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BackgroundDysregulated systemic inflammation is the primary driver of mortality in severe coronavirus disease 2019 (COVID-19) pneumonia. Current guidelines favour a 7–10-day course of any glucocorticoid equivalent to dexamethasone 6 mg daily. A comparative randomised controlled trial (RCT) with a higher dose and a longer duration of intervention was lacking.MethodsWe conducted a multicentre, open-label RCT to investigate methylprednisolone 80 mg as a continuous daily infusion for 8 days followed by slow taperingversusdexamethasone 6 mg once daily for up to 10 days in adult patients with COVID-19 pneumonia requiring oxygen or noninvasive respiratory support. The primary outcome was reduction in 28-day mortality. Secondary outcomes were mechanical ventilation-free days at 28 days, need for intensive care unit (ICU) referral, length of hospitalisation, need for tracheostomy, and changes in C-reactive protein (CRP) levels, arterial oxygen tension/inspiratory oxygen fraction (PaO2/FIO2) ratio and World Health Organization Clinical Progression Scale at days 3, 7 and 14.Results677 randomised patients were included. Findings are reported as methylprednisolone (n=337)versusdexamethasone (n=340). By day 28, there were no significant differences in mortality (35 (10.4%)versus41 (12.1%); p=0.49) nor in median mechanical ventilation-free days (median (interquartile range (IQR)) 23 (14)versus24 (16) days; p=0.49). ICU referral was necessary in 41 (12.2%)versus45 (13.2%) (p=0.68) and tracheostomy in 8 (2.4%)versus9 (2.6%) (p=0.82). Survivors in the methylprednisolone group required a longer median (IQR) hospitalisation (15 (11)versus14 (11) days; p=0.005) and experienced an improvement in CRP levels, but not inPaO2/FIO2ratio, at days 7 and 14. There were no differences in disease progression at the prespecified time-points.ConclusionProlonged, higher dose methylprednisolone did not reduce mortality at 28 days compared with conventional dexamethasone in COVID-19 pneumonia.

Welfare of farmed fish is a key aspect of sustainable and ethical aquaculture. At European level, the EU Commission has recently published studies on welfare of farmed fish and has reported on fish welfare during transport and at slaughter to the EU Council and Parliament (SANTE, 2017; SCAR FISH, 2018; COM 87 final (2018). The European Aquaculture Advisory Council has adopted in 2017 the position paper ‘Farmed Fish Welfare During Slaughter in the European Union’ and in June 2019 has provided a new recommendation for optimising fish welfare at slaughter (AAC, 2019). Producers’ Associations, certification bodies and NGOs are searching for suitable operational welfare indicators (OWIs) for measuring the welfare status of farmed fish and improving standard and welfare friendly practices. The PerformFISH consortium (http://performfish.eu/) is working on welfare of European sea bass and gilthead sea bream and is validating a set of OWIs and a welfare scoring system specifically designed for sea bass and sea bream, in collaboration with fish Producers’ Associations and Mediterranean aquaculture companies. This paper presents the results of the PerformFISH Fish Welfare Survey for the selection of most suitable OWIs for sea bass and sea bream aquaculture.&nbsp

Background : Peach fruit can cause allergic reactions with symptoms ranging from mild to very severe. With the aim of contributing to the improvement of allergy diagnosis at the molecular level, the specific objective of this study was the characterization of ENEA, a still un-known peach IgE- binding protein. Method : ENEA protein was isolated from the natural source by chro-matographic separations, identified by direct protein sequencing and produced as a recombinant molecule by cloning the sequence of the homologous protein from apricot, that shows a 97% identity. The structural characterization revealed that ENEA is an intrinsically disordered protein showing similarities with the major allergen Hev b 5 from rubber latex and with Man e 5 from manioc. The immuno-logical properties were investigated by dot blotting, the ABA system and the FABER test. Results : The data obtained indicated that ENEA is recognized by specific IgE, but the prevalence of positive patients is lower com-pared to the homologous allergen Hev b 5. In addition, 78% of the ENEA IgE positive patients were positive also to Hev b 5 and both the natural and recombinant molecules partially inhibit the IgE bind-ing to the latex allergen. Conclusion : ENEA, a new IgE binding protein from peach has been identified and characterized. Studies are needed to understand whether ENEA is involved in a latex- associated peach and apricot allergy. A recombinant form of ENEA has been produced and char-acterized and will be used for diagnostic purposes using a multiplex test.

