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Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hey b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hey b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hey b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hey b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hey b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hey b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hey b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/ IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

Allergy to pomegranate is often associated with severe symptoms. Two allergens have previously been described: 9k-LTP Pun g 1 and pommaclein Pun g 7. This study describes the isolation of a chitinase III, identified by direct protein sequencing and mass spectrometry. It is a 29-kDa protein showing 69% sequence identity with the latex hevamine and IgE binding in dot blotting, immunoblotting and FABER(center dot) test. Chitinase-specific IgE were detected in 69 of 357 patients sensitized to one or more pomegranate allergenic preparations present on the FABER(center dot) test. Using this test, 19.2% of the patients sensitized to kiwifruit chitinase IV were also sensitized to pomegranate chitinase III, rather than to latex chitinase I (7.2%) with which it shares the N-terminal hevein-like domain. In conclusion, a new allergen has been identified, contributing to improving food allergy diagnosis. This study reveals the important role of chitinases III and IV as allergy sensitizers and prompts further investigations.

Kiwifruit is an important cause of allergy. Although several allergens have been identified in this food ( www.allergome.org ), data on the number and clinical relevance of kiwifruit allergenic proteins are still fragmentary. With the aim of providing a contribution to the description of the complete kiwifruit allergome, we have investigated the immunological features of pectin methylesterase (PME) and its inhibitor (PMEI) purified from the natural source. Specific IgE to PME (Act d 7) and PMEI (Act d 6) were recorded in patients with kiwifruit allergies by immunoblotting, multiplex microarray-based immunoassay and skin testing (ST). Compared to the results obtained by the microarray system, the percentage of subjects detected positive to Act d 6 was much higher in the immunoblotting. Conversely, a similar percentage of subjects positive to Act d 7 was estimated in both the in vitro tests. Few subjects compared to in vitro testing gave a positive response on ST. In the case of Act d 7 this could be ascribed to the glycan side chain recognized by cross-reactive carbohydrate determinants (CCD)-specific IgE. In fact, Act d 7 fell in the cluster of glycoallergens (including Api m 1, Cup a 1, Hor v 17, Ole e 1, Pla a 2) immobilized on the microarray and recognized by CCD-specific IgE. A comparative analysis of different kiwifruit allergens (Act d 1, 2, 5, 6, 7, 11) revealed that only Act d 7 fell in this cluster, whereas Act d 2, although reported to be glycosylated, fell outside, thus suggesting the lack of sugars bound to this molecule. The subsequent biochemical characterization confirmed that Act d 2 is not a glycoallergen. In conclusion, Act d 7 and 6 seem to be rarely involved in causing symptoms in kiwifruit allergic patients. Nevertheless they are useful to understand a single patient profile and the mechanism of protein allergenicity. In addition, in this study the use of a multiplex test system has provided more information than expected, thus contributing to both the immunological and biochemical characterization of the immobilized allergens.

Diagnostic tests to detect allergic sensitization were introduced at the end of the nineteenth century but only in the late 1990s did the advent of molecular allergology revolutionize the approach to the allergic patient. Personalized Medicine, a medical procedure that separates patients into different groups with different medical decisions, practices and interventions has sanctioned this change. In fact, in the last few years molecular allergology and the observation that not every patient has the same allergic profile, even when allergic to the same allergenic source, has originated the concept “one size does not fit all”. This new approach requires the identification of still unknown allergens, but also the more detailed investigation of those already known. In depth studies of the structure–function relationships in allergenic molecules can reveal the structural determinants involved in the IgE-binding. Then, the knowledge of the epitope profile of each allergen and of the environmental/experimental conditions affecting the exposure of IgE-binding epitopes can provide important contributions to the understanding of cross-reaction processes and to the improvement of diagnosis, immunotherapy and the overall patient treatment. The evolution of diagnostic systems cannot ignore these new needs in this field.

