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Alergia; Anafilaxia; Asma Allergy; Anaphylaxis; Asthma Al·lèrgia; Anafilaxi; Asma Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the “EAACI Molecular Allergology User's Guide” (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be “classical” cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.

Background Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. Objective To analyse the allergenic composition of the basic peanut protein (BPP) fraction. Methods A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. Results Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. Conclusion The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.

Crossreactivity of IgE antibodies are occasionally unexpected and confusing. Moreover, the implications are not always the same. Some types of serologic crossreactivities usually produce clinical symptoms, whereas other do not. Even the latter type may be relevant, as it may contribute to the induction of immune responses to the crossreactive antigens ('crossreactive priming'). For the interpretation of IgE antibody assays it is important to appreciate that finding IgE antibodies to some allergen does not imply that the patient has ever been exposed to that allergen.

Background It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co-vaccination with cellular Bordetella pertussis vaccine. Methods In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co-vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2- or 5-component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65). Results One month after vaccination, D-IgE was detected in 10% sera of DTPw-vaccinated children, whereas vaccination in the absence of whole-cell pertussis resulted in 50%-60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D-IgG and T-IgG. No immediate-type allergic reactions were observed. Conclusion Cellular (but not acellular) B pertussis vaccine downregulates IgE to co-vaccinated antigens in infants. We assume that the absence of immediate-type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Introduction IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory condition characterised by an abundance of IgG4+ antibodies in the serum and tissue of involved organs. IgG glycosylation plays an important role in many chronic inflammatory and autoimmune conditions. We sought to assess the glycosylation status in patients with IgG4-RD and correlate with disease activity, damage and response to treatment. Methods IgG Fc and Fab glycosylation status was assessed in patients with IgG4-RD involving the bile ducts (IgG4-sclerosing cholangitis, IgG4-SC) and pancreas (autoimmune pancreatitis) (n=22), disease controls with primary sclerosing cholangitis (DC n=22) and healthy controls (HC n=22). Disease activity, organ damage and response to treatment were assessed serially using the IgG4-responder index. Serum IgG and subclasses were quantified using an ELISA and nephelometry. IgG and subclass Fc glycosylation was analysed by mass spectrometry and Fab glycosylation by lectin (SNA) affinity chromatography. Statistics were performed using Prism. Results IgG4-SC and AIP patients exhibited reduced total IgG Fc galactosylation and IgG1 Fc bisection, and increased IgG4 Fc fucosylation and IgG2/3 Fc hybrid compared with HC. There was recovery of IgG1 Fc bisection (increase) and IgG2/3 Fc hybrid (decrease) upon corticosteroid treatment. IgG Fc galactosylation and IgG2/3 Fc hybrid correlated with disease activity. IgG Fab glycosylation was higher in IgG4-RD patients, with an increase in IgG4-specific, and to a lesser extent IgG1-specific, Fab glycosylation compared to HC and DC. Conclusions In the first study to assess glycosylation status in IgG4-RD, we demonstrated alterations in both IgG Fc and Fab glycosylation, which may play a role in pathophysiology and serve as a biomarker of disease.