Background:Mammalian serum albumins (SA) are thermolabileallergens present in milk, meat and animal dander. The sensitizationcan occur through ingestion, inhalation and an IgE co-recognitionmight be detected. Objectives: To explore the IgE sensitization pro-files to SA in a population of 6113 patients. Method:The sera of 6113 patients, average age 33 years, weretested by means of the FABER®multiplex IgE detection assay. Theanalysis was focused on 10 SA (Bos d 6, Equ c 3, Ovi a 6, Ory c 6,Sus s 1, Can f 3, Fel d 2, Mus m 4, Rat n 4, Hom s HSA), 6 milkextracts (Bos d, Bub b, Cam d, Cap h, Equ c, Equ as), 4 cow's milkproteins (Bos d 4, Bos d 5, Bos d 8, Bos d LF), 4 Meat Extracts (Bosd, Ory c, Ovi a, Sus s), 8 epithelium extracts (cat, dog, guinea pig,hamster, horse, mouse, rabbit, rat) and 7 genuine animal allergenicproteins (Can f 1, Can f 2, Can f 5, Fel d 1, Mus m 1, Rat n 1) Results:None of the tested sera was positive for HSA. Out of the6113 patients, 433 (7%) are SA+patients, sensitized to at least oneSA. The distribution by age shows a peak of 180 patients in the firstdecade (41.6%), progressively decreasing with age. Bos d 6 is glob-ally the most recognized, 54% of the SA+, followed by Ovi a 6(46%) and Fel d 2 (45%), with a peak in the first decade of 83% fol-lowed by Ovi a 6 (52%) and Sus m 1 (39%). Within the first decade94% of patients sensitized to Bos d 6 are also sensitized to at leastone milk protein, this prevalence becomes 92% when consideringthe entire SA population. Concerning the other SA, the prevalenceout of the 433 SA+are as follows: Sus s 1 35%, Ory c 6 33%, Equ c3 29%, Mus m 428%, Rat n 4 28%. SA+subjects were more fre-quently positive for milk, meat, and epithelium extracts regardlessreal exposures. Genuine markers of milk sensitization and of epithe-lia were recorded positive in the SA+subjects helping defining theirfull profiles.216| Conclusion:Sensitization to Bos d 6 seems to be driven by milkingestion, both globally and within first decade. Further studies arerequired to detect the real driver of the sensitization to the otherSA. Genuine allergens help in the definition of single patient sensiti-zation profiles. The specific advantage of FABER®relies on thechance of testing patients to a broad panel of proteins and extracts.Interpreting the sensitization profiles with an accurate patient's his-tory could provide improvement to the allergic patient's life.

Background:The consumption of pomegranate is increasing as itis considered a human health-promoting food. However, it can trig-ger mild to severe allergic reactions. So far several allergens havebeen identified in its red pulp, including Pun g 1 (LTP), Pun g 7 (pea-maclein like-protein) and Pun g 14 (chitinase), whereas any possibleallergens of the pomegranate seeds are still unknown. Objectives.The aim of this study was the investigation of possible allergens con-tained in pomegranate seeds. Method:2S albumin was isolated from the fruit seeds, analyzed bydirect protein sequencing and identified by mass spectrometry. Tenpatients allergic to pomegranate were analyzed by IgE-immunoblot-ting and 4537 randomly-selected subjects were tested for IgE bind-ing to a pomegranate seed extract by the FABER®test. Results:Of these 4537 patients analyzed by FABER®test, 266(6%) proved to be sensitized to at least one of the 5 pomegranateallergenic preparations, Pun g [Seed], Pun g 1, Pun g 14, Pun g 7and Pun g 5 with a prevalence of 54%, 54%, 23%, 13% and 6%, respectively. Ten subjects allergic to pomegranate and analyzed byimmunoblotting showed IgE recognition of a main protein compo-nent of the seed extract. Therefore, this IgE-binding molecule waspurified by chromatographic separations and characterized. Directprotein sequencing revealed a blocked N-terminal residue. Massspectrometry analysis showed a molecular mass of 16 496 Da andpeptidemassfingerprinting allowed its identification as the proteinreported in the Uniprot database with the accession numberA0A218XU94 and classified as uncharacterized protein. The homol-ogy search performed in the Allergome database displayed a similar-ity with 2S albumin storage proteins, such as the allergens Cor a 14,Jug n 1 and Pis v 1. In line with the well-known low degree of aminoacid sequence conservation among 2S albumins, the overall identityobserved between the pomegranate protein (Pun g 2S albumin) andother homologs is quite low, whereas a high identity is limited tospecific protein regions. Conclusion:Pomegranate seed is a sensitizing source. The mainprotein component, Pun g 2S albumin, is a newly identified IgE-bind-ing protein of about 16.5 kDa. Its future inclusion in the FABER®multiplex test will allow us to establish if it represents the main aller-gen in pomegranate seed and the relationships with other seed-derived 2S albumins, thus improving the allergy diagnosis to plant-derived foods.

