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The standard rapid approach for the diagnosis of coronavirus disease 2019 (COVID-19) is the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The detection of specific anti-SARS-CoV-2 immunoglobulins is crucial for screening people who have been exposed to the virus, whether or not they presented symptoms. Recent publications report different methods for the detection of specific IgGs, IgMs, and IgAs against SARS-CoV-2; these methods mainly detect immunoglobulins in the serum using conventional techniques such as rapid lateral flow tests or enzyme-linked immunosorbent assay (ELISA). In this article, we report the production of recombinant SARS-CoV-2 spike protein and the development of a rapid, reliable, cost-effective test, capable of detecting immunoglobulins in serum and saliva samples. This method is based on interferometric optical detection. The results obtained using this method and those obtained using ELISA were compared. Owing to its low cost and simplicity, this test can be used periodically for the early detection, surveillance, detection of immunity, and control of the spread of COVID-19.

The aim of this work is to study the effect of High Pressure Processing (HPP) on the allergenicity of the main protein involved in peach allergy (Pru p 3). Results obtained showed that most pressure/time combinations slightly enhanced in vitro IgE-binding to Pru p 3 and peach extract. Moreover, additional tests were carried out by means of skin prick tests on peach allergic patients. Different from in vitro results, in vivo effects evidenced that HPP (600 MPa/5 min) can either reduce or increase Pru p 3 allergenicity, depending on the particular sensitisation of each patient. Notwithstanding this variability, it is highly remarkable that the skin response to pressurized peach extract was stronger in more than half of individuals. These results would suggest a higher risk of HPP-treated peach products to elicit an allergenic reaction. However, it has been also proved that matrix plays an important role in peach allergenicity modification. Consequently, further investigations are needed before extrapolating results to more complex products. Industrial relevance Peach allergy is one of the most frequent allergies in Mediterranean countries among adult patients. However, up to date, there is no information about the effect of HPP on peach allergenicity. This study increases understanding about the impact of processing on an important fruit allergen in order to advance knowledge-based ways to managing allergens risks in industry, as well as it opens new opportunities of research in other technologies or strategies in the attempt to reduce peach allergenicity.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses.OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis.METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples.RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating soluble TWEAK levels were increased in patients with anaphylaxis, and plasma from those patients increased Fn14 expression in ECs.CONCLUSION: The TWEAK/Fn14 axis participates in anaphylactic reactions. Inhibition of TWEAK/Fn14 interaction could be efficacious in anaphylaxis therapy.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Background Pru p 3 is the major peach allergen and the most frequent cause of food allergy in adults in the Mediterranean area. Although its allergenicity is well characterized, its ability to generate a T-cell response is not completely known. Objective To investigate the influence of Pru p 3 allergen on dendritic cell (DC) maturation and specific T-cell response (T H 1/T H 2) in peach allergic patients. Methods Peach allergic patients (n = 11) and tolerant controls (n = 14) were included in the study. Monocyte-derived DC maturation after incubation with Pru p 3 was evaluated by the increase of maturational markers (CD80, CD86, and CD83) by flow cytometry. Lymphocyte proliferation was evaluated by coculturing monocyte-derived DCs and 5,6-carboxyfluorescein diacetate N -succinimidyl ester–stained lymphocytes with different concentrations of Pru p 3 (25, 10, and 1 μg/mL) by flow cytometry and cytokine production. Results Pru p 3 induced a significant increase in the CD80, CD86, and CD83 expression on stimulated DCs from patients compared with controls. The lymphocyte proliferative response after Pru p 3 stimulation was also significantly higher along with an increase in interleukin 8 in patients compared with tolerant controls. Conclusion Pru p 3 allergen induces changes in DC maturational status mainly in peach allergic patients. An increase in lymphocyte proliferative response accompanied with a different cytokine pattern was also observed compared with healthy controls.

