen extract was fractionated by SDS-PAGE and the allergenic profile was determined by IgE-immunoblotting using twelve S. kali allergic patients. Protein identification was carried out by the means of mass spectrometry. Using degenerated primers, two DNA fragments encoding N- and C-terminal domain of Sal k 3 were amplified by PCR, then cloned into the PTZ57R/T vector and sequenced. The open reading frame of Sal k 3 fragments were subcloned in the pET-32b(+) vector, expressed in E. coli, and purified by Ni2+ affinity chromatography. The IgE-binding capacity of rSal k 3 fragments was then studied by IgE-immunoblotting, inhibition assays, and skin prick tests. A 45-kDa allergen was identified as a fragment of the cobalamin-independent methionine synthase (MetE) by mass spectrometry and was detected in the sera of 8/12 (66.6%) of S. kali allergic patients. Moreover, inhibition assays demonstrated that the purified rSal k 3 fragments were similar to their counterparts in the crude extract. Sal k 3 represents a new allergen of S. kali pollen and seems to be an important allergenic compound in S. kali pollen.

Sal k 4, a new allergen of Salsola kali, is profilin: a predictive value of conserved conformational regions in cross-reactivity with other plant-derived profilins.

The aim of this study was to investigate a new allergen of Salsola kali, Sal k 4, and to investigate the predictive value of the conserved conformational regions in cross-reactivity with other plant-derived profilins. The Sal k 4-coding sequence was cloned, expressed, and purified by one-step Ni2+ affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted conserved conformational regions among rSal k 4 and other plant-derived profilins. Immunodetection and inhibition assays using 30 individual sera from S. kali allergic patients indicated that purified rSal k 4 might be the same as that in the crude extract. The results of inhibition assays among rSal k 4 and other plant-derived profilins were in accordance with the homology of the predicted conserved conformational regions. Amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins might depend on the predicted conserved conformational regions.

Allergenic differences among pollens of three Salsola species.

BACKGROUND: Sensitivity to Chenopodiaceae is a frequent cause of allergic respiratory diseases in geographic areas where sensitization to Salsola kali and Chenopodium album has been reported. The objective of this study was to evaluate the pattern of sensitization to 3 Salsola species in patients residing on the Mediterranean coast of south-eastern Spain. METHODS: S. kali, S. vermiculata and S. oppositifolia pollen extracts were prepared. Patients reporting respiratory and/or cutaneous symptoms were skin prick tested with the 3 Salsola extracts. Individuals with positive skin prick tests to at least 1 of the 3 Salsola species were included. Specific IgE was determined by direct ELISA. SDS-PAGE and 2-D analysis were conducted to elucidate the protein profile. The allergenic profile was investigated by immunoblot. Inhibition experiments were conducted to establish cross-reactivity between different species. RESULTS: 246 patients were included. 237 patients (96.3%) tested positive to S. oppositifolia, 189 (76.8%) to S. kali and 185 (75.2%) to S. vermiculata. Protein profile and immunoblot demonstrated similar patterns in all extracts, except in low-molecular-weight allergens of S. oppositifolia. Immunoblot inhibition experiments demonstrated that most high-molecular-weight allergens of S. oppositifolia were inhibited by S. kali whereas low-molecular-weight allergens were totally inhibited only by C. album. CONCLUSIONS: This study confirms the allergenic importance of other Salsola species, especially S. oppositifolia. We have demonstrated that the 3 species show a high degree of cross-reactivity, but S. oppositifolia shares more allergenic similarities with C. album than S. kali.

In vitro cross-reactivity between tomato and other plant allergens.

BACKGROUND: Cross-reactivity among fruits and different pollen and fruit species has been extensively reported. OBJECTIVES: To investigate the in vitro cross-reactivity between tomato and pollen, fruit, and latex extracts and to identify the proteins involved. METHODS: A serum pool was prepared from 18 individuals residing on the Spanish Mediterranean coast (9 men and 9 women; mean [SD] age, 27.4 [10.1] years) who had positive skin prick test reactions to tomato peel. Extracts from 10 pollens, 12 fruits, and latex were tested. Levels of specific IgE to each extract were measured. The allergenic profile was evaluated by means of immunoblot. The percentage of inhibition between extracts and tomato peel extract was analyzed by means of CAP inhibition, and the allergens implicated were elucidated by immunoblot inhibition. RESULTS: For pollens, the highest specific IgE values were obtained for grasses. Most pollen extracts showed a capacity of inhibition similar to that of tomato peel extract; high percentages were obtained with Artemisia vulgaris and Poa pratensis. The most strongly inhibited allergens in tomato corresponded to bands of 32 and 45 kDa. For fruits, the highest value of specific IgE was detected for peach. High percentages of inhibition were obtained with peach and hazelnut. No inhibition was detected with latex. Peach, chestnut, and melon inhibited high molecular weight bands (32 and 45 kDa) and a band of approximately 10 kDa. CONCLUSIONS: Cross-reactivity between tomato and pollen and fruit extracts has been demonstrated. Allergens with a high molecular weight range seem to be responsible in pollen extracts. A 10-kDa band seems to be responsible in Platanus acerifolia, Salsola kali, peach, chestnut, and melon.

