EAACI Molecular Allergology User's Guide.

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.

A new framework for the interpretation of IgE sensitization tests.

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 coexisting 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 (pretest probability). The presence of each of these patient-specific factors may mean that a patient is more or less likely to have clinical allergy with a given test result (post-test probability). We present two approaches to include pretest probabilities in the interpretation of results. These approaches are currently limited by a lack of data to allow us to derive pretest probabilities for diverse setting, regions and allergens. Also, cofactors, 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.

Moving from peanut extract to peanut components: towards validation of component-resolved IgE tests.

BACKGROUND: Replacement of peanut extracts by recombinant peanut components is an important step in allergy serologic testing. Criteria are needed for the unbiased inclusion of patients into a study to validate such a replacement. METHODS: Plasma samples from 64 peanut-positive children (42 reactors, 22 nonreactors in a double-blind, placebo-controlled food challenge) were used to compare IgE reactivity to six recombinant peanut allergens with reactivity to natural peanut proteins extracted at neutral or low pH. We tested the hypothesis that poor extractability of Ara h 9 and other basic allergens at neutral pH leads to under-representation of patients with such sensitization. RESULTS: IgE reactivity to the components did not fully explain IgE reactivity to peanut extract in 5 of 32 reactors with IgE to peanut extract ≤100 kUA /l. IgE reactivity to components was stronger than to the extract in 11 plasma samples, which was largely due to a low Ara h 8 reactivity of the extract. IgE reactivity to Ara h 9 was much lower than reactivity to other basic proteins, some of which bound IgE well in the RAST, but lost IgE reactivity upon immunoblotting. CONCLUSIONS: Conventional peanut extracts are deficient in significant IgE-binding components. The inclusion of patients for a validation study should be based on serology performed with improved peanut reagents to avoid a bias against these under-represented, potentially important allergens. To judge clinical relevance of an allergen, the reagent used for inclusion of patients needs to be efficient in detecting IgE to this component.

IgE-binding epitopes: a reappraisal.

Here, we discuss various questions related to IgE epitopes: What are the technical possibilities and pitfalls, what is currently known, how can we put this information into hypothetical frameworks and the unavoidable question: how useful is this information for patient care or allergenicity prediction? We discuss the information obtained by (i) 3D structures of allergen-antibody complexes; (ii) analysis of allergen analogues; (iii) mimics without obvious structural similarity; (iv) mAbs competing with IgE; (v) repertoire analysis of cloned IgEs, and other developments. Based on limited data, four suggestions are presented in the literature: (i) IgE might be more cross-reactive than IgG; (ii) IgE might be more often directed to immunologically 'uninviting' surfaces; (iii) IgE epitopes may tend to cluster and (iv) IgE paratopes might have a higher intrinsic flexibility. While these are not proven facts, they still can generate hypotheses for future research. The hypothesis is put forward that the IgE repertoire of switched B-cells is less influenced by positive selection, because positive selection might not be able to rescue IgE-switched B cells. While this might be of interest for the discussion about mechanisms leading to allergen-sensitization, we need to be modest in answering the 'clinical relevance' question. Current evidence indicates the IgE-epitope repertoire is too big to make specific IgE epitopes a realistic target for diagnosis, treatment or allergenicity prediction. In-depth analysis of a few selected IgE epitope-peptides or mimitopes derived from allergen-sequences and from random peptide libraries, respectively, might well prove rewarding in relation to diagnosis and prognosis of allergy, particularly food allergy.

The role of IgG antibodies in allergy and immunotherapy.

In specific immunotherapy (SIT), a beneficial response is associated with an increase in allergen-specific IgG(4) . This does not indicate that IgE-producing B cells have switched to IgG(4) production, because in human DNA, IgE is downstream from IgG(4) . Thus, by conventional switching, B cells should produce IgG(4) before IgE. This presentation discusses three possible hypotheses explaining the favourable association between IgG(4) and SIT, including that SIT causes B cells to become regulatory. Regulatory B cells may produce cytokines like IL-10 that promote IgG(4) production. IgG(4) can undergo in vitro switching to create bispecific antibodies that recognize more than one antigen, which has important consequences for reducing IgE-allergen complexes in immunotherapy.

Differentiating the cellular and humoral components of neuromuscular blocking agent-induced anaphylactic reactions in patients undergoing anaesthesia.