International audience; No abstract available

Seit vielen Jahren setzen Allergologen auf IgE-Tests wie Hautpricktests und die Messung der spezifischen IgE-Antikorper im Serum gegen das vollstandige Allergen, um IgE-vermittelte, klinisch relevante Allergien zu diagnostizieren. Diese Tests haben einen wesentlichen Nachteil: Sie stellen zwar eine Sensibilisierung fest, diese hangt aber nur indirekt mit der klinisch relevanten Allergie zusammen. Mit vielen Patienten mussen daher Provokationstests durchgefuhrt werden, die haufig teuer und mit potenziell schwerwiegenden Reaktionen verbunden sind, um eine endgultige Diagnose zu stellen. Die Wahrscheinlichkeit fur eine klinisch relevante Allergie kann durch die Ergebnisse eines IgE-Tests semi-quantitativ bestimmt werden. Wie belastbar diese Korrelation ist, variiert je nach Alter, ethnischer Herkunft und Art der vermeintlich allergischen Reaktion des Patienten, und wird durch den Grad der Pollenexposition, die Ernahrung sowie bestehende klinische Erkrankungen, etwa Ekzeme, beeinflusst. Werden diese individuellen Faktoren bei der Auswertung eines IgE-Tests berucksichtigt, kann die Wahrscheinlichkeit fur das Auftreten einer klinisch relevanten Allergie praziser abgeschatzt werden (Pratestwahrscheinlichkeit). Beruhend auf den Testergebnissen weist dann das Vorhandensein jeder dieser patientenspezifischen Charakteristika darauf hin, ob bei einem Patienten mit hoherer oder geringerer Wahrscheinlichkeit eine klinisch relevante Allergie vorliegt (Posttestwahrscheinlichkeit). Die Einbeziehung von Ko-Faktoren, wie zum Beispiel sportliche Betatigung, konnten auch erforderlich sein, um sensitivere und genauere Testresultate zu erzielen. In dieser Arbeit werden zwei Ansatze vorgestellt, wie Pratestwahrscheinlichkeiten bei der Interpretation der IgE-Testergebnisse eingebunden werden konnen. Obwohl diese erhebliche Vorteile bieten, speziell in der Klinik, muss noch mehr in diesen Forschungsbereich investiert werden, um kunftig die Pratestwahrscheinlichkeiten fur verschiedene Bedingungen, Lebensregionen und Allergene ableiten zu konnen. Ebenfalls muss noch genauer geklart werden, welchen Stellenwert die Testergebnisse in den verschiedenen Populationen haben. Kliniker mussen dafur sorgen, dass die Patienten die Testergebnisse klar verstehen, um ihnen Selbstvertrauen zum Selbstmanagement zu geben.

Highlights • Room temperature structure of human IgG4-Fc solved from crystals analysed in situ. • Structure reveals changes in crystal packing at different temperatures. • Structure reveals physiologically relevant conformation of a key Fcγ receptor binding loop., The Fc region of IgG antibodies (Cγ2 and Cγ3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fcγ receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fcγ receptor interactions is the FG loop in the Cγ2 domain. High resolution cryogenic IgG4-Fc crystal structures have revealed a unique conformation for this loop, which could contribute to the particular biological properties of this subclass. To further explore the conformation of the IgG4 Cγ2 FG loop at near-physiological temperature, we solved a 2.7 Å resolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The Cγ2 FG loop in one chain differs from the cryogenic structure, and adopts the conserved conformation found in IgG1-Fc; however, this conformation participates in extensive crystal packing interactions. On the other hand, at room temperature, and free from any crystal packing interactions, the Cγ2 FG loop in the other chain adopts the conformation previously observed in the cryogenic IgG4-Fc structures, despite both conformations being accessible. The room temperature human IgG4-Fc structure thus provides a more complete and physiologically relevant description of the conformation of this functionally critical Cγ2 FG loop.

IgE sensitization tests, such as skin prick testing and serum specific IgE, have been used to diagnose IgE-mediated clinical allergy for many years. Their prime drawback is that they detect sensitization which is only loosely related to clinical allergy. Many patients therefore require provocation tests to make a definitive diagnosis; these are often expensive and potentially associated with severe reactions. The likelihood of clinical allergy can be semi-quantified from an IgE sensitization test results. This relationship varies though according to the patients’ age, ethnicity, nature of the putative allergic reaction and co-existing clinical diseases such as eczema. The likelihood of clinical allergy can be more precisely estimated from an IgE sensitization test result, by taking into account the patient's presenting features (pre-test probability). The presence of each of these patient specific factors may mean that a patient is more or less likely to have clinically allergy with a given test result (post-test probability). We present two approaches to including pre-test probabilities in the interpretation of results. These approaches are currently limited by a lack of data to allow us to derive pre-test probabilities for diverse setting, regions and allergens. Also, co-factors, such as exercise, may be necessary for exposure to an allergen to result in an allergic reaction in specific IgE positive patients. The diagnosis of IgE-mediated allergy is now being aided by the introduction of allergen component testing which may identify clinically relevant sensitization. Other approaches are in development with basophil activation testing being closest to clinical application.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

In addition to the original description of the atopic march as a fixed sequence of clinical features, data from birth cohorts suggest an immunological counterpart. The development of atopic sensitization has been found to loosely follow a pattern starting with immunoglobulin E (IgE) to foods (milk and, unexpectedly, egg), then indoor airborne allergens (particularly mites), and finally, outdoor allergens (typically from pollen). For the different allergen source materials, the aim is to identify allergens that are prone to initiate sensitization. For house dust mites, no convincing initiator has been identified. For grass pollen, group 1 allergen was the probable initiator in 75% of children. A plausible scenario for the subsequent expansion of repertoire is that mucosal inflammation induced by allergen–IgE–mast cell reactions creates a milieu that facilitates new sensitizations. Although the recent cohort data do not yet add convincing support for this hypothesis, it remains an important topic for further investigation.