Allergy to Salsola Kali in a Salsola incanescens-rich area: role of extensive cross allergenicity.

BACKGROUND: Pollens from the Salsola spp. are an important source of respiratory allergy in tropical countries. Our aim was to characterize the IgE binding proteins of S. incanescens pollen extract and study its cross-reactivity with S. kali pollen allergens. METHODS: Prick tests with S. kali and S. incanescens pollen extracts were performed on eight respiratory allergy patients from Mashhad, Northeast Iran. The antigenic profiles and IgE-binding patterns of S. kali and S. incanescens pollen extracts were compared by SDS-PAGE and Western blotting, using individual sera from the salsola pollen-sensitive patients. Cross-reactivity of proteins in the two weeds was assessed by IgE- immunoblotting inhibition. RESULTS: S. kali and S. incanescens pollen extracts showed similar IgE-binding profiles in Western blotting. The IgE binding components of 39, 45, 66 and 85 kDa were detected in both pollen extracts. Furthermore, inhibition of the immunoblots revealed extensive inhibition of IgE binding to proteins and a close relationship between these two weeds allergens. CONCLUSIONS: S. incanescens pollen is a potent allergen source with several IgE binding components that shows a close allergenic relationship with S. kali. Our results suggest that in S. incanescens-rich areas, S. kali pollen extracts could be used as a diagnostic reagent for allergic patients to S. incanescens pollen.

A pectin methylesterase as an allergenic marker for the sensitization to Russian thistle (Salsola kali) pollen.

BACKGROUND: Chenopodiaceae pollen is considered the main cause of pollen allergy in desert countries and its incidence is world-wide increasing by the desertization of extensive zones. Although the correlation between the sensitization to Chenopodium album and Salsola kali pollens of patients suffering from allergy to Chenopodiaceae pollens is high, a significant number of patients exhibited IgE sensitivity exclusively towards S. kali. OBJECTIVE: To analyse this differential reactivity and to purify, clone and characterize the putative responsible allergen. METHODS: Immunoblotting was used to analyse the IgE binding to pollen extract for S. kali and C. album. The protein was isolated by two chromatographic steps and characterized by Edman degradation, mass spectrometry, finger print analysis and Concanavalin A lectin staining. Specific cDNA was amplified by polymerase chain reaction, cloned in Escherichia coli and sequenced. Immunologic characterization was performed by immunoblotting, enzyme-linked immunoassay detection and inhibition experiments using sera from 11 patients allergic to S. kali pollen. RESULTS: cDNA codifies for a mature protein of 339 amino acids plus a putative signal peptide of 23 residues and it belongs to the plant pectin methylesterase (PME) family. It is a mildly basic and polymorphic protein and was recognized by the IgE from all the patients allergic to S. kali included in the study, and was called Sal k 1. The protein was not recognized in the C. album pollen extract using the sera of these patients. CONCLUSION: Sal k 1 is a protein from the PME family with a high allergenic relevance. Considering this allergen as responsible for the different sensitization between S. kali and C. album pollen, it may be a useful marker to classify patients allergic to Chenopodiaceae allowing a safer and more specific immunotherapy.

Double-blind, placebo-controlled study with a modified therapeutic vaccine of Salsola kali (Russian thistle) administered through use of a cluster schedule.