To cite this article: Schulten V, Nagl B, Scala E, Bernardi ML, Mari A, Ciardiello MA, Lauer I, Scheurer S, Briza P, Jurets A, Ferreira F, Jahn-Schmid B, Fischer GF, Bohle B. Pru p 3, the nonspecific lipid transfer protein from peach, dominates the immune response to its homolog in hazelnut. Allergy 2011; 66: 1005–1013. Abstract Background: Nonspecific lipid transfer proteins (nsLTPs) are important food allergens. Often, patients allergic to the nsLTP in peach suffer from allergy to hazelnuts. We aimed to analyse the T-cell response to Cor a 8, the nsLTP in hazelnut and its immunological cross-reactivity with the nsLTP in peach, Pru p 3. Methods: Cor a 8-reactive T-cell lines (TCL) established from patients allergic to hazelnut and peach were stimulated with 12-mer peptides representing the complete amino acid sequence of Cor a 8 to identify its T-cell-activating regions and with Pru p 3 to investigate cellular cross-reactivity. T-cell clones specific for different major T-cell-activating regions of Pru p 3 were stimulated with Cor a 8. Both nsLTPs were subjected to endolysosomal degradation assays. Immunoglobulin E (IgE) cross-reactivity between Cor a 8 and Pru p 3 was assessed in inhibition enzyme-linked immunosorbent assay. Results: No major T-cell-activating region was found among 26 T-cell-reactive peptides identified in Cor a 8. Although generated with Cor a 8, 62% of the TCL responded more strongly to Pru p 3. This cross-reactivity was mediated by T cells specific for the immunodominant region Pru p 361–75. Peptide clusters encompassing this region were generated during lysosomal degradation of both nsLTPs. Cor a 8 was more rapidly degraded by lysosomal proteases than Pru p 3. Pre-incubation of sera with Pru p 3 completely abolished IgE binding to Cor a 8, which was not the case vice versa. Conclusions: T-cell reactivity to Cor a 8 is predominantly based on cross-reactivity with Pru p 3, indicating that the latter initiates sensitisation to its homolog in hazelnut. The limited allergenic potential of Cor a 8 seems to be associated with rapid lysosomal degradation during allergen processing and the lack of major T-cell-activating regions.

To cite this article: D’Avino R, Bernardi ML, Wallner M, Palazzo P, Camardella L, Tuppo L, Alessandri C, Breiteneder H, Ferreira F, Ciardiello MA, Mari A. Kiwifruit Act d 11 is the first member of the ripening-related protein family identified as an allergen. Allergy 2011; 66: 870–877. Abstract Background: Kiwifruit is an important cause of food allergy. A high amount of a protein with a molecular mass compatible with that of Bet v 1 was observed in the kiwifruit extract. Objective: To identify and characterize kirola, the 17-kDa protein of green kiwifruit (Act d 11). Methods: Act d 11 was purified from green kiwifruit. Its primary structure was obtained by direct protein sequencing. The IgE binding was investigated by skin testing, immunoblotting, inhibition tests, and detection by the ISAC microarray in an Italian cohort and in selected Bet v 1-sensitized Austrian patients. A clinical evaluation of kiwi allergy was carried out. Results: Act d 11 was identified as a member of the major latex protein/ripening-related protein (MLP/RRP) family. IgE binding to Act d 11 was shown by all the applied testing. Patients tested positive for Act d 11 and reporting symptoms on kiwifruit exposure were found within the Bet v 1-positive subset rather than within the population selected for highly reliable history of allergic reactions to kiwifruit. Epidemiology of Act d 11 IgE reactivity was documented in the two cohorts. IgE co-recognition of Act d 11 within the Bet v 1-like molecules is documented using the microarray IgE inhibition assay. Conclusions: Act d 11 is the first member of the MLP/RRP protein family to be described as an allergen. It displays IgE co-recognition with allergens belonging to the PR-10 family, including Bet v 1.

Summary Background Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. Objective To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. Methods With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. Results The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. Conclusions Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests. Cite this as: M. Bublin, S. Dennstedt, M. Buchegger, M. Antonietta Ciardiello, M. L. Bernardi, L. Tuppo, C. Harwanegg, C. Hafner, C. Ebner, B. K. Ballmer-Weber, A. Knulst, K. Hoffmann-Sommergruber, C. Radauer, A. Mari and H. Breiteneder, Clinical & Experimental Allergy, 2011 (41) 129–136.

Summary Background Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen–IgE interaction sites is worthy to be investigated. Objective To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system. Methods Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects. Results CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions. Conclusions and Clinical Relevance The conformation and IgE reactivity of Act d 5 are affected by the physico-chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes. Cite this as: M. L. Bernardi, D. Picone, L. Tuppo, I. Giangrieco, G. Petrella, P. Palazzo, R. Ferrara, M. Tamburrini, A. Mari and M. A. Ciardiello, Clinical & Experimental Allergy, 2010 (40) 1819–1826.