Exposure to certain substances in the workplace may lead to sensitization and increased respiratory symptoms.To evaluate the frequency of work-related specific sensitization and respiratory symptoms in carpentry apprentices with occupational exposure to wood dust and diisocyanates.Apprentices (n=101) completed an occupational and symptoms questionnaire. Spirometry and skin prick tests to aeroallergens and to a battery of 14 different woods were performed in all the participants. Blood samples were collected for total IgE measurement and detection of specific IgE to diisocyanates.Half the participants (56%) had work-related respiratory symptoms: 54% due to wood dust, 15% due diisocyanates, and 9% to both. Participants with respiratory symptoms related to wood dust exposure had a significantly lower forced expiratory volume in 1 second compared with symptomatic individuals due to diisocyanates and asymptomatic individuals (P.05). A history of rhinitis or asthma was associated with a 2.1- or 2.8-fold increase, respectively, in the likelihood of having respiratory symptoms due to wood dust exposure. Sensitization to wood was detected in 9% of participants, all of whom were atopic with a history of rhinitis and a high total IgE level (P.05). Sensitization to diisocyanates was detected in 2% of exposed participants.Work-related respiratory symptoms are common in carpentry apprentices and are more frequently related to exposure to wood dust than to diisocyanates. Symptomatic participants due to wood dust exposure had a lower forced expiratory volume in 1 second. Individuals with a history of rhinitis or asthma had an increased risk of respiratory symptoms. Sensitization to wood was more common in atopic apprentices with a history of rhinitis and a high total IgE level.

Plant non-specific LTPs (lipid transfer proteins) form a protein family of basic polypeptides of 9 kDa ubiquitously distributed throughout the plant kingdom. The members of this family are located extracellularly, usually associated with plant cell walls, and possess a broad lipid-binding specificity closely related to their three-dimensional structure. The nsLTP fold is characterized by a compact domain composed of 4 alpha-helices, firmly held by a network of 4 conserved disulphide bridges. This fold presents a large internal tunnel-like cavity, which can accommodate different types of lipids. nsLTPs are involved in plant defence mechanisms against phytopathogenic bacteria and fungi, and, possibly, in the assembly of hydrophobic protective layers of surface polymers, such as cutin. In addition, several members of the nsLTP family have been identified as relevant allergens in plant foods and pollens. Their high resistance to both heat treatment and digestive proteolytic attack has been related with the induction by these allergens of severe symptoms in many patients. Therefore, they are probably primary sensitizers by the oral route. nsLTP sensitization shows an unexpected pattern throughout Europe, with a high prevalence in the Mediterranean area, but a low incidence in Northern and Central European countries.

We have cloned a human cDNA encoding a new serine protease that has been called polyserase-2 (polyserine protease-2) because it is the second identified human enzyme with several tandem serine protease domains in its amino acid sequence. The first serine protease domain contains all characteristic features of these enzymes, whereas the second and third domains lack one residue of the catalytic triad of serine proteases and are predicted to be catalytically inactive. This complex domain organization is also present in the sequences of mouse and rat polyserase-2 and resembles that of polyserase-1, which also contains three serine protease domains in its amino acid sequence. However, polyserase-2 lacks additional domains present in polyserase-1, including a type II transmembrane motif and a low-density lipoprotein receptor A module. Enzymatic analysis demonstrated that both full-length polyserase-2 and its first serine protease domain hydrolyzed synthetic peptides used for assaying serine proteases. Nevertheless, the activity of the isolated domain was greater than that of the entire protein, suggesting that the two catalytically inactive serine protease domains of polyserase-2 may modulate the activity of the first domain. Northern blot analysis showed that polyserase-2 is expressed in fetal kidney; adult skeletal muscle, liver, placenta, prostate, and heart; and tumor cell lines derived from lung and colon adenocarcinomas. Finally, analysis of post-translational processing mechanisms of polyserase-2 revealed that, contrary to those affecting to the membrane-bound polyserase-1, this novel polyprotein is a secreted enzyme whose three protease domains remain as an integral part of a single polypeptide chain.