BACKGROUND: The significance of IgE antibodies to neuromuscular blocking agent (NMBA)-induced anaphylactic reactions during anaesthesia is unclear. We investigated the relevance of IgE to rocuronium using an in vitro technique. METHODS: Serum samples from 61 patients with anaphylactic reactions during anaesthesia were investigated. On the basis of clinical history, allergy to NMBA was considered likely in 48 patients, further assessed using intradermal skin tests for several commonly used NMBAs, including rocuronium, vecuronium, and succinylcholine. To determine the presence of rocuronium IgE in human serum, a rocuronium-human serum albumin (rocHSA) conjugate was coupled to a solid phase and a radioallergosorbent test performed. The biological effects of patient serum NMBA-IgE on histamine release were investigated using in vitro sensitized basophils from healthy blood donors. RESULTS: IgE to rocuronium was found in 23 of 48 serum samples (48%) with NMBA allergy, although only two of these were able to sensitize basophils to release histamine in response to rocHSA. IgE-responsiveness in the basophil test was only observed with conjugated rocHSA and not with unconjugated rocuronium or the other NMBAs evaluated. However, unconjugated rocuronium inhibited the histamine release induced by rocHSA. Correlation between skin-test reactivity to rocuronium and IgE to rocHSA was low (P>0.1). In contrast, striking correlation between IgE to rocuronium and skin-test reactivity to succinylcholine was found (P<0.001). CONCLUSIONS: Our results indicate that NMBA-related anaphylaxis requires not only IgE NMBA reactivity, but also altered cellular reactivity in the patient. The latter may be demonstrable by testing basophils from the patient, a skin test with (steroidal) NMBA, or both.

IgG4 antibodies against rodents in laboratory animal workers do not protect against allergic sensitization.

BACKGROUND: The modified Th2 response, defined as an IgG4 response in the absence of IgE, is suggested to protect against the development of allergic sensitization. However, studies suggesting this protective effect all had a cross-sectional design, making it impossible to study the development of both responses. AIM OF THE STUDY: We aimed to study the dynamics in IgG4 antibodies in relation to allergic sensitization in an occupational cohort of starting laboratory animal workers. Moreover, we studied the relation between exposure, antibody responses, atopy and self reported allergic symptoms. METHODS: A total of 110 starting animal workers were followed for 2 years. IgG4 antibodies against rats and mice were assessed. Workers were tested for allergic sensitization and exposure to animal allergens was estimated. Symptom status was assessed using questionnaires. RESULTS: Rat and mouse specific IgG4 antibodies were present before the development of allergy and did not significantly change over time. Allergic sensitization was related to exposure and atopic status but high levels of IgG4 showed no protective effect. In contrary, workers that developed mouse specific sensitization during follow up had higher levels of mouse specific IgG4. Symptoms were related to allergic sensitization and IgG4 levels did not influence that relationship. CONCLUSIONS: IgG4 antibodies are present before IgE antibodies develop and IgG4 levels are stable over time. In our occupational cohort, the modified Th2 response had no protective effect on development of sensitization or allergic symptoms.

Dynamics in cytokine responses during the development of occupational sensitization to rats.

BACKGROUND: Occupational allergy forms an attractive model to study the development of allergic responses, as in some occupations it has a high incidence and develops quickly. In a cohort of starting laboratory animal workers, we previously found 20% sensitization to animal allergens within 2 years. METHODS: We compared cellular responses of incident laboratory animal workers who developed rat-specific sensitization (cases, n = 18) during 2 years of follow-up to control animal workers matched for atopic status but without sensitization after follow-up (controls, n = 18). Practically, this is a case-control study, nested within the cohort. Rat-specific IgE antibodies were measured in sera, and allergen-specific and nonspecific cytokine responses were measured in whole blood and in isolated peripheral blood mononuclear cells. RESULTS: Self-reported allergic symptoms were related to the presence of rat-specific IgE (P ≤ 0.01). Cases developed a rat allergen-specific interleukin (IL)-4 response during sensitization, while controls did not show an increased IL-4 response (at visit D: 33 vs 5 IL-4 producing cells/10(6) cells, P < 0.001). The IL-4 response was related to the levels of rat-specific IgE in cases (visit D: rho = 0.706, P < 0.001). By contrast, allergen-specific IL-10 and interferon γ (IFNγ) responses as well as nonspecific cytokine responses were comparable between cases and controls. CONCLUSION: This study is the first to show the development of an allergen-specific IL-4 response in adult human subjects during allergen-specific sensitization. This IL-4 response was quantitatively associated with the development of the specific IgE antibodies. Allergen-specific or nonspecific IL-10 and IFNγ responses showed no protective effect on the development of allergic sensitization.