A common reaction from anyone confronted with allergy is the question: what prevents universal allergy? We will discuss recent findings in the mouse system that have provided us with clues on why allergy is not more common. We will also address one crucial aspect of atopic allergy in humans, which is absent in most mouse model systems, an IgG/IgE ratio

Molecular allergen-based component-resolved diagnostic IgE antibody tests have emerged in the form of singleplex assays and multiplex arrays. They use both native and recombinant allergen molecules, sometimes in combination with each other, to supplement allergen extract-based IgE antibody analyses. The total number of available allergenic molecules has reached a diagnostically useful level; however, more molecules are needed to cover all the clinically important allergen specificities. Thus, for the foreseeable future, molecular allergen-specific IgE analyses will remain a supplement for initial allergen extract-based IgE antibody analyses in the diagnostic workup of the allergic patient. As a spin-off, it will enable manufacturers to improve the quality of extracts for in vitro testing. The 2 most exciting diagnostic developments linked to component-resolved diagnostic tests are the possibility to increase diagnostic sensitivity by the inclusion of allergens that are underrepresented in the current extracts and in vitro assays and to increase the diagnostic specificity by taking the information on allergen cross-reactivity into account. Particularly the latter application is still under development. This requires additional studies on the clinical relevance of serological cross-reactivity.

with a positive skin test to shrimp extract, of whom 58 were established to be allergic. IgE to a panel of 10 recombinant crustacean allergens (mostly muscle proteins) was tested in a dot-blot assay. As expected, tropomyosin was the dominating component being positive not only in 83% of the 58 allergic subjects but also in 44% of the 16 tolerant subjects. Its false-negativity rate of 17% is disappointing and intriguing. Of the other components tested, arginine kinase had the highest sensitivity (48%), followed by myosin light chain (38%) and sarcoplasmic calcium-binding protein (34%). However, even with the complete panel the false-negativity rate was still 12%. This deficiency of the panel could be due to technical problems with the expression of the recombinant proteins, but could also reflect species differences. Lured by the high cross-reactivity of tropomyosin, we might be tempted to assume erroneously that our immune system is unable to distinguish invertebrates. An illustrative example to the contrary is the limited cross-reactivity of the major mite allergens. THE 4 DIMENSIONS OF ALLERGY DIAGNOSIS The first dimension is diagnostic sensitivity: the ability to identify more than 95% of the patients with allergy to the source in question. Cross-reactivity is convenient for shrimp allergy diagnosis, because of the many different shrimp species that patients may be exposed to. Without cross-reactivity, we would need to test for reactivity to all these species. From this perspective, it is a positive feature that the cross-reactivity amongst invertebrate tropomyosins is very high. On the other hand, more than 10% of the shrimp-allergic patients were found to be negative for tropomyosin. Because positivity to shrimp extract was the inclusion criterion, the potential exclusion of some patients may even have resulted in a too optimistic estimate of test sensitivities. It might be worthwhile to look for other cross-reactive shrimp allergens that might be used alone or in combination with tropomyosin to achieve a higher diagnostic sensitivity. The other 3 dimensions are all related to specificity. The second dimension is the ability to distinguish between sensitized subjects with allergy and those without allergy. Exposure to a cross-reactive substance is often (but not always, see below) better tolerated than exposure to the sensitizing substance. In the present study population 11 (as in other studies 4 ), almost all shrimp-allergic patients were sensitized to mites. It would be interesting to know how many of these mite-sensitized patients were mite-tolerant and negative for the major group 1 and group 2 mite allergens. The third dimension is the ability to distinguish between allergen source materials that do or do not induce symptoms. In the case of shellfish allergy, it is generally assumed that consumption of all species of invertebrates should be avoided. In view of the promotion of invertebrate meat sources as an alternative for mammalian meat, the general validity of this approach is under scrutiny. In a fish allergy study, consumption of crossreactive fish species was in some cases well tolerated. 12