IgE-mediated allergic diseases are among the most prevalent diseases worldwide. The use of extracts in the skin test and the additional use of IgE testing still represent the current basis for the diagnostic work-up. During the past 30 years, knowledge of the molecular structure of allergens has increased dramatically, and the characterization and production of allergenic molecules, as natural purified compounds or recombinant products, is allowing us to approach the allergy diagnostic work-up differently. Much of this is based on the adoption of microtechnology since the first release of a biochip for IgE detection. Its use has prompted the development of new concepts linked to the diagnosis of allergic diseases. This review describes the background of allergy diagnosis and the tools currently used for specific IgE detection. It gives insight into the most recent advancement in the field of biotechnology leading to allergenic molecule availability, microtechnology leading to the routine use of protein biochips for IgE detection, and how they should be combined with information technology.

Summary Background The availability of allergenic molecules and high-throughput microtechnologies allow the collection of a large number of IgE results at the same time in a single test. This can be carried out applying the test in the routine diagnostic work-up. Objective The aim of this study was to make a cross-sectional evaluation of the raw prevalence of IgE reactivity to allergenic molecules in serum samples from a cohort of Italian patients using an innovative tool. Methods The ISAC, a microarray system, has been used for specific IgE detection using 75 different allergenic molecules. Sera were collected from 23 077 unselected consecutive individuals complaining about any allergic disease. Results Sixteen thousand four hundred and eight of 23 077 patients had IgE to at least one of 75 allergenic molecules. The top-ranked molecules in this cohort were Cup a 1 (42.7%), Der f 2 (38.7%), and Phl p 1 (37.9%), whereas all the other allergens tested scored in a range between 36.8% and 0.04%, including the first food allergen, Pru p 3, ranked 15th (9.79%). Prevalence varied quite markedly depending on the age range considered, and showing a different behaviour in the lifetime sensitization process. Unsupervised two-way hierarchical clustering analysis generated distinctive patterns of reactivity as the result of IgE recognition of either homologous allergens belonging to different biological sources or non-homologous belonging to the same biological source. Conclusions Allergen-based microarray is a tool for the detection of IgE-related sensitization to panels of allergens and gives a more precise and comprehensive evaluation for an IgE-based epidemiology. This insight brings data for better understanding of the sensitization process.

Background: FABER 244 is a new in vitro multiplex test for specific IgE detection using 122 molecular allergens and 122 allergenic extracts, coupled to chemically activated nanoparticles. Allergenic preparations, either produced in house or obtained from commercial providers, are individually coupled to nanobeads by means of optimized protocols to achieve maximum test performance. Aim: To measure the diagnostic accuracy of FABER 244-122-122 01 by comparing with the ImmunoCAP ISAC 112 (TFS, Sweden) in patients allergic to LTP proteins shared by both tests. Methods: A real life study as been set by analyzing the clinical records, and the FABER and ISAC test results from 94 consecutive patients referred to CAAM from March 2015 to August 2016. Data were extracted from the electronic medical record InterAll. The diagnostic accuracy measurement and test comparison were performed on 3 LTP proteins (Cor a 8, Jug r 3, Pru p 3) adopting the patients' symptoms as gold standard and MedCalc as software for statistical analysis. ISAC test Limit of Detection (LoD) was set to 0.3 ISU whereas FABER LoD was set to 0.01 FIU. In the overall analysis attention has been put on additional information coming from non-shared LTPs, Tri a 14 and Ara h 9 for ISAC, and Act d 10, Pun g 1, Sola l 6, Tri a 7k-LTP, Zea m 14 plus peanut and wheat extracts available on FABER. Results-Discussion: Both tests appear to be accurate and well aligned to each other for all three analyzed LTP proteins. In terms of sensitivity FABER performs better than ISAC on Cor a 8 (100% vs 73%), same as ISAC on Jug r 3 (100% both) and FABER better than ISAC on Pru p 3 (96% vs 91%). In terms of specificity FABER performs better than ISAC on Cor a 8 (96% vs 94%), same as ISAC on Jug r 3 (96%), whilst ISAC performs better on Pru p 3 (97% vs 92%). IgE detection by ISAC Ara h 9 and Tri a 14 was compensated by the peanut and wheat extracts, whereas info on additional food LTPs could be obtained from the FABER extended panel including 25 extracts from plant-derived foods bearing LTPs. Conclusions: FABER test is a new in vitro test for specific IgE detection, including molecules and extracts. Considering the food allergen group belonging to the LTP, FABER appears to be accurate and in good agreement with ISAC results. The specific advantage of FABER relies on the chance of testing patients to a broader panel of LTP as well as to a large number of extracts, complementing the results on single allergenic molecules.

