Understanding of the Molecular Mechanisms of Allergy.

The prevalence of allergic disease has dramatically increased over the past 30 years in Westernized countries. It is unlikely that the rapid increase in the prevalence of allergic disease is the result of genetic changes, which highlights the importance of environmental factors in the development of allergic disease. The "hygiene hypothesis" was put forward in 1989 and focused attention on the notion that exposure to microbes and their products in early life can modify the risk for development of allergic disease. Infections were thought to polarize the immunological response toward a Th2-mediated immune response causing allergic disease. However, it is likely that the Th1/Th2 imbalance is too simplistic to explain the increased prevalence of allergic disease. Current research is focusing on understanding the role of T-regulatory cells in inducing a state of tolerance and the resulting modified Th2 response observed in natural and induced tolerance.

Measurement of Specific IgG Anti-Fel d 1 Antibodies.

There is currently considerable interest in the role of specific IgG antibodies in allergy. Several studies suggest that specific IgG antibodies may play a protective role in allergy. Successful immunotherapy is associated with increases in allergen-specific IgG antibodies which correlate with clinical outcome. Other studies have identified an inverse relationship between exposure to cat and sensitization, which was associated with high-titer-specific IgG and IgG4. This immune response was described as a modified Th2 response, since both IgE and IgG4 require Th2 cytokine IL-4 for their production. A modified Th2 response was described with laboratory animal allergy, where there was almost a twofold reduction in the risk of developing work-related chest symptoms.In this chapter, we review the major factors to be considered in the development of an ELISA for the determination of specific IgG and IgG4 antibodies.

Laboratory animal allergy is preventable in modern research facilities.

BACKGROUND: Historical data suggest 15% of laboratory animal workers develop IgE sensitisation and 10% symptoms of laboratory animal allergy (LAA), including occupational asthma. Individually ventilated cages (IVCs) are replacing conventional open cages; we sought to evaluate their impact on the development of LAA. METHODS: We surveyed 750 laboratory animal workers and measured airborne Mus m 1 (mouse allergen) levels in seven UK institutions. We compared the prevalence of sensitisation to mouse proteins (by specific IgE assay or skin prick test) and of work-related allergic symptoms in IVC-only and open cage units. RESULTS: Full-shift Mus m 1 levels were lower in IVC than open cage units (geometric mean 1.00 (95% CI 0.73-1.36) versus 8.35 (95% CI 6.97-9.95) ng·m-3; p<0.001), but varied eight-fold across the IVC units (geometric mean range 0.33-4.12 ng·m-3). Primary analyses on data from 216 participants with ≤3 years exposure to mice revealed a lower prevalence of sensitisation in those working in IVC units compared with conventional cage units (2.4% (n=2) versus 9.8% (n=13); p=0.052). Sensitisation in IVC units varied from 0% to 12.5%; the use of fitted respiratory protection was less common in IVC units where prevalence of sensitisation was higher. Work-related allergy symptoms were more frequently reported by mouse-sensitised individuals (46.7% versus 10.9%; p<0.001) and only by those working in open cage units. CONCLUSION: In contemporary practice, LAA is now largely preventable with the use of IVC systems and the judicious use of appropriate respiratory protection.

Immunological methods for diagnosis and monitoring of IgE-mediated allergy caused by industrial sensitizing agents (IMExAllergy).

Industrial sensitizing agents (allergens) in living and working environments play an important role in eliciting type 1 allergic disorders including asthma and allergic rhinitis. Successful management of allergic diseases necessitates identifying their specific causes (ie, identify the causative agent(s) and the route of contact to allergen: airborne, or skin contact) to avoid further exposure. Identification of sensitization by a sensitive and validated measurement of specific IgE is an important step in the diagnosis. However, only a limited number of environmental and occupational allergens are available on the market for use in sIgE testing. Accordingly, specific in-house testing by individual diagnostic and laboratory centers is often required. Currently, different immunological tests are in use at various diagnostic centers that often produce considerably divergent results, mostly due to lack of standardized allergen preparation and standardized procedures as well as inadequate quality control. Our review and meta-analysis exhibited satisfactory performance of sIgE detection test for most high molecular weight (HMW) allergens with a pooled sensitivity of 0.74 and specificity of 0.71. However, for low molecular weight (LMW) allergens, pooled sensitivity is generally lower (0.28) and specificity higher (0.89) than for HMW tests. Major recommendations based on the presented data include diagnostic use of sIgE to HMW allergens. A negative sIgE result for LMW agents does not exclude sensitization. In addition, the requirements for full transparency of the content of allergen preparations with details on standardization and quality control are underlined. Development of standard operating procedures for in-house sIgE assays, and clinical validation, centralized quality control and audits are emphasized. There is also a need for specialized laboratories to provide a custom service for the development of tests for the measurement of putative novel occupational allergens that are not commercially available.