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.

Background Lentils are among the main plant foods causing allergic reactions in pediatric patients in the Mediterranean area and in many Asian communities. However, very few reports have been devoted to identifying lentil allergens. Seed storage proteins of the vicilin family have been characterized as major allergens in several seed legumes and tree nuts. Objective We sought to evaluate the role of lentil vicilins as food allergens. Methods A serum pool and individual sera from 22 patients with lentil allergy were used in different IgE-binding assays. Mature lentil vicilin was isolated by means of cation-exchange chromatography, followed by reverse-phase HPLC, and characterized by means of N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis, complex asparagine-linked glycan detection, specific IgE immunodetection with individual sera, and ELISA inhibition assays. Complete cDNAs encoding lentil vicilin variants were isolated by means of PCR with primers based on the amino acid sequence of the allergen. Results A major IgE-binding component of approximately 50 kd was detected in lentil extracts. This component was isolated and characterized, showing a single N-terminal amino acid sequence homologous to those of legume vicilins and a broad peak (maximum at 48,613 d) in MALDI analysis. The purified allergen was recognized by 77% (17/22) of the individual sera from patients with lentil allergy and reached up to 65% inhibition of the IgE binding to the crude lentil extract. The allergen showed 3 isoforms varying in their degree of N-glycosylation. Two cDNA clones encoding different allergen variants were isolated. The amino acid sequences deduced from both clones (415 and 418 residues; 47.4 and 47.8 kd) showed greater than 50% identity with major peanut (Ara h 1) and soybean (con-glutinin subunits) allergens belonging to the vicilin family. Furthermore, these sequences included those of the previously characterized lentil allergen Len c 1.02 (108 amino acid residues of the C-terminal domain) and those of a novel lentil IgE-binding protein of 26 kd. Conclusion The mature 48-kd lentil vicilin, designated Len c 1.01, is a major allergen. Two of its processing fragments, corresponding to subunits of 12 to 16 kd (previously named Len c 1) and 26 kd, are also relevant lentil IgE-binding proteins. The sequence homology of Len c 1.01 to those of major allergens from peanut, soybean, walnut, and cashew can help to investigate potential cross-reactions among these plant foods.

Background Resistance to pepsin digestion has been claimed to be a characteristic of food allergens that can induce severe adverse reactions. Moreover, pepsin treatment is included in protocols to evaluate the potential allergenicity of transgenic foods. Allergenic plant class I chitinases, such as avocado Prs a 1, are the panallergens involved in the latex-fruit syndrome. Previous reports indicated their susceptibility to simulated gastric fluid (SGF) digestion. Objective We sought to evaluate the IgE-binding capacity and the in vivo reactivity of the SGF products of the avocado allergen Prs a 1. Methods Patients with a clinical history of latex-fruit allergy syndrome, a positive skin prick test (SPT) response to Prs a 1, and specific IgE to avocado were selected. Untreated and SGF-digested Prs a 1 samples were analyzed by means of IgE and IgG immunoblotting, IgE immunoblotting and ELISA-inhibition assays, and SPTs. Peptides from SGF-digested samples were fractionated by means of HPLC, characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization analysis, and tested for in vivo reactivity with SPTs. Results Neither protein staining nor IgE immunoblotting with a pool of sera from allergic patients resulted in the detection of any band after SDS-PAGE separation of an SGF-digested sample of Prs a 1. However, this sample showed a similar inhibitory potency to that of untreated Prs a 1 in both immunoblot- and ELISA-inhibition assays (up to 70% inhibition of the IgE binding to crude avocado extract) and induced positive SPT responses in 5 of 8 allergic patients. Peptides from SGF-digested Prs a 1 were separated by means of HPLC, and 4 of them reached more than 50% inhibition values when using avocado extract as the solid phase in ELISA-inhibition assays. Reactive peptides were located both in the N-terminal hevein-like domain and in the catalytic domain of Prs a 1. Those corresponding to the hevein-like domain (approximately 5100 d) produced positive SPT responses in 5 of 8 allergic patients, whereas 2 peptides located in the catalytic domain (approximately 1400 and 2500 d) were reactive in 2 or 3 of the 8 patients. Conclusion Prs a 1 was extensively degradated when subjected to SGF digestion. However, the resulting peptides, particularly those corresponding to the hevein-like domain, were clearly reactive both in vitro and in vivo.