Introduction:Laboratory allergy testing systems for IgE detectionare evaluated for standard parameters and performances. Multiplexis the new generation of multiplex IgE detection systems based onallergen preparations coupled on nano-beads using molecules andextracts. The best way to understand the performances of a newsystem is to compare with others.Objectives:To report the comparative evaluation of the multiplexIgE test versus the most used singleplex IgE testing systems.Results:The evaluation of the IgE binding was obtained by usingan Allergen IgE (BL-IgE), a standard polyclonal commercial product.BL-IgE is supplied after being tested on the 3 IgE testing systems.IgE mean values and ranges are provided. The product data sheetshows IgE values for 15 allergen extracts. 12 were used: Alt a, Ara h,Art v, Asp f, Bet v, Bos d, Can f, Der p, Equ c, Fel d, Gal d, Phl p.BL-IgE was tested on 22 consecutive multiplex batches, and extractto extract comparison was performed when the same was availableon FABER. FABER IgE, expressed as arbitrary units (FIU) gave thefollowing results: Ara h, overlapping with CAP-IMM-HYT; Art v,slightly below CAP, overlapping with IMM, above the HYT; Bet v,slightly below CAP-IMM; Bos d, above CAP-IMM-HYT; Can f,slightly below CAP-IMM, overlapping with HYT; Fel d, reproduciblebut below the three systems; Gal d, below IMM, overlapping withCAP-HYT; Phl p, overlapping with CAP-IMM. Alt a 1 performed bet-ter than CAP-HYT, overlapping with IMM. The 6 Der p FABER aller-gens gave overlapping results with the 3. Although reproducibleresults were record with Equ c FABER extract, average values alwaysfell below the range of the three systems. Asp f values were hardlyreproducible and always below the three systems, but the use of asimilar extract, Asp n, and the Asp r 1 allergen supported the Asp fIgE detection. A plus value of our study was to disclose IgE bindingto allergens not declared in the BL-IgE data sheet (e.g. Cup a 1, Prup 3), mostly all the molecule detected specificities (e.g. mite aller-gens) and all the IgE co-recognized preparations (e.g. eggs).Conclusions:The three systems having different reference stan-dards do not overlap each other. FABER IgE measurements performsvery well with most allergens, but improving the quality of someextracts will lead to better FABER performances. The multiplex IgEdetection is useful to disclose unknown sensitizations.

Introduction:A seed is a reproductive structure that possessesembryonic plant, stored material, and a protective coat. Its contentof proteins belonging to different allergenic families is high with pos-sible but not sure phenomena of cross-reactivity. Several seeds havebeen increasingly added in human diet with consequent risk of hy-persensitivity reactions that are often severe. The test is a newnanobead-based IVD test for specific IgE detection using 122 molec-ular allergens and 122 allergenic extracts, coupled to chemically acti-vated nano-particles.Objectives:To investigate profiles of seeds sensitization by meansof test. The tested allergenic preparations, all spotted on the chip,were 28 allergenic extracts (almond, amaranth, barley, bean, brazilnut, buckwheat, cashew, chestnut, carob, corn, chickpea, hazelnut,kamut, kiwi, lentil, linseed, lupine, peanuts, pine nut, pistachio, qui-noa, rice, sesame, soy, tomato seed, walnut, wheat, white mustard)and 23 allergenic proteins (Act d 10, Ana o 3, Ara h 1, Ara h 2, Arah 3, Ara h 6, Ara h Agglutinin, Cor a 8, Cor a 9, Cor a 14, Gly m1,Gly m Agglutinin, Gly m TI, Jug r 2, Jug r 3), along with markers forIgE-CCD reactivity and plant allergens belonging to other groups.Results:Out of the 1751 routinely tested patients 519 (29.64%)turned out to be sensitized to at least one allergenic preparation.The most recognized extract are buckwheat and carob with 50% and49% sensitization prevalence respectively. The least recognizedextract is soy with 0.8% sensitization prevalence. The most recog-nized protein is Jug r 3 with 19.5% sensitization prevalence. Theleast recognized proteins are Jug r 2 and Gly m 1 with 0.2% sensiti-zation prevalence. It is worth noting that within the 519 patientssensitized to at least one allergenic preparation 19% were sensitizedto Bet v 1-like protein, 18% to profilins, 33% to CCD, 33% to LTP.The cluster analysis shows several patterns of sensitization consider-ing molecules and extracts together.Conclusions:The results confirm differences in sensitizationtowards different allergenic sources from seeds and seeds allergenicproteins regardless if they are consumed as such or processed. Fil-tering the sensitization results by positive tests, taking into accountthe chance of co-sensitization to panallergens and CCD, the decisionmaking on which seeds have to be excluded from the diet becomeseasier with the Multiplex IgE test.