Metal worker's lung: spatial association with Mycobacterium avium.

BACKGROUND: Outbreaks of hypersensitivity pneumonitis (HP) are not uncommon in workplaces where metal working fluid (MWF) is used to facilitate metal turning. Inhalation of microbe-contaminated MWF has been assumed to be the cause, but previous investigations have failed to establish a spatial relationship between a contaminated source and an outbreak. OBJECTIVES: After an outbreak of five cases of HP in a UK factory, we carried out blinded, molecular-based microbiological investigation of MWF samples in order to identify potential links between specific microbial taxa and machines in the outbreak zone. METHODS: Custom-quantitative PCR assays, microscopy and phylogenetic analyses were performed on blinded MWF samples to quantify microbial burden and identify potential aetiological agents of HP in metal workers. MEASUREMENTS AND MAIN RESULTS: MWF from machines fed by a central sump, but not those with an isolated supply, was contaminated by mycobacteria. The factory sump and a single linked machine at the centre of the outbreak zone, known to be the workstation of the index cases, had very high levels of detectable organisms. Phylogenetic placement of mycobacterial taxonomic marker genes generated from these samples indicated that the contaminating organisms were closely related to Mycobacterium avium. CONCLUSIONS: We describe, for the first time, a close spatial relationship between the abundance of a mycobacterium-like organism, most probably M. avium, and a localised outbreak of MWF-associated HP. The further development of sequence-based analytic techniques should assist in the prevention of this important occupational disease.

Occupational allergy to fruit flies (Drosophila melanogaster) in laboratory workers.

OBJECTIVES: Drosophila melanogaster (the 'fruit fly') is commonly used in genetic research, but there is only one report of IgE-associated allergy in exposed workers. 4 newly identified cases prompted us to examine the extent of this problem in a university laboratory. Our aim in this study is to determine the prevalence and determinants of sensitisation to fruit flies in a population of exposed workers. METHODS: In a cross-sectional study, we surveyed 286 employees working in a department carrying out research involving D. melanogaster. Sensitisation was assessed by specific IgE measurement in serum and examined in relation to symptoms and to estimated exposure to fruit flies. RESULTS: The overall prevalence of specific sensitisation was 6% with a clear relationship to increasing frequency/intensity of exposure (p trend<0.001). Work-related eye/nose, chest or skin symptoms were reported by substantial proportions of participants but for most of these there was no evidence of specific sensitisation to fruit fly. The overall prevalence of any work-related symptoms and sensitisation was 2.4%, rising to 7.1% in those working in high exposure groups. CONCLUSIONS: We were able to demonstrate, for the first time, a clear exposure-response relationship between fruit fly exposure and specific sensitisation. Facilities housing fruit flies should carefully consider methods to reduce exposure levels in the workplace.

Association of pulse wave velocity with total lung capacity: A cross-sectional analysis of the BOLD London study.

BACKGROUND: Low lung function, measured using spirometry, has been associated with mortality from cardiovascular disease, but whether this is explained by airflow obstruction or restriction is a question that remains unanswered. OBJECTIVES: To assess the association of total lung capacity (TLC), forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) with several cardio-metabolic and inflammatory markers. METHODS: In the follow up of the Burden of Lung Disease (BOLD) study in London, acceptable post-bronchodilator spirometric, pulse rate, pulse wave velocity and blood pressure data were obtained from 108 participants. Blood samples for measurement of cardio-metabolic and inflammatory markers were also collected from these participants. Association of lung function and volume with the different biomarkers was examined in multivariable linear regression models adjusted for potential confounders. RESULTS: Following adjustment for age, sex, height, and ethnicity, TLC (adjusted coefficient = -1.53; 95% CI: -2.57, -0.49) and FVC (adjusted coefficient = -2.66; 95% CI: -4.98, -0.34) were inversely associated with pulse wave velocity, and further adjustment for smoking status, pack-years and body mass index (BMI) did not materially change these results. FEV1 was inversely associated with systolic blood pressure, and adjustment for smoking status, pack-years and BMI made this association stronger (adjusted coefficient = -9.47; 95% CI: -15.62, -3.32). CONCLUSION: The inverse association of pulse wave velocity, which is a marker of cardiovascular disease, with TLC suggests that the association of the former with low FVC is independent of airflow obstruction. The association between FEV1 with systolic blood pressure after adjustment for FVC suggests an association with airflow obstruction rather than with restricted spirometry.