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.

Background Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro. Objective We sought to locate major IgE-binding epitopes of Pru p 3. Methods A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immu-nodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by 125 I–anti-IgE immunodetection. Results Pru p 3 showed a 3-dimensional structure comprising 4 α-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immu-nodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes. Conclusions Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an α-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.

Background: Cross-reactions between latex and plant foods (mainly fruits) have been widely reported. Although the cross-reactive components have not been well identified, class I chitinases seem to be the most credible candidates in chestnut, avocado, and banana. Objective: We sought to evaluate the potential role of chitinases and complex glycans as cross-reactive determinants linked to latex-food allergy. Methods: Extracts from 20 different plant foods and from latex were obtained. These preparations were immunodetected with anticomplex glycans and antichitinase sera raised in rabbits, as well as with sera from patients with latex-fruit allergy and sera from patients allergic to latex without food allergy. Immunoblot inhibition assays were carried out by using a purified class I chitinase from avocado or latex extract as inhibitors. Results: Reactive proteins of approximately 30 to 45 kd (putative class I chitinases) were recognized by both specific polyclonal antibodies to chitinases and sera from patients with latex-fruit allergy in chestnut, cherimoya, passion fruit, kiwi, papaya, mango, tomato, and flour wheat extracts. Prs a 1, the major allergen and class I chitinase from avocado, and the latex extract strongly or fully inhibited IgE binding by these components when tested in immunoblot inhibition assays. Additional bands of 16 to 20 kd, 23 to 28 kd, and 50 to 70 kd were detected by the antichitinase serum but not with the patients' pooled sera. The putative 30- to 45-kd chitinases present in different food extracts did not react with a pool of sera from subjects allergic to latex but not to fruit. Very different immunodetection patterns were produced with the anticomplex glycan serum and the sera from allergic patients. Conclusions: Putative class I chitinases seem to be relevant cross-reactive components in foods associated with the latex-fruit syndrome, but do not play a specific role in allergy to latex but not to fruit. Cross-reactive carbohydrate determinants are not important structures in the context of latex-fruit cross-sensitization. (J Allergy Clin Immunol 1999;104:681-7.)

Background: Latex-fruit cross-sensitization has been fully demonstrated. However, the antigens responsible for this "latex-fruit syndrome" have not been identified. We have recently shown that class I chitinases are relevant chestnut and avocado allergens. Objective: We sought to evaluate the in vivo and in vitro reactions of purified chestnut and avocado chitinases in relation to the latex-fruit syndrome. Methods: From a latex-allergic population, eighteen patients allergic to chestnut, avocado, or both were selected. Skin prick tests (SPTs) were performed with crude chestnut and avocado extracts, chitinase-enriched preparations, and purified class I and II chitinases from both fruits. CAP-inhibition assays with the crude extracts and purified proteins were carried out. Immunodetection with sera from patients with latex-fruit allergy and immunoblot inhibition tests with a latex extract were also performed. Eighteen subjects paired with our patients and 15 patients allergic to latex but not food were used as control groups. Results: The chestnut class I chitinase elicited positive SPT responses in 13 of 18 patients with latex-fruit allergy (72%), and the avocado class I chitinase elicited positive responses in 12 of 18 (67%) similarly allergic patients. By contrast, class II enzymes without a hevein-like domain did not show SPT responses in the same patient group. Each isolated class I chitinase reached inhibition values higher than 85% in CAP inhibition assays against the corresponding food extract in solid phase. Immunodetection of the crude extracts and the purified class I chitinases revealed a single 32-kd band for both chestnut and avocado. Preincubation with a natural latex extract fully inhibited the IgE binding to the crude extracts, as well as to the purified chestnut and avocado class I chitinases. Conclusion: Chestnut and avocado class I chitinases with an N-terminal hevein-like domain are major allergens that cross-react with latex. Therefore they are probably the panallergens responsible for the latex-fruit syndrome. (J Allergy Clin Immunol 1999;103:507-13.)