Introduction:Cow's milk (CM) allergy is the most common foodallergy in infancy. The overall allergic reactions to CM as well as thespecific sensitization pattern to each CM protein are variable frompatient to patient. Specific IgE detection can help to determinewhether all foods containing milk should be eliminated from thepatient's diet or some milks, sharing proteins homologues but notidentical to CM, might be used for feeding.Objectives:To investigate the milk and meat IgE sensitization pro-files in a population of 1751 allergic patients by means of FABER, anew nanobead-based IVD test for specific IgE detection using 122molecular allergens and 122 allergenic extracts, all coupled to chemi-cally activated nano-particles.Results:The tested allergenic preparations, all spotted on theFABER chip, were 7 milk extracts from different species used inhumans, 4 CM proteins, 4 meat extracts, 3 meat proteins and 5serum albumins shared between milk and meat. Out of the 1751patients 105 (5.99%), the majority of them in the first decade of life,turned out to be sensitized to at least one of the 11 Faber allergenicpreparations available for routine testing. The sensitization preva-lence was as follows: for milk extracts cow 73%, buffalo 70%, sheep69%, jenny 69%, goat 67%, mare 47%, camel 43%, whilst for CMproteins Bos d 4 41%, Bos d 5 20%, Bos d 8 35%, Bos d Lactoferrin52%. 75 patients are sensitized to at least one of the 7 Faber aller-genic preparations made by meat extracts and proteins. The sensiti-zation prevalence was: for meat extracts lamb 68%, cow 59%, rabbit31%, pork 29%, whilst for meat proteins Bos d CA 35%, Bos d Gela-tin 9%, Equ c Myoglobin 1%. Serum albumin sensitization prevalenceappears very similar between milk and meat sensitized groups, hav-ing Bos d 6 the highest IgE binding prevalence and Equ c 3 the leastone.Conclusions:The study results confirm differences in sensitizationtowards the tested milk and meat allergenic sources. Filtering thesensitization results with the patient's history and oral food chal-lenges, when needed, could improve the allergic patient's diet. Forthe first time FABER allows to simultaneously test the patients to abroad panel of animal milk, meat extracts and CM proteins, providingat the same time data on sensitizations to the largest number ofother inhalant, food, latex, animal and insect allergens, thus providingdata for alternative feeding.

Introduction:Pomegranate, Punica granatum L., is one of the old-est cultivated fruit trees. The fruit contains the arils, which are seedscovered by a red pulp, that is a juice sac. The arils are surroundedby the white and fleshy mesocarp. Pomegranate can trigger allergicreactions, but the allergenic pattern of this fruit is still poorly charac-terized and only one allergen, the LTP Pun g 1, was reported.Objectives:The aim of this study was the investigation of theallergen pattern in pomegranate tissues and cultivars.Results:Seven different pomegranate types were selected for thisstudy: 2 cultivars, Dente di Cavallo and Zanna Bianca; 3 accessions,CREA-FRC4, CREA-PR1 and CREA-PR2; 1 commercial sampleimported from Israel. Protein extracts, prepared from the mesocarp,juice and seeds, were analyzed by SDS-PAGE and RP-HPLC. Someprotein components were purified, identified by protein sequencingand their IgE binding capacity was investigated by dot blotting,immunoblotting and FABER®test. A variable amount of proteinswas measured in the juice of the different cultivars/accessions,whereas the mesocarp was devoid of detectable amounts of them.SDS-PAGE and RP-HPLC separations highlighted a different proteinprofile for each cultivar. Four proteins were purified from pomegra-nate juice, namely chitinase, punein (homologous to Bra r 2), pom-maclein (homologous to Pru p 7) and the 9k-LTP Pun g 1. All the 4isolated proteins displayed positive signals in IgE dot blotting andimmunoblotting. Specific IgE towards chitinase, pommaclein and Pung 1 were recorded in 5 (0.31%), 6 (0.37%) and 44 (2.74%) patientsout of 1606, using the FABER nanotech test. In addition, 20 patients(1.25%) were positive to the seed extract.Conclusions:Three new IgE binding proteins were identified inpomegranate juice. It was observed that seed proteins provide a sig-nificant contribution to the pomegranate allergenicity, whereas themesocarp could represent a rich and safe source of nutraceuticalsalso for allergic subjects.

Introduction:Allergy diagnosis is currently based on the use ofpurified allergenic proteins and protein extract. Diagnosis can be per-formed in vivo using a limited number of extracts, whereas all aller-genic preparations can be tested in vitro by measuring specific IgE.In addition to third generation singleplex lab tests, multiplex testingtools are available since 10 years, allowing to have many IgE resultsfrom a single sample by testing allergens as micro spots on a rigidsurface. Recently a new generation of lab diagnostic device has beenreleased, the FABER test, adopting modern nanotechnologies,namely nanobeads, for allergen immobilization.Objectives:To report the general set up of a multiplex test andthe evaluation of some of its performances.Results:The test is based on 244 molecules (122) and extracts (122),coupled to nano-particles. Particles are arrayed to a solid phase matrix,to form a one-step comprehensive array-based testing solution, using120ll of serum per test. Each allergen particle population can be indi-vidually optimized to achieve the maximum testing performance. Theproof of the IgE binding for the 244 allergen preparations has beenobtained by using routine serum samples from the sera bank. The eval-uation of the IgE binding was obtained by using a standard polyclonalcommercial preparation obtained by pooling human sera. The test wasused on 22 consecutive batches. 243 allergen preparations, even therarest one in term of prevalence in the general population, gave posi-tive IgE result using single serum samples. Negative results arerecorded for HSA which is the negative control test. 174 out of 244allergens gave positive IgE results. Average CV values were between15 and 25%. Allergen specificities having values in the lower IgE rangehad a lower reproducibility rather than those in the higher range, any-how never dropping to negative IgE detection. The multiplex testreproducibility performance was not related to the kind of allergenpreparations, namely purified molecule or verified extracts.Conclusions:This test is a new lab test for multiplex specific IgEdetection using allergenic molecules and extracts at the same timeshowing good performances. All steps in assembling the test are ver-ified and the present study reports that all allergens bind IgE. Evalua-tion of missing allergen specificities is performed with in-housemade serum pools and performances could be further improved.ABSTRACTS|1