Prevalence of antibodies against Ascaris suum and its association with allergic manifestations in 4-year-old children in The Netherlands: the PIAMA birth cohort study.

The association between helminth infections and childhood atopic diseases remains controversial. The majority of studies have been carried out in tropical areas, whereas less information is available from western countries with low intensity of helminth infections. In the Netherlands, the infection of pigs with Ascaris suum is very common, particularly on pig farms with outdoor facilities. This helminth can also infect humans, causing visceral larva migrans. This study aims at determining the prevalence of antibodies against A. suum and its association with allergic symptoms and sensitisation in a population of 4-year-old children living in The Netherlands. Blood samples from 629 children from the prospective birth cohort Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study were examined for Ascaris antibodies. Data on allergic symptoms and sensitisation were collected using questionnaires and radioallergosorbent tests (RAST). A total of 45 out of 629 (7%) were found to be Ascaris-seropositive. In addition, a positive association between Ascaris seropositivity and wheeze in the last year, doctor-diagnosed asthma and food and aero-allergen sensitisation was found. These results support the hypothesis that low-level or transient infection with helminths enhances allergic reactivity.

Breast feeding, parental allergy and asthma in children followed for 8 years. The PIAMA birth cohort study.

BACKGROUND: It is unclear how the association between breast feeding and asthma develops with age of the child and how this association over time is influenced by maternal or paternal allergy. These factors--the age of the child and maternal or paternal allergy--might partly explain the conflicting results observed in cross-sectional studies. METHODS: The study population consisted of 3115 Dutch children born in 1996/1997 who participated in the PIAMA (Prevention and Incidence of Asthma and Mite Allergy) birth cohort study. Data on breast feeding and asthma (based on wheeze, dyspnoea and prescription of inhaled steroids) were collected by yearly questionnaires. At 8 years, specific immunoglobulin E (IgE) to airborne allergens and bronchial responsiveness were measured. Data were analysed by logistic regression and generalised estimating equations (GEEs), and stratified by maternal and paternal allergic status. RESULTS: 35% (n = 1081) of the children were breast fed for >16 weeks. At 8 years of age, 12.6% (n = 392) had asthma. Breast feeding (>16 weeks vs no breast feeding) was significantly associated with a lower asthma prevalence from 3 to 8 years of age, in children of both non-allergic and allergic mothers. The inverse association between breast feeding and sensitisation to airborne allergens at 8 years was non-significant. Breast feeding was not associated with bronchial hyper-responsiveness. No interaction between breast feeding and gender, maternal allergy or paternal allergy was observed in any of the associations. CONCLUSIONS: Breast feeding is associated with a lower asthma risk in children until 8 years of age without evidence of attenuation and regardless of the family history of allergy.

Effects of pets on asthma development up to 8 years of age: the PIAMA study.

BACKGROUND: Recall bias may provide discrepant relationships of pet exposure with sensitization and asthma development. We studied prospectively effects of pets at home on development of sensitization, asthma and respiratory symptoms from birth up to age 8 years. METHODS: Event history analysis was performed on annually registered data of 2951 children, participating in the PIAMA birth cohort study. RESULTS: Children with a cat or dog at home at 3 months of age had a significantly lower prevalence of sensitization to inhalant allergens at age 8, but not of asthma. A cat decreased the risk of house dust mite sensitization at age 8 [odds ratio (OR) = 0.68, 95% confidence interval (CI) 0.49-0.95], a dog of pollen sensitization (OR = 0.49, 95% CI: 0.29-0.83). A cat or dog at home did not significantly affect asthma incidence in each subsequent year. From 2 years of age onwards, the incidence of wheeze (OR = 1.52, 95% CI: 1.12-2.05) and a dry cough at night (OR = 1.28, 95% CI: 1.05-1.57) was higher in children with a dog, whereas removal of a dog increased the risk of developing asthma symptoms. Comparing analyses using prospectively and retrospectively collected data on diagnosed asthma showed important recall bias. CONCLUSIONS: Our prospective study shows a protective effect of early presence of pets at home on sensitization to inhalant allergens, but no prevention of asthma development. Furthermore, children with pets had more frequent transient or intermittent asthma symptoms. Parental report of asthma by recall may provide spurious results of these associations.

Immunoglobulin G4: an odd antibody.

Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also referred to as 'Fab-arm exchange', results usually in asymmetric antibodies with two different antigen-combining sites. While these antibodies are hetero- bivalent, they will behave as monovalent antibodies in most situations. Another aspect of IgG4, still poorly understood, is its tendency to mimic IgG rheumatoid factor (RF) activity by interacting with IgG on a solid support. In contrast to conventional RF, which binds via its variable domains, the activity of IgG4 is located in its constant domains. This is potentially a source of false positives in IgG4 antibody assay results. Because regulation of IgG4 production is dependent on help by T-helper type 2 (Th2) cells, the IgG4 response is largely restricted to non-microbial antigens. This Th2-dependency associates the IgG4 and IgE responses. Another typical feature in the immune regulation of IgG4 is its tendency to appear only after prolonged immunization. In the context of IgE-mediated allergy, the appearance of IgG4 antibodies is usually associated with a decrease in symptoms. This is likely to be due, at least in part, to an allergen-blocking effect at the mast cell level and/or at the level of the antigen-presenting cell (preventing IgE-facilitated activation of T cells). In addition, the favourable association reflects the enhanced production of IL-10 and other anti-inflammatory cytokines, which drive the production of IgG4. While in general, IgG4 is being associated with non-activating characteristics, in some situations IgG4 antibodies have an association with pathology. Two striking examples are pemphigoid diseases and sclerosing diseases such as autoimmune pancreatitis. The mechanistic basis for the association of IgG4 with these diseases is still enigmatic. However, the association with sclerosing diseases may reflect an excessive production of anti-inflammatory cytokines triggering an overwhelming expansion of IgG4-producing plasma cells. The bottom line for allergy diagnosis: IgG4 by itself is unlikely to be a cause of allergic symptoms. In general, the presence of allergen-specific IgG4 indicates that anti-inflammatory, tolerance-inducing mechanisms have been activated. The existence of the IgG4 subclass, its up-regulation by anti-inflammatory factors and its own anti-inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions.

Asthma at 8 years of age in children born by caesarean section.

BACKGROUND: Caesarean section might be a risk factor for asthma because of delayed microbial colonisation, but the association remains controversial. A study was undertaken to investigate prospectively whether children born by caesarean section are more at risk of having asthma in childhood and sensitisation at the age of 8 years, taking into account the allergic status of the parents. METHODS: 2917 children who participated in a birth cohort study were followed for 8 years. The definition of asthma included wheeze, dyspnoea and prescription of inhaled steroids. In a subgroup (n = 1454), serum IgE antibodies for inhalant and food allergens were measured at 8 years. RESULTS: In the total study population, 12.4% (n = 362) of the children had asthma at the age of 8 years. Caesarean section, with a total prevalence of 8.5%, was associated with an increased risk of asthma (OR 1.79; 95% CI 1.27 to 2.51). This association was stronger among predisposed children (with two allergic parents: OR 2.91; 95% CI 1.20 to 7.05; with only one: OR 1.86; 95% CI 1.12 to 3.09) than in children with non-allergic parents (OR 1.36; 95% CI 0.77 to 2.42). The association between caesarean section and sensitisation at the age of 8 years was significant only in children of non-allergic parents (OR 2.14; 95% CI 1.16 to 3.98). CONCLUSIONS: Children born by caesarean section have a higher risk of asthma than those born by vaginal delivery, particularly children of allergic parents. Caesarean section increases the risk for sensitisation to common allergens in children with non-allergic parents only.

Repeated measurements of mite and pet allergen levels in house dust over a time period of 8 years.

BACKGROUND: Studies of the association between indoor allergen exposure and the development of allergic diseases have often measured allergen exposure at one point in time. OBJECTIVE: We investigated the variability of house dust mite (Der p 1, Der f 1) and cat (Fel d 1) allergen in Dutch homes over a period of 8 years. METHODS: Data were obtained in the Dutch PIAMA birth cohort study. Dust from the child's mattress, the parents' mattress and the living room floor was collected at four points in time, when the child was 3 months, 4, 6 and 8 years old. Dust samples were analysed for Der p 1, Der f 1 and Fel d 1 by sandwich enzyme immuno assay. RESULTS: Mite allergen concentrations for the child's mattress, the parents' mattress and the living room floor were moderately correlated between time-points. Agreement was better for cat allergen. For Der p 1 and Der f 1 on the child's mattress, the within-home variance was close to or smaller than the between-home variance in most cases. For Fel d 1, the within-home variance was almost always smaller than the between-home variance. Results were similar for allergen levels expressed per gram of dust and allergen levels expressed per square metre of the sampled surface. Variance ratios were smaller when samples were taken at shorter time intervals than at longer time intervals. CONCLUSION: Over a period of 4 years, mite and cat allergens measured in house dust are sufficiently stable to use single measurements with confidence in epidemiological studies. The within-home variance was larger when samples were taken 8 years apart so that over such long periods, repetition of sampling is recommended.