Laboratory Animal Allergy in the Modern Era.

Laboratory animal workers face a high risk of developing laboratory animal allergy as a consequence of inhaling animal proteins at work; this has serious consequences for their health and future employment. Exposure to animal allergen remains to be the greatest risk factor although the relationship is complex, with attenuation at high allergen exposure. Recent evidence suggests that this may be due to a form of natural immunotolerance. Furthermore, the pattern of exposure to allergen may also be important in determining whether an allergic or a tolerant immune response is initiated. Risk associated with specific tasks in the laboratory need to be determined to provide evidence to devise a code of best practice for working within modern laboratory animal facilities. Recent evidence suggests that members of lipocalin allergens, such as Mus m 1, may act as immunomodulatory proteins, triggering innate immune receptors through toll-like receptors and promoting airway laboratory animal allergy. This highlights the need to understand the relationship between endotoxin, animal allergen and development of laboratory animal allergy to provide a safe working environment for all laboratory animal workers.

Causes of discordance between thyroglobulin antibody assays.

BACKGROUND: Anti-thyroglobulin (Anti-Tg) assays show poor concordance. METHODS: We have investigated concordance and the causes of discordance between Abbott, Roche and Immulite Anti-Tg assays in 606 patients followed up for differentiated thyroid cancer (DTC). The reference range (RR) or lower reporting limit (LRL) was used to classify samples as negative or positive. RESULTS: Anti-Tg prevalence ranged between 6% and 55% depending on the method and cut-off. Concordance was 45% using LRL and 75% using RR. Specimens between the RR and LRL using the Immulite and Roche assays were identified that were positive by the Abbott assay and showed poor recovery of Tg in the Tg assay. This suggests misclassification using the RR. Anti-Tg International Reference Preparation (IRP) concentrations measured by the Roche and Abbott methods agreed well but patient samples did not. This is likely to be due to the heterogeneity of Anti-Tg. The Immulite assay appeared less sensitive than the Abbott and Roche based on investigations using the IRP and the low prevalence of Anti-Tg in the DTC patients (6-8%). Interference by Tg (>1000 µg/L) in the Roche assay was also identified as a cause of assay discordance. CONCLUSIONS: Anti-Tg is used as a tumour marker for DTC and to predict interference in Tg assays themselves and hence inform clinicians of reported Tg concentrations. We have identified several causes of Anti-Tg assay discordance. This includes variation in assay sensitivity and interference from Tg, the heterogeneity of Anti-Tg and the use of different cut-offs to classify samples as antibody-positive or -negative.

Association of HLA class II alleles with sensitization to cow dander Bos d 2, an important occupational allergen.

Allergic sensitization results from a complex interaction between genetic and environmental factors. Earlier studies have shown that highly polymorphic HLA genes are associated with a variety of allergies. Several important respiratory allergens belong to the family of lipocalin proteins. These include occupational sensitizers, such as cow Bos d 2 or rat Rat n 1, and prevalent indoor sensitizers, such as dog Can f 1 or cockroach Bla g 4. HLA associations with sensitization to lipocalin allergens are incompletely known. In the present study we have investigated an association between HLA alleles and sensitization to the major cow allergen Bos d 2. The HLA-DR/DQ genotypes of 40 Bos d 2-sensitized subjects having occupational asthma were determined by polymerase chain reaction (PCR) and the results were compared with the genotypes of 151 unrelated Finnish subjects. The frequencies of HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501 were significantly higher among Bos d 2-sensitized than among control subjects. In addition, the allergic subjects expressed significantly lower frequencies of HLA-DRB1*0301 and DQB1*0201 alleles than did the control subjects. These data suggest that the HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501, and the haplotypes that include them, are associated with sensitization to the major cow allergen Bos d 2, whereas HLA-DRB1*0301 and DQB1*0201 are dissociated with it. Amino acid analysis provides a biologically plausible explanation for the HLA associations.