Introduction:Dermatophagoides pteronyssinus, Dermatophagoidesfarinae, Euroglyphus Maynei, Blomia tropicalis are house dust mites(HDM), representing the main sources of indoor allergens, with con-centration highly dependent upon the domestic environmental con-ditions and external climate. 37 proteins from HDM's described asallergens.Objectives:To define the sensitization patterns to HDM allergensand extract available in the test in a population of 1751 patients,tested for routine allergy diagnosis.Results:Der f 1, Der f 2, Der p 1, Der p 2, Der p 7, Der p 9, Der p10, Der p 23, Eur m 2 and Blomia tropicalis extract were availablefor testing in a nanobead-based IVD test for specific IgE detectionbearing 122 molecular allergens and 122 allergenic extracts, all cou-pled to chemically activated nanoparticles. Out of the 1751 analyzedpatients 641 (36.61%) turned out to be sensitized to at least oneHDM protein. The sensitization prevalence of each allergen was asfollows: Der p 2 88%, Der f 2 85%, Eur m 2 52%, Der p 23 48%,Der p 1 44%, Der f 1 37%, Der p 7 24%, Der p 9 7%, Blo t extract13%. Pattern of sensitization and specific IgE concentration were dif-ferent, resulting in different patient clustering. Analyzing the data bypatients'age, all proteins behave quite similarly, with sensitizationpeaks in the second and third decades of life.Conclusions:Although the proteolytic activity of mite group oneallergens is claimed as a cause of the high prevalence of HDM sensiti-zation, Der p 1 and Der f 1 do not represent the most common sensiti-zation in mite allergic patients. This evidence is further supported bythe low sensitization prevalence to Der p 9, another proteases. GroupDer p 2 and Der p 23, whose biological function is almost unknown,represent the most common sensitizers. From a therapeutic perspec-tive and considering the need of a personalized medicine, knowledgeabout single patient IgE profile leads to the selection of the most suit-able extract for immunotherapy. This implies that the allergy investiga-tion should be carried out in the most extensive and comprehensivemanner starting with mite allergy representing one of the most com-mon allergy worldwide. The test is the newest in vitro test for specificIgE detection, including molecules and extracts, actually allowing totest the patients'responses to the broadest spectrum of availableHDM allergens in the routine setting.

Introduction:Parvalbumin is an allergenic protein from fish whitemuscle responsible for more than 95% of food allergies induced byfish. The content of parvalbumin is higher in bottom dwelling fishspecies such as cod and solea whereas is lower in the active fishes(rich in dark muscle) such as tuna, sardine, salmon. The parvalbumincontent also depends on muscle parts: higher levels in rostral partthan in caudal part. Parvalbumin sequences from different speciesare homologous but not identical. FABER test is a new nanobead-based IVD test for specific IgE detection using 122 molecular aller-gens and 122 allergenic extracts, all coupled to chemically activatednano-particles. The array contains Mer mr 1 parvalbumin from Euro-pean hake along with five allergenic extracts from Atlantic cod (Gadm), Atlantic salmon (Sal s), Sardine (Sar m), Common sole (Sol so) andYellowfin tuna (Thu a).Objectives:To investigate the IgE profiles of fish sensitizedpatients.Results:Out of 1751 routinely tested patients, 47 (2.68%) turnedout to be sensitized to at least one fish allergenic preparation. Thesensitization prevalence of each specific fish extract within the 47patients was as follows: Sardine 66%, Sole 62%, Cod 34%, Salmon26%, Tuna 19%. The sensitization prevalence of Mer mr 1 wasdetected 43%. It is worth noting that 25 out of the 47 patients weremonosensitized, with sensitization concentrated exclusively on Sar-dine extract, Sole Extract and Mer mr 1 parvalbumin. Co-reactivityto most or all fish extracts was always present when IgE levels weredetected high or very high. Few single fish species isolated sensitiza-tion have been detected.Conclusions:The presence on the FABER test of several fishextracts together with Mer mr 1 parvalbumin allows to improve thepatient's sensitization interpretation. The specific advantage ofFABER relies on the chance of testing patients to a broad panel offish extracts complementing the results on the single allergenic pro-tein, thus non missing any non-parvalbumin IgE reactivity. Interpret-ing the sensitization profiles with an accurate patient's historycombined with specific oral food challenges could provide improve-ment to the allergic patient's diet.