In most HIV-1-infected patients, highly active antiretroviral therapy (HAART) reduces plasma viral load to

Background: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Objectives: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Methods:Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Results: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. Conclusion: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

SUMMARY To investigate whether defective costimulatory signals could be involved in the loss of T lymphocyte functions during HIV-1 infection, we tested the effect of CD28 costimulation on both T cell receptor/CD3 and HIV-1 antigen-induced proliferative responses. Although CD3-mediated responses significantly decreased with more advanced stages of HIV-1 infection, the ability of potentiating the responses through CD28 costimulation was maintained at all stages and did not differ from that of HIV-1− subjects. When CD28 costimulation was studied in lymphocyte cultures stimulated with HIV-1 gp160 or p24, potentiation was seen only when a significant response was present without additional CD28 triggering, namely in subjects receiving active immunization with recombinant gp160. These results confirm the integrity of the CD28 pathway of costimulation during HIV-1 infection, and suggest that lymphocytes responding to soluble HIV-1 antigen are not deleted in HIV-1-infected patients, but do not receive significant priming during the natural course of the infection.

SUMMARYIn vitro lymphoproliferative responses to HIV-1 recombinant antigens (gp160, p24, and Rev protein) were studied in 83 patients with asymptomatic HIV-1 infection (CDC groups 11 and III) and circulating CD4 lymphocyte numbers > 400/mm3. Significant response to at least one of the three antigens was detected in 52.4% of the subjects, but the responses were weak, and concordance of the response to the three antigens was rare, the frequency of individuals responding to each antigen not exceeding 22.4%. Increasing frequencies of response were observed when recall antigens (tetanus toxoid and Candida albicans glycomannoprotein) (65.5%) and anti-CD3 MoAb (76.6%) were used as stimuli. Although a significant association between lymphocyte response to p24, but not gp160, and steadiness of CD4 lymphocyte numbers before the assay was observed, no predictive value for lack of CD4 cell decrease was confirmed for either antigen, and fluctuation of the responses to HIV antigens was seen during subsequent follow up. The panel of T cell assays used could be regarded as appropriate for monitoring both HIV-specific responses and T lymphocyte function during immunotherapy with soluble HIV antigens.

Background From patients’ reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow’s milk (CM) allergic patients. Objective and Methods To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), β-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. Results The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly αS1-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. Conclusions 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.

Methods Pulp and seeds of green kiwifruit were manually separated and homogenized. Act d 10 was purified from seed extracts by ion exchange chromatography and RPHPLC, and sequenced by an automatic amino acid sequencer. Act d 10 was evaluated in vivo by skin prick test (SPT) and in vitro by detecting IgE using streptavidin-conjugated CAP (Phadia, Sweden). Act d 10 was spotted on a customized ISAC microarray (PMD, Austria) for evaluating IgE prevalence in a larger cohort of allergic patients.

SummaryBackground In the temperate climate zone of the Northern hemisphere, Fagales pollen allergy represents the main cause of winter/spring pollinosis. Among Fagales trees, pollen allergies are strongly associated within the Betulaceae and the Fagaceae families. It is widely accepted that Fagales pollen allergies are initiated by sensitization against Bet v 1, the birch pollen major allergen, although evidence is accumulating that the allergenic activity of some Bet v 1-like molecules has been underestimated. Objective To investigate the allergenic potential of the clinically most important Fagales pollen allergens from birch, alder, hazel, hornbeam, hop-hornbeam, oak, beech and chestnut. Methods To obtain the full spectrum of allergens, the three previously unavailable members of the Bet v 1-family, hop-hornbeam Ost c 1, chestnut Cas s 1 and beech Fag s 1, were identified in the respective pollen extracts, cloned and produced as recombinant proteins in E. coli. Together with recombinant Bet v 1, Aln g 1, Car b 1, Cor a 1 and Que a 1, the molecules were characterized physicochemically, mediator release assays were performed and IgE cross-reactivity was evaluated by ELISA and Immuno Solid-phase Allergen Chip (ISAC) IgE inhibition assays. Results All allergens showed the typical Bet v 1-like secondary structure elements, and they were all able to bind serum IgE from Fagales allergic donors. Strong IgE binding was observed for Betuloideae and Coryloideae allergens, however, cross-reactivity between the two subfamilies was limited as explored by inhibition experiments. In contrast, IgE binding to members of the Fagaceae could be strongly inhibited by serum pre-incubation with allergens of the Betuloideae subfamily. Conclusions and Clinical Relevance The data suggest that Bet v 1-like allergens of the Betuloideae and Coryloideae subfamily might have the potential to induce IgE antibodies with different specificities, while allergic reactions towards Fagaceae allergens are the result of IgE cross-reactivity.