Background: Several foods associated with the latex-fruit syndrome present relevant allergens of around 30 kd. Neither these components nor any other responsible for the reported cross-reactions have been identified and purified. Objective: We sought to isolate and characterize the 30 kd allergens from avocado fruit and chestnut seed, two of the main allergenic foods linked with latex allergy. Methods: Sera from patients allergic to chestnut and avocado were selected according to clinical symptoms, specific IgE levels, and positive skin prick test responses. Class I and II chitinases were purified by affinity and cation-exchange chromatography and characterized by specific IgE and anti-chitinase immunodetection, immunoblot inhibition assays, enzymatic activity tests, and N-terminal sequencing. Results: Relevant 32 kd allergens were detected by specific IgE immunodetection in both avocado and chestnut crude extracts. The same bands, together with others of 25 kd, were revealed by a monospecific antiserum against class II chitinases. Purification and characterization of the 32 kd allergens from both plant sources allowed their identification as class I chitinases with an N-terminal hevein-domain. The purified allergens fully inhibited IgE binding by the corresponding crude extract when tested in immunoblot inhibition assays. Highly related 25 kd class II chitinases that lack the hevein-like domain were also isolated from the same protein preparations. No IgE-binding capacity was shown by these class II enzymes. Conclusion: Class I chitinases are relevant allergens of avocado and chestnut and could be the panallergens responsible for the latex-fruit syndrome. The hevein-like domain seems to be involved in their allergenic reactivity. (J Allergy Clin Immunol 1998;102:127-33.)

S U N D A Y 313 Baked Milk Oral Food Challenges: A Single-Center Pediatric Hospital Experience Rushani W. Saltzman, MD, Mary E. Kelleher, RN, CPN, AE-C, Jonathan M. Spergel, MD, PhD, FAAAAI; The Children’s Hospital of Philadelphia, Voorhees, NJ, Children’s Hospital of Philadelphia, Philadelphia, PA, Perelman School of Medicine at Univ of Pennsylvania. RATIONALE: Cow’s milk (CM) allergy is the most common food allergen in children. Recent research has shown that a majority of children with unheated or native cow’s milk (N-CM) allergy can tolerate the extensively heated form (H-CM) due to epitope structure changes from thermal processing. Furthermore, those H-CM tolerant appear to have accelerated development of tolerance to N-CMwhen compared with those strictly avoiding N-CM. METHODS: Graded, open baked milk oral food challenges (OFC) were performed for CM allergic patients [clinical history and/or positive CM skin prick testing (SPT)]. A standard protocol (1 cup CM baked in standard cake recipe, 3508Fahrenheit, minimum 30 minutes) was followed. Baked milk OFCs conducted at our institution (July 2011-early September 2012) were evaluated by retrospective chart review. Baseline demographics and characteristics were analyzed. RESULTS: Eighteen baked milk OFCs were performed and 61% were baked milk tolerant (ingested 2 gm baked CMwithout reaction). Male sex, younger age, history of other food allergies, history of atopic dermatitis, and larger median CM SPTwheal size were trends noted in the baked CM reactive cohort. Median cumulative dose eliciting clinical reaction was 0.34 gm baked CM. CONCLUSIONS: Our study confirms that a majority of CM allergic patients can tolerate baked milk. Dietary introduction of baked milk after successful completion of a supervised OFC improves one’s quality of life through a broadened diet. Additionally, there is likely accelerated development of tolerance to N-CM. Further studies are needed to elucidate such food allergy phenotypes and establish baked milk OFC patient selection criteria.