Understanding of the molecular mechanisms of allergy.

The prevalence of allergic disease has dramatically increased over the past 30 years in Westernised countries. It is unlikely that the rapid increase in the prevalence of allergic disease is the result of genetic changes, which highlights the importance of environmental factors in the development of allergic disease. The 'hygiene hypothesis' was put forward in 1989 and focused attention on the notion that exposure to microbes and their products in early life can modify the risk for development of allergic disease. Infections were thought to polarize the immunological response towards a Th2-mediated immune responses causing allergic disease. However it is likely that the Th1/Th2 imbalance is too simplistic to explain the increased prevalence of allergic disease. Current research is focusing on understanding the role of T regulatory cells in inducing a state of tolerance and the resulting modified Th2 response observed in natural and induced tolerance.

Measurement of specific IgG anti-Fel d 1 antibodies.

There is currently considerable interest in the role of specific IgG antibodies in allergy. Several studies suggest that specific IgG antibodies may play a protective role in allergy. Successful immunotherapy is associated with increases in allergen-specific IgG antibodies which correlate with clinical outcome. Other studies have identified an inverse relationship between exposure to cat and sensitization, which was associated with high titer specific IgG and IgG(4). This immune response was described as a modified Th2 response, because both IgE and IgG(4) require Th2 cytokine IL-4 for their production. A modified Th2 response was described with laboratory animal allergy, where there was almost a twofold reduction in the risk of developing work-related chest symptoms.In this chapter, we review the major factors to be considered in the development of an ELISA for the determination of specific IgG and IgG(4) antibodies.

Exposure-response in occupational allergy.

PURPOSE OF REVIEW: This review examines the relationship between exposure to workplace allergens and the risk of developing occupational allergy. RECENT FINDINGS: Evidence suggests that the risk of developing occupational allergy increases with allergen exposure; however, with some occupational allergens, this exposure-response relationship is more complex. In laboratory animal workers, the risk of developing occupational allergy increases with exposure, except at high allergen exposure when there is a reduction in sensitization. This attenuation of specific immunoglobulin E antibody is associated with increased specific immunoglobulin G4 antibodies, which are likely to play a protective role, leading to a form of natural tolerance. Exposure-response relationships are also very dependent on the genetic susceptibility of the individual. The interaction between genes, occupational allergens and other cofactors in the environment, such as endotoxin, are also important risk factors in the development of sensitization and asthma. SUMMARY: Occupational allergy provides a good opportunity to understand the complex relationships between exposure to allergens in the workplace, interaction with genes and the coexposures with other factors in the working environment and the increased risk of developing occupational allergy.

Identification of the major allergens of Indian scad (Decapterus russelli).

The purpose of this study was to characterize major allergens of Indian scad (Decapterus russelli) which is among the most commonly consumed fish in Malaysia. Raw and cooked extracts of the fish were prepared. Protein profiles and IgE binding patterns were produced by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from subjects with fish allergy. The major allergens of the fish were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry of the peptide digests. The SDS-PAGE of the raw extract revealed 27 protein fractions over a wide molecular weight range, while the cooked extract demonstrated only six protein fractions. The 1-DE immunoblotting detected 14 IgE-binding proteins, with a molecular weight range from 90 to < 6.5 kDa. Three protein fractions with molecular weights of approximately 51, 46 and 12 kDa were identified as the major allergens of this fish. The approximately 12 kDa band was a heat-resistant protein while the approximately 51 and 46 kDa proteins were sensitive to heat. The 2-DE gel profile of the raw extract demonstrated > 100 distinct protein spots and immunoblotting detected at least 10 different major IgE reactive spots with molecular masses as expected and isoelectric point (pI) values ranging from 4.0 to 7.0. A comparison of the major allergenic spot sequences of the 12 kDa proteins with known protein sequences in databases revealed extensive similarity with fish parvalbumin. In conclusion, this study demonstrated that a parvalbumin which is similar to Gad c 1 is the major allergen of Indian scad. Interestingly, we also detected heat-sensitive proteins as major allergenic components in our fish allergy patients.