Introduction:Pomegranate, Punica granatum L., is a temperate cli-mate species, mainly cultivated in the Mediterranean area, SouthernAsia, and in several countries of North and South America. The con-sumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, some-times severe. The LTP, Pun g 1, is the only pomegranate allergenreported so far.Objectives:Aim of this study is the detection of still unknownallergens in Pomegranate fruit.Results:Pommaclein, was isolated from the fruit juice, identified bydirect protein sequencing and characterized as IgE-binding proteinby immunoblotting, dot blotting and FABER test. Nineteen patientswith a reliable clinical history of allergic reactions to pomegranatefruit and/or sensitization to Pru p 3 and Pru p 7 were selected forthis study. Pommaclein is a 7 kDa protein, registered in the Uni-protKB under the accession number C0HKC0. It displays high struc-tural similarities with the peach allergen Peamaclein, Pru p 7. Amongthe nine patients tested with Pru p 7 by SPT, four resulted IgE posi-tive to Pru p 7 and Pommaclein and one to Pommaclein only. Twopatients out of 19 were monosensitized to Pommaclein, whereasfour patients were positive to both pomegranate and peach homolo-gous proteins. When IgE were detected in parallel for Pru p 7 andPommaclein by FABER nanotech test, 16 sera out of 1751 werefound positive for the former and 6 for the latter.Conclusions:A new allergenic protein, Pommaclein, Pun g 7, wasidentified. It is a homolog of the peach allergen Pru p 7, but theimmunological properties of the pomegranate allergen are not com-pletely shared with those of the peach allergen. Pun g 7 can con-tribute to improve the allergy diagnosis to plant-derived foods andprofiling the allergic patient IgE reactivity using panel of homologousmolecules as already done for other allergen groups.

Introduction:The plant LTP family consists of ubiquitous proteinstypically involved in defense mechanisms against the attack of bacte-ria, fungi and viruses. They constitute an important cause of allergicsensitization in the Mediterranean area. A new nanobead-based IVDtest for specific IgE detection using 122 molecular allergens and 122allergenic extracts, all coupled to chemically activated nano-particles.Eight LTP purified from kiwi, hazelnut, pomegranate, peach, walnut,corn, tomato and wheat are immobilized together with 25 extractsfrom plant-derived foods bearing, along with other allergens, theirLTP.Objectives:To describe the LTP sensitization in a population of1751 patients by using molecules and extracts.Results:Out of the 1751 routinely tested patients 208 (11.88%)turned out to be sensitized to at least one LTP. The sensitizationprevalence of each specific LTP within the 208 patients wasdetected as follows: Pru p 3 94%, Jug r 3 49%, Cor a 8 30%, Zea m14 30%, Pun g 1 23%, Act d 10 21%, Tri a 7k-LTP 3%, Sola l 6 2%.Pru p 3 reaches 100% prevalence in pediatric patients between 0 to10 years old population. The 6% Pru p 3 negative subjects turnedout to be mono-sensitized to one of the other tested LTP. The avail-ability of extracts from many other vegetables, fruits and seedsallowed the definition of the sensitization to a broader number ofrelevant LTP. An IgE reactivity was detected to allergenic sourceswhose content in LTP is still unknown. A different IgE reactivity hasbeen recorded for the 9 k-LTP and the 7 k-LTP. Patients recognizingthe latter group have a broader recognition pattern and higher IgElevels.Conclusions:The specific advantage of a MULTIPLEX isle testrelies on the chance of testing patients to a broader panel of LTP aswell as to a large number of extracts, complementing the results onthe single allergenic molecules.The availability of the MULTIPLEX test of allergens belonging to theBet v 1-like proteins, Profilins, CCD markers and Anti-Microbial Pep-tide groups allows to greatly improve the patient sensitization profil-ing. The use of extracts to consumed food or newly introduced onesincreases our knowledge on allergens.