Mouse allergen-specific immunoglobulin G4 and risk of mouse skin test sensitivity.

BACKGROUND: High serum levels of cat-specific IgG and IgG4 are associated with protection against allergic sensitization to cat, but whether this association applies to other animal allergens remains unclear. OBJECTIVE: To determine if high levels of mouse-specific IgG and IgG4 are associated with a decreased risk of mouse skin test sensitivity. METHODS: Two hundred and sixty workers of a mouse facility underwent skin prick testing and completed a questionnaire. Serum levels of mouse-specific IgG and IgG4 were quantified by solid-phase antigen binding assays. Room air samples were collected and airborne Mus m 1 was quantified by ELISA. RESULTS: Forty-nine participants had a positive skin prick test to mouse. Mouse-specific IgG was detected in 219 (84%) participants and IgG4 was detected in 72 (28%) participants. A detectable mouse-specific IgG4 level was associated with an increased risk of mouse skin test sensitivity (odds ratios (OR) 6.4, 95% confidence intervals (CI) 3.3-12.4). Mouse-specific IgG and IgG4 were both positively correlated with mouse allergen exposure (r(s)=0.31, P=0.0001, and r(s)=0.27, P=0.0006, respectively). The odds of skin test sensitivity peaked at moderate levels of IgG4, but decreased at the highest levels of mouse-specific IgG4. In contrast, the odds of skin test sensitivity increased monotonically with IgG levels. CONCLUSIONS: A detectable level of mouse-specific IgG4 is associated with an increased risk of skin test sensitivity to mouse. However, the highest IgG4 levels appear to be associated with an attenuated risk of mouse skin test sensitivity, suggesting that induction of high levels of IgG4 through natural exposure may protect against the development of allergic sensitization.

The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor.

Healthy volunteers are hyperimmunized with RhD-positive red cells in order to obtain plasma containing high titres of anti-D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti-D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti-D titres despite re-immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig-gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD-binding phages from both libraries, except one, had heavy chains with IGH-VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3-positive B cells. We show the presence of clonally related RhD-specific B cells in a hyperimmunized anti-D donor who had declining anti-D titres and who was unresponsive to re-immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti-D donors.

Mouse allergen-specific immunoglobulin G and immunoglobulin G4 and allergic symptoms in immunoglobulin E-sensitized laboratory animal workers.

BACKGROUND: High levels of allergen-specific IgG have been associated with clinical efficacy in immunotherapy studies, but whether this antibody isotype is associated with clinical tolerance in the setting of environmental exposure remains unclear. OBJECTIVE: To determine if mouse allergen-specific IgG (mIgG) and IgG4 (mIgG4) levels are associated with mouse-related symptoms among IgE-sensitized laboratory workers. METHODS: Fifty-eight workers with either skin test or serologic evidence of IgE-mediated mouse sensitization were studied. Symptom data were obtained by a questionnaire. Serum levels of mouse-specific IgG, IgG4, and IgE were quantified by a solid-phase antigen-binding assay (IgG) and RAST (IgG4 and IgE), and the relationships between mouse-specific serologic responses and mouse-related symptoms were analysed. RESULTS: Twenty-three (39.7%) participants reported mouse-related symptoms. Mouse-specific IgG and IgG4 levels were not associated with mouse-related symptoms among the study population as a whole. Among the 29 (50%) participants with detectable mouse-specific IgE (mIgE), higher mouse-specific IgG and IgG4 levels were associated with a decreased risk of symptoms, after adjusting for mIgE level (odds ratio (OR) 0.3, 95% confidence interval (CI): 0.1-1.4, and OR 0.3, 95% CI: 0.04-2.6, respectively). Higher levels of mIgG and mIgG4 remained associated with a decreased risk of symptoms after additional adjustment for sex and handling of mice (OR 0.1, 95% CI: 0.02-0.7, and OR 0.2, 95% CI: 0.02-2.1, respectively). Higher mIgG : IgE and mIgG4 : IgE ratios were also associated with a decreased risk of symptoms after adjusting for these confounders (OR 0.1, 95% CI: 0.02-0.7, and OR 0.2, 95% CI: 0.02-0.92, respectively). CONCLUSION: Among workers with detectable mIgE, higher mIgG and mIgG4 levels are associated with a decreased risk of mouse-related symptoms. High serum levels of mIgG or mIgG4 may be markers for clinical tolerance among laboratory mouse workers with detectable mIgE, but these findings need to be confirmed in larger, prospective studies.