BACKGROUND: The inhalation of Salsola kali pollen is a common cause of respiratory diseases in Europe and North America. OBJECTIVE: To evaluate the efficacy and safety of a depigmented and glutaraldehyde-polymerized therapeutic vaccine of S kali. METHODS: The trial was randomized, double-blind, and placebo-controlled using a rush protocol in the build-up phase. Sixty patients with rhinoconjunctivitis (19 also had mild asthma) were randomly allocated to receive either active treatment (polymerized extract) or placebo. The final distribution was 41 patients in the active and 19 in the placebo group. Side effects were registered. Symptom and medication scores and the number of days free of symptoms during the pollen season were assessed to evaluate the clinical efficacy. A Rhinoconjunctivitis Quality of Life Questionnaire was completed in the previous pollen season (before treatment) and during the pollen season 1 year later (in the trial). Dose-response skin tests were performed at baseline and at the end of the trial. RESULTS: There was a significant difference (P < .05) in symptom and medication scores between both groups during the pollen season, with the active group the one that had fewer symptoms and lower intake of medication. The number of days without symptoms was higher in the active group (P < .05). This group also had a significant improvement in the Rhinoconjunctivitis Quality of Life Questionnaire and a reduction in skin sensitivity. No moderate or severe systemic reactions were registered. CONCLUSION: Immunotherapy with this modified vaccine of S kali pollen is safe and efficacious to treat patients clinically sensitive to this pollen. CLINICAL IMPLICATIONS: Patients allergic to S kali (Russian thistle) can be successfully treated with immunotherapy to improve symptoms of allergic rhinitis and asthma, reduce medication use, and improve quality of life parameters.

Tolerance of a Salsola kali extract standardized in biological units administered by subcutaneous route. Multicenter study.

BACKGROUND: Sensitivity to Salsola kali is a frequent cause of allergic respiratory disease in various regions of Spain. However, there are very few articles in which this allergen has been studied. METHODS AND RESULTS: In order to evaluate the tolerance of this extract, a prospective study has been performed. This study was observational, multi-centred and open, involving 88 patients with allergic respiratory disease due to sensitivity to Salsola, aged between 5 and 52 years. The administration of the extract was performed subcutaneously, through one of two treatment schedules: cluster (8 doses in 4 visits) or conventional (13 doses in 12 visits). A total of 42 adverse reactions were registered, in 26 patients (35 local reactions in 21 patients and 7 systemic reactions in 6 patients). Among the 7 systemic reactions, 4 were registered with the cluster protocol and 2 with the conventional protocol (p = 0.329). In no patients were serious adverse reactions registered. CONCLUSION: The subcutaneous administration of a Salsola extract is safe and well tolerated, both when administered using a conventional schedule and when using a cluster schedule.

Tolerance of a Salsola kali extract standardized in biological units administered by subcutaneous route. Multicenter study

Background: Sensitivity to Salsola kali is a frequent cause of allergic respiratory disease in various regions of Spain. However, there are very few articles in which this allergen has been studied. Methods and Results: In order to evaluate the tolerance of this extract, a prospective study has been performed. This study was observational, multi-centred and open, involving 88 patients with allergic respiratory disease due to sensitivity to Salsola, aged between 5 and 52 years. The administration of the extract was performed subcutaneously, through one of two treatment schedules: cluster (8 doses in 4 visits) or conventional (13 doses in 12 visits). A total of 42 adverse reactions were registered, in 26 patients (35 local reactions in 21 patients and 7 systemic reactions in 6 patients). Among the 7 systemic reactions, 4 were registered with the cluster protocol and 2 with the conventional protocol (p = 0.329). In no patients were serious adverse reactions registered. Conclusion: The subcutaneous administration of a Salsola extract is safe and well tolerated, both when administered using a conventional schedule and when using a cluster schedule

Antecedentes: La sensibilización a Salsola kali es una causa frecuente de enfermedad alérgica respiratoria en varias zonas de España. Sin embargo, apenas existen publicaciones en las que se estudie este alergeno. Métodos y resultados: Para valorar la tolerancia de este extracto, se ha realizado un estudio prospectivo, observacional, multicéntrico y abierto, en el que se han incluido 88 pacientes, de edad entre 5 y 52 años, con enfermedad alérgica respiratoria por sensibilización a Salsola. La administración del extracto se ha realizado por vía subcutánea, mediante dos esquemas de tratamiento: agrupada (8 dosis en 4 visitas) o convencional (13 dosis en 12 visitas). Se han registrado un total de 42 reacciones adversas en 26 pacientes (35 locales en 21 pacientes y 7 sistémicas en 6 pacientes). De las 7 reacciones sistémicas, 4 se registraron con la pauta agrupada y 2 con la convencional (p = 0,329). No se registró ninguna reacción adversa grave. Conclusión: La administración subcutánea de un extracto de Salsola es segura y bien tolerada, tanto cuando se administra con una pauta convencional como con una pauta agrupada

Correlation between Chenopodiacea/Amaranthacea pollen counts and allergic symptoms in Salsola kali monosensitized patients.