ntroduction:Tropomyosins are allergenic proteins present ininvertebrate muscle and non-muscle cells, involved in muscle con-traction together with actin and myosin. A great number of aller-genic tropomyosins have already been described in invertebrate(crustaceans, molluscs, snails, mites, insects) whose IgE cross-reactiv-ity is related to the high structural similarity in amino acidsequences. The test is a new nanobead-based IVD test for specificIgE detection using 122 molecular allergens and 122 allergenicextracts, all coupled to chemically activated nano-particles.Objectives:To analyze the sensitization profiles of tropomyosinsensitized patients by means of the multiplex test, considering aller-genic molecules and extracts from crustaceans and mollusks.Results:The tested allergenic preparations, all immobilized on thechip, were 10 invertebrate extracts (Anisakis pegreffi, Anisakis sim-plex, calamari, clam, German and American cockroach, mussel, octo-pus, shrimp, snail), and seven tropomyosins (Ani s 3, Der p 10, Hel as 1, Lit v 1, Per a 7, Uro du 1, Ven ga 1). Out of the 1751 analyzedpatients 115 (6.58%) turned out to be sensitized to at least one ofthe 17 tested allergenic preparations. The most recognized extractwas clam with 36% prevalence, the least recognized is Americancockroach with 5% prevalence. The most recognized tropomyosin isVen ga 1 with 29% prevalence, the least recognized is Hel as 1 with20% prevalence. The patients recognizing at least one of the seventropomyosins are 49, 33% of them having IgE to all seven tropomyo-sins, 16% of them recognizing one only.Conclusions:The study results confirm differences in sensitizationpatterns toward the tested invertebrates extracts and tropomyosins.The single patient defined IgE profile, supported by clinical data andoral food challenge if needed, might lead to a better selection ofwhich of the involved foods should be excluded from the diet. Any-how, highly similar tropomyosins seems to behave in a patient-related manner. Food and non-food extracts from allergenic sourceswith known or still unknown tropomyosins help to broad the IgEprofile definition. Testing extracts from vertebrate species and fishesseems not to bring evidence of any IgE cross-reactivity.

Introduction:IgE-mediated Allergic diseases are progressivelyaffecting more people globally. The allergenic sources vary from onegeographic area to another. To compare the prevalence of IgE sensi-tization between Countries the diagnostic tests should be pre-defined in terms of allergenic preparations and be as comprehensiveas possible. The use of a broad panel of allergens facilitates thestudy design. The FABER test for IgE detection bearing 244 aller-gens, both molecules (122) and extracts (122), is the most advancedtool for this purpose including inhalant and food allergens.Objectives:A pilot study has been performed comparing IgE sensi-tization in three European Countries (Croatia, HR; Romania, RO;Italy, IT). FABER 244 version has been used to detect specific IgE.Serum samples were all tested in a single lab for the routine diagnos-tic workup. The three groups of 68 (HR), 60 (RO) and 69 (IT)patients were matched for both age (0.6-75 yo, average: 28 yo) andgender distribution (F/M=1.11:1).Results:The sensitization for all dust mite allergens appears to behomogeneous in the three groups with the highest values in RO forDer p 2 (45%) and the lowest in HR for Der p 7 (1.5%). Der p 23,showed a significant difference in prevalence between IT (26.1%)and RO (8.3%). Grass, Fagales, Parietaria, Olive and Cypress pollenmarker allergens were as follows: Bet v 1 (HR 7%, IT 10%, RO 5%),Par j 2 (HR 6%, IT 16%, RO 2%), Ole e 1 (HR 18%, IT 22%, RO 5%),Cup a 1 (HR 29%, IT 46%, RO 23%), Phl p 1 (HR 13%, IT 36%, RO13%), Phl p 5 (HR 7%, IT 17%, RO 2%), and Amb a 1 (HR 9%, IT 0%,RO 15%). The allergenic molecules bearing CCD appear to be morerecognized by RO patients, whereas LTP sensitization was recordedhigher in RO (15%) compared to IT (13%) and HR (9%). Other aller-gen sensitizations were: Tropomyosin (HR 6%, IT 3%, RO 3%); Pro-filin (HR 4%, IT 7%, RO 5%); Parvalbumin (HR 6%, IT 3%, RO 2%).Conclusions:IgE detection by means of the FABER multiplex testallows to precisely detect sensitization prevalence to inhalant andfood allergens using data from the routine diagnosis. FABER testingpreliminary discloses differences and similarities among differentCountries having different climates and eating habits. Due to the354|ABSTRACTS small number of observations, unwanted biases could be introducedin the patient enrolled in this preliminary study. To draw any conclu-sion the study using the same FABER multiplex tool on largercohorts of patients will be performed.

Background: Multiplex tests allow to detect specific IgE to several different preparations at once. They allow patient's profiling tailoring decisions for interventions. The last ten years have seen the availability of new technologies and when combined can lead to increase diagnostic information from allergy tests. Aim: To report about the FABER nanotechnology-based test in food allergy diagnosis. Methods: FABER 244 IgE test is a new multiplexed in vitro test for specific IgE measurement having 122 molecular allergens and 122 allergenic extracts. Allergenic molecules and extracts, produced in house or obtained from top quality providers in the field, are coupled to chemically activated nanoparticles. Coupling is individually optimized to achieve maximum test performance providing high diagnostic accuracy for each spotted allergenic item. Once coupled they are arrayed to a solid phase matrix to form a one-step comprehensive array based testing solution, using 100 ul of patient serum or plasma. Results-Discussion: Extracts from 91 food-borne allergenic sources (fruits, vegetables, eggs, milks, meats, fishes, crustacean, mollusks, snails, mushrooms, anisakis) are arrayed together with 66 allergenic proteins obtained from the same sources. CCD-bearing proteins are included as markers to support test result interpretation, as well as allergenic molecular groups which cross-sectionally belong to food and inhalant sources. Extracts on FABER244 expand the panel overcoming missing of any not yet identified or available allergenic molecule, increasing diagnostic accuracy and comprehensiveness. Test interpretation is supported by CAAM Digital Reporting System (CDRS), a unique online tool available worldwide, allowing visualization on mobile devices of FABER test results. CDRS has been developed for patients to familiarize with the new extended molecule-based results. To be patient-friendly it uses local languages taking advantage of the Allergome platform as the multi-language source. Data on CDRS are shown with tables, graphs, images; comments are generated real time by experts using the Allergome and its external modules, InterAll and ReTiME. Conclusions: FABER 244 is the most advanced in vitro test for specific IgE detection, including molecules and extracts. It makes available to the molecular allergist an unprecedented quantity of data. The inclusion of allergenic extracts is strategic to confirm or complement results obtained with the single allergenic molecules.

