Maria Basagaña

Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes

UNLABELLED : Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mutations and included in the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been detected in ≈35% of patients with CINCA. However, no data are currently available regarding the relevance of this mechanism in other CAPS phenotypes. OBJECTIVE To evaluate somatic NLRP3 mosaicism as the disease-causing mechanism in patients with clinical CAPS phenotypes other than CINCA and NLRP3 mutation-negative. METHODS NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. RESULTS A variable degree (5.5-34.9%) of somatic NLRP3 mosaicism was detected in 12.5% of enrolled patients, all of them with a MWS phenotype. Six different missense variants, three novel (p.D303A, p.K355T and p.L411F), were identified. Bioinformatics and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. All patients treated with anti-interleukin1 drugs showed long-lasting positive responses. CONCLUSIONS We herein show somatic NLRP3 mosaicism underlying MWS, probably representing a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed definitive diagnoses of these patients, which had serious implications for gaining access to anti-interleukin 1 treatments under legal indication and for genetic counselling. The detection of somatic mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of modern genetic tools.

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

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

Involvement of Can f 5 in a Case of Human Seminal Plasma Allergy

Background: The existence of IgE binding to dog dander extract without IgE antibodies against the described dog allergens (Can f 1, 2, 3 and 4) implies the presence of other dog allergens yet to be identified. Recently, an IgE-binding protein was isolated from dog urine and identified as prostatic kallikrein; it has been named Can f 5. Cross-reactivity between a dog dander allergen and human prostate-specific antigen (PSA) has been described. The aim of this study was to identify the dog dander allergen that presents cross-reactivity with PSA and demonstrate its clinical relevance in our patient with human seminal plasma allergy. Methods: SDS-PAGE immunoblotting and inhibition tests were performed. Mass spectrometry was carried out to identify the protein involved in the allergy reactions. Results: SDS-PAGE immunoblotting-inhibition with an IgE-binding protein from dog prostatic secretion showed total IgE binding inhibition to a 28-kDa IgE-reactive band identified as PSA. The electroeluted protein from dog prostatic secretion was identified by mass spectrometry as Can f 5. IgE immunoblotting of human seminal plasma incubated with the serum of the patient revealed two IgE-binding bands (28 and 32.7 kDa). Both SDS-PAGE immunoblotting inhibition assays, with human seminal plasma or purified PSA in solid phase, showed complete IgE binding inhibition when the serum of the anaphylactic patient was preincubated with dog dander extract or recombinant Can f 5. Conclusions: The dog dander allergen that shows cross-reactivity with human PSA has been characterized and turns out to be the recently described Can f 5. We demonstrated the clinical relevance of this cross-reactivity in a patient.

DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes

Background: Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection‐ or stress‐associated stimuli, activating both caspase‐1–mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. Objective: We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. Methods: Inflammasome‐related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT‐PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor &kgr;B to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA–mediated downregulation, and pharmacologic inhibition. Results: We observed that inflammasome‐related genes are rapidly demethylated in both monocyte‐to‐macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor &kgr;B are downregulated. We analyzed DNA methylation levels of inflammasome‐related genes in patients with cryopyrin‐associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti–IL‐1 drugs display methylation levels similar to those of healthy control subjects. Conclusion: Our study is the first to demonstrate the involvement of DNA methylation–associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers.

Conjunctivitis and Total IgE in Lacrimal Fluid: Lacrytest Screening

Total tear IgE has been considered to play an important role in allergic conjunctivitis, and measurement has been considered useful for diagnosis. The aim of this study was to ascertain whether Lacrytest®, a new commercialised method to detect IgE levels in lacrimal fluid, could constitute a screening test for the diagnosis of allergic conjunctivitis. This was a cross-sectional study. Patients with seasonal and perennial allergic conjunctivitis, vernal keratoconjunctivitis and a control group were included. Clinical history, ophthalmic examination, skin prick test and conjunctival provocation test were obtained. Lacrytest® was later performed in all groups. Fifty-four patients were enrolled: thirty with IgE-mediated conjunctivitis and, nine with vernal keratoconjunctivitis and fifteen controls. Lacrytest® was negative in all controls, positive in 20% of the IgE-mediated conjunctivitis group and in 88.9% of the vernal keratoconjunctivitis group. Global statistically-significant differences were found among the three groups (P = .003). Sensitivity of the test in the IgE-mediated conjunctivitis group was 20%, specificity 100%, positive predictive value 100%, and negative predictive value 38.46%, while in VKC sensitivity was 88.88%, specificity 100%, positive predictive value 100%, and negative predictive value 93.75%. Our data confirm that this test is not useful for screening allergic conjunctivitis. Lacrytest®, while not providing any useful information to an allergist, could be helpful for ophthalmologists to confirm an IgE-mediated or VKC conjunctivitis.

Food allergy in Catalonia: Clinical manifestations and its association with airborne allergens

BACKGROUND Food allergy affects around 6% of the European population and its prevalence worldwide has been increasing in the last decades, but studies focused on investigating food allergy epidemiology in Europe are lacking. OBJECTIVE The Cibus project was created to register the main culprit foods and their clinical manifestations in food allergic patients in Catalonia. METHODS A specific online database was designed. Allergists from eight different Catalan hospitals registered the new diagnoses of food allergy. RESULTS 618 food allergic patients were included. Egg and milk were the main elicitors in the early ages, while fruits and nuts were the most frequent in patients >14 years old. Fish was more frequent in children, while seafood and Anisakis allergy were more frequent in the >14-year-old group. Overall, peach was the most prevalent food eliciting an allergic reaction (10%). Food allergy diagnosis was reached using compatible clinical history and positive skin prick test to the involved food in 98% of cases. Globally, urticaria was the most frequently reported manifestation in our population (48.2%), followed by oral allergy syndrome (25.6%) and anaphylaxis (24.8%). CONCLUSIONS The Cibus project gives a full overview of the profile of food allergic patients in Catalonia and reinforces the predominance of plant food allergies in the Mediterranean area.

Register of food and latex allergy in the Allergy Units of Catalonia

Results 278 patients, 232 adults (83.5%) and 46 children (16.5%) have been analyzed. The mean age in adults was 34 years (IQR: 26-44) and in children 5 years (IQR: 3-7). 57.2% were women and 42.8% men. 70% had a personal history of atopic disease. Milk and egg, 28.3% each, were the more frequently implicated foods in children. In adults; nuts (39.4%), rosaceae fruits (29.9%) and shellfish (11.6%) were the main causes of food allergy. Egg and banana allergy was more frequent in men and anisakis allergy in women. Urticaria (55.4%) was the most frequent clinical manifestation followed by anaphylaxis (32.4%) and oral allergy syndrome (31.7%). 97.4% of the anaphylactic reactions were presented in adults. Nuts caused 51% of the anaphylactic reactions, rosacea fruits the 35.6% and shellfish the 16.7%. Some foods like shellfish, fruits and nuts caused almost always symptoms while legumes and mustard sensitizations were asymptomatic in most patients. Eleven patients had latex allergy and 4 were sensitized to latex without symptoms. Urticaria was the clinical manifestation in most of the latex allergic patients (72%). Sensitization to airborne allergens was frequent in patients with food allergy 51% of the patients were sensitized to mites, 26.5% to grass, 24.5% to Olea, 18.6% to Platanus, 17.6% to Artemisia and 8.8% to Parietaria pollens.