UNLABELLED: We performed a prospective observational study to establish a relationship between pollen counts of Chenopodiacea/Amaranthacea and clinical symptoms of rhinoconjunctivitis and asthma in a group of monosensitised patients. MATERIAL AND METHODS: A total of 60 patients (19 with asthma) were included in the study. All patients collected daily symptom scores during the summer months of 1999, 2000 and 2001. The questionnaire included ocular, nasal and pulmonary symptoms. Pollen counts were expressed as pollen grains/m3. Symptom scores and pollen counts were correlated using correlation coefficients and Log transformed variables. RESULTS: In the 3 seasons studied we identified a peak of pollen and clinical symptoms in the second half of August and first half of September. In 1999, there was a significant positive correlation between total symptoms and daily pollen grains/m3 (p<0.005, r = 0.347). This correlation was not significant for the summers of 2000 and 2001. After further analysis, and by displacing one of both variables between 11 to 17 days, the correlation coefficients for total symptoms, improved for 1999 (r = 0. 744; p < 0.0001) and became significant for 2000 (r = 0. 521; p < 0.0001) and 2001 (r = 0.635; p < 0.0001). CONCLUSION: We identified a significant time lag between pollen counts and symptom scores in S. kali monosensitized patients.

Salsola pollen as a predominant cause of respiratory allergies in Kuwait.

BACKGROUND: Respiratory allergies are common in Kuwait, and the role of certain allergens has been previously documented. OBJECTIVE: To evaluate the results of skin prick tests to a range of allergens that were considered relevant to the vegetation surveys and aerobiological studies performed in Kuwait. METHOD: New patients attending our center during August 2002 to February 2003 with asthma or allergic rhinitis underwent skin prick tests to a battery of allergens. RESULTS: A total of 451 patients aged 5 to 60 years (mean age, 29.5 years) were tested. Of these patients, 403 (89.4%) had a positive test result to at least one allergen and were considered allergic. A total of 76.7% of the allergic patients had a positive reaction to Salsola pollen, with a mean wheal diameter of 8.25 mm (median, 8 mm). Chenopodium album was positive in 57.6% and Bermuda grass was positive in 38.2% of the allergic cases. Indoor allergens seemed to play a lesser role than pollens: Dermatophagoides pteronyssinus was positive in only 37.5%, and American and German cockroaches were positive in 33.2% and 22.3%, respectively. All the allergens other than Salsola elicited a mean wheal diameter of less than 6.25 mm (median, < or = 6 mm). CONCLUSIONS: Indoor allergens seem to play a lesser role in respiratory allergies in Kuwait. Most allergic patients become sensitized to pollens; the strongest and most frequent reaction is from Salsola pollen. Salsola imbricata is found growing extensively in most areas of the country, flowering mainly in autumn, when the most common pollen is of the Chenopod-Amaranth type and when most patients with seasonal allergic rhinitis become symptomatic.

Immunochemical characterization of Russian thistle (Salsola kali) pollen extracts. Purification of the allergen Sal k 1.

BACKGROUND: Salsola kali (Russian thistle) is a weed which belongs to the Chaenopodiacea family. It is widely distributed along the coasts of Europe, North Africa, USA and Australia. The objectives of this study were to study the allergenic composition of S. kali pollen and to purify an important allergen from the pollen extracts of this plant. METHODS: A population of 66 individuals with specific IgE-mediated allergic symptoms and positive skin tests to S. kali were included in the study. Specific IgE to S. kali was determined by direct enzyme-linked immunosorbent assay (ELISA). The antigenic and allergenic profile of S. kali was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF) and immunoblot. Allergen purification was conducted by preparative SDS-PAGE. The allergenicity of the protein was evaluated by skin testing, direct ELISA, ELISA inhibition and immunoblots. RESULTS: Specific IgE to S. kali was detected in 39 of the 66 individuals (59%). An allergen with a molecular weight of approximately 43 kDa was purified. This allergen was termed Sal k 1. A partial sequencing was obtained and no homology was found with other known proteins/allergens. The allergenicity of Sal k 1 was tested in vitro and in vivo. Of the 39 individuals with a positive specific IgE determination to S. kali, 26 (66.6%) had detectable specific IgE to Sal k 1. Twenty of these 39 individuals were skin-prick tested with the purified allergen (0.5 mg/ml) and all of them had a positive skin test to the purified allergen. Ten additional individuals, used as negative controls, had a negative response. CONCLUSIONS: Sal k 1, an important allergen of S. kali, is recognized, in vitro, by approximately 67% of the patients sensitized to S. kali. Twenty patients with a positive skin test to a standardized S. kali extract had a positive reaction to the purified allergen.