CIBSE/ASHRAE Building Sustainability, Value and Profit Conference, September 24-26th 2003, Edinburgh, Scotland

"Background: Food 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. Objective: Using 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. Methods: Two 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. Results: Alignment 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. Conclusion: The 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."

Le caratteristiche biochimiche e nutrizionali di un frutto sono legate al suo proteoma che dipende dal patrimonio genetico. Cultivar diverse di una stessa specie possono essere caratterizzate da variazioni qualitative e quantitative, più o meno evidenti, a carico di diverse componenti. In questo lavoro sono descritti i dati ottenuti in un'analisi comparativa del quadro delle proteine totali e della quantità della proteina allergenica "lipid transfer protein" (LTP) in alcune cultivar di pesco. E', inoltre, riportata un'analisi delle possibili componenti allergeniche nell'estratto proteico di una delle cultivar, effettuata mediante separazione elettroforetica su gel 2D e IgE immunoblot, utilizzando sieri di pazienti allergici alla pesca. Le cultivar in esame [cv Vista Rich (nettarina a polpa gialla), cv Stark Saturn (pesca platicarpa a polpa bianca), cv Neve (nettarina a polpa bianca), cv Crimson Lady (pesca a polpa gialla), cv Ionia (percoca), cv Maria Cristina (pesca a polpa bianca)] provenivano da un campo sperimentale del CRA-FRC di Caserta. I risultati ottenuti mostrano che la composizione del quadro proteico e la quantità dell'allergene LTP differiscono significativamente in relazione alla cultivar. Fra le cultivar analizzate la nettarina Neve presenta la minor concentrazione di LTP/g di sostanza secca mentre la concentrazione più elevata è stata ritrovata nella cv Stark Saturn. Gli esperimenti di IgE immunoblot hanno evidenziato che nella pesca sono presenti proteine allergeniche non ancora descritte.

Proteomic is the study of the overall protein content of a cell, a tissue or an organism, aiming to identification and qualification of all the expressed proteins. Many studies, mainly linked to the development of new medicines or therapies, have so far been focused on the analysis of human proteins. On the other hand, little attention has been given to plant tissues, and in particular to fruit ones. In this study, a proteomic approach was used for the analysis of biodiversity of fruits of some Mediterranean species, and in particular: apple, cv Annurca Rossa del Sud, cv Annurca tradizionale and cv Golden Delicious; peach, cv Percoco di Tunisi; lemon, cv Femminello Siracusano; persimmon, cv Calfuyu; orange, cv Washington Navel; grape, cv Moscato Bianco; prickly pear, cv Gialla. For each sample, the total protein concentration of the saline extract of the pulp was determined, and the protein electrophoretic pattern of the extract was analysed by SDS-PAGE. The fruits under investigation showed a variable protein content, from 20mg/100g of prickly pear to 0.54mg/100g of grape. For each species, a low number of abundant protein, having different molecular weights, was shown. In some cases, a significant variability, dependent on the post-harvesting time, was found. Proteins present in high amounts were analysed by N-terminal amino acid sequencing, and the obtained data were compared to sequences available in Data Banks. The results showed that most of the abundant proteins are involved in mechanisms of plant defence against water and temperature stress, pathogen attack, chemical agents, etc.

When making design decisions, which can impact on building capital cost and performance, the design team has to be confident that the simulation tool is generating reliable output. Confidence can be gained via several mechanisms. In all cases data quality is of prime importance. Recognising the quality of data, or the lack of quality, and how this impacts on the predictions of the simulation tool is crucial to making informed design decisions. This paper describes information sources and their use with respect to simulation. To elaborate the process, inputs and outputs are examined from an example Supported Technology Deployment. The example demonstrates effective simulation use for assessing the refurbishment of an existing building. Many information sources were available to the design team, including data collected from site visits and from interviews with the building’s occupants. The example also demonstrates how simulation can be a cost effective solution for a client., Building Simulation '03, August 11-14th, 2003, Eindhoven, Netherlands

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.

The Arctic fish Anarhichas minor, a benthic sedentary species, displays high emoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.