BackgroundFood allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.ObjectiveUsing the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.MethodsTwo new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.ResultsAlignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.ConclusionThe biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

To cite this article: Ciardiello MA, Palazzo P, Bernardi ML, Carratore V, Giangrieco I, Longo V, Melis M, Tamburrini M, Zennaro D, Mari A, Colombo P. Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry. Allergy 2010; 65: 597–605. Abstract Background: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. Methods: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). Results: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3+ subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. Conclusions: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs.

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

The case of a 20-year-old patient, affected by X-linked agammaglobulinaemia (XLA), who developed severe pneumonia from Pneumocystis jiroveci (formerly Pneumocystis carinii) (PCP), is reported. This infection usually affects patients with AIDS, children affected by severe combined immunodeficiency or hypogammaglobulinaemia with hyperimmunoglobulin M, or patients undergoing severe immunosuppression. The XLA patient developed PCP during therapy with steroids and cyclosporine A, carried out for several months, due to an extended skin vasculitis, accompanied by general symptoms. The pneumonia had a severe clinical course, requiring a long hospitalization. At the diagnosis of PCP, immunosuppressive therapy was suspended and the patient recovered after a long-term trimethoprim/sulfamethoxazole therapy. Immunological studies revealed an unexpected normal number of CD4+ and CD8+ T cells. The two subsets had an exclusive naïve phenotype (95% CD4+CD45RA+CD62L+ and 89% CD8+CD45RA+CD62L+ cells), with an absence of primed cells. Lymphoproliferative responses to P. carinii and recall antigens as well as to mitogens were extremely deficient. During the follow-up, memory cells appeared with recovery of the lymphoproliferative response to mitogens and maintained defective responses to antigens. This is one of the few reported XLA cases experiencing severe PCP. In this patient, the infection became clinically evident during immunosuppressive therapy. We believe that the absence of functional activities, despite a normal level of T lymphocyte counts, sustained this long-lasting infection. Thus, the CD4+ and CD8+ T cell count evaluation, without functional studies, may not be per se sufficient for predicting the risk of a severe clinical course of PCP in patients undergoing immunosuppression.

During HIV disease an increased in vitro apoptosis of peripheral blood mononuclear cells has been demonstrated. This can be reversed in vitro by interleukin (IL)-2. Recent trials with highly active antiretroviral therapy (HAART) and IL-2 in HIV-1-infected patients showed promising immunological and clinical results. Here we investigated the effects of subcutaneous low-dose IL-2 administration in combination with HAART on in vitro apoptosis and the relationship between apoptosis, CD4 + counts, and HIV replication. Twenty-two asymptomatic HIV patients were randomized for HAART (arm I) and HAART plus IL-2 (arm II). Spontaneous apoptosis was decreased in both arms after 28 weeks of therapy but the reduction was highly significant only in arm II ( P = 0.05 vs P = 0.001). As the percentage of apoptosis decreased, there was a significantly higher increase of both CD4 + and CD4 + naive T cells in arm II vs arm I. HIV plasma viremia was reduced in all patients after therapy. Our data suggest that intermittent therapy with low-dose subcutaneous IL-2 in addition to HAART induces a positive immunomodulation in asymptomatic HIV-infected patients.

Objectives: Evaluation of immunological reconstitution after 2 years of highly active antiretroviral therapy (HAART) in AIDS patients. Design: Previous data showed the effectiveness of HAART but conflicting evidence of immune reconstitution has been found in severely immunocompromised patients. Therefore, T-cell subsets and functions were analysed during 24 months of HAART in 21 AIDS patients (mean baseline CD4 cell count, 20¥106/l). Methods: Subjects were tested at baseline and after 4, 12 and 24 months of therapy for clinical symptoms and the following investigations were carried out: plasma HIV RNA, T-cell subsets and lymphoproliferative responses to mitogens (phytohaemagglutinin, anti-CD3), and recall antigens (Candida mannoprotein, tetanus toxoid and recombinant glycoprotein 160). Results: Increase in body weight, improvement of Karnofsky‚s score and reduction of opportunistic infections were observed. All patients showed an initial increase in the CD4 memory subset, whereas naive CD4 cells consistently increased only after 1 year. The magnitude of immune recovery was stronger in patients showing a significant reduction in viral load. However seven out of 21 patients who did not reach a sustained suppression of viral load showed also an increase in T-cell subsets. The majority of patients recovered lymphoproliferative responses to mitogens, whereas only four subjects showed a functional response to Candida mannoprotein. No patients showed a response to HIV recombinant glycoprotein 160 or tetanus toxoid. Conclusions: The immune recovery observed is slower and not complete in severely immunocompromised patients. Our data suggest that HAART may be continued also in the absence of a significant HIV RNA decrease if alternative drugs are not available.