Modified Th2 responses at high-dose exposures to allergen: using an occupational model.

RATIONALE: The relationships between allergen exposures and allergy and asthma are complex. High exposure levels to cat allergen are associated with IgG- and IgG(4)-specific antibody responses without sensitization or risk of asthma, a process described as a "modified Th2 response." Attenuation of risk of allergy and asthma at high exposure levels has been reported in longitudinal studies of both childhood and occupational asthma. OBJECTIVES: To investigate, using an occupational model, the relationships among estimated exposure to aeroallergens, the production of specific IgE, IgG and IgG(4) antibodies, and the prevalence of associated symptoms. METHODS: Cross-sectional survey of employees exposed to rats at work on six pharmaceutical sites across the United Kingdom. A total of 689 (89%) provided a blood sample and completed a questionnaire. MEASUREMENTS AND MAIN RESULTS: At highest exposure to rats, there was an attenuation of the exposure response for sensitization and symptoms. In contrast, the frequency of individuals producing high quantities of specific IgG and IgG(4) increased with exposure intensity. Ratios of IgG(4)/IgE were highest in those handling the greatest number of rats. Risk of developing work-related chest symptoms was lower for those who produced both specific IgE and IgG(4) compared to those with specific IgE only. CONCLUSIONS: High exposure to rats is associated with lower rates of specific IgE and symptoms but an increased frequency of high specific IgG and IgG(4) production. Specific IgG(4) produced together with specific IgE may reduce the risk of developing work-related chest symptoms compared with when specific IgE is produced alone.

Is occupational asthma to diisocyanates a non-IgE-mediated disease?

BACKGROUND: Exposure to diisocyanates in the workplace is an important cause of occupational asthma. The majority of patients with diisocyanate-induced asthma have no detectable diisocyanate-specific IgE antibodies in serum. There has been much debate as to whether this is due to diisocyanate-induced asthma being mediated by non-IgE mechanisms or whether it is the result of using inappropriate conjugates. OBJECTIVE: We sought to determine whether RNA message for Cepsilon, IL-4, and other associated inflammatory markers could be detected locally within the bronchial mucosa after diisocyanate challenge. METHODS: Fiberoptic bronchoscopic bronchial biopsy specimens were obtained at 24 hours after both a control and an active challenge in 5 patients with positive and 7 patients with negative inhalation test responses to diisocyanates. Using both immunohistochemistry and in situ hybridization, we determined mRNA for Cepsilon, IL-4, IL-5, and other associated inflammatory markers. RESULTS: There was a striking absence of Cepsilon and IL-4 mRNA-positive cells in bronchial biopsy specimens from patients challenged with diisocyanate (Cepsilon median of 0 and interquartile range of 0-1.85; IL-4 median of 0 and interquartile range of 0-0.85). In contrast, there were increased numbers of IL-5-, CD25-, and CD4-positive cells and a trend toward an increase in eosinophils after active challenge with diisocyanate. CONCLUSION: We found a striking absence of both bronchial Cepsilon and IL-4 RNA message after inhalation challenge with diisocyanates, irrespective of whether the challenge test response was positive or negative. We propose that diisocyanate-induced asthma is a non-IgE-mediated disease, at least in patients in whom specific IgE antibodies to diisocyanates are undetectable.

HLA-C and killer cell immunoglobulin-like receptor genes in idiopathic bronchiectasis.

RATIONALE: In idiopathic bronchiectasis, lung inflammation and chronic bacterial infection lead to progressive lung damage. A possible role for natural killer (NK) cells is suggested by the observation that familial bronchiectasis occurs in a rare group of individuals with impaired HLA class I expression and consequent NK cell dysfunction. OBJECTIVE: Because the HLA-C locus and killer cell immunoglobulin-like receptors (KIRs) are of key importance for NK cell recognition, we analyzed HLA-C/KIR combinations by genotyping patients with idiopathic bronchiectasis. METHODS: Genomic DNA from 96 individuals with idiopathic bronchiectasis and 101 control subjects was analyzed by polymerase chain reaction with sequence-specific primers. High-resolution HLA-C genotyping was performed in addition to KIR analysis. RESULTS: HLA-Cw*03 alleles and, in particular, HLA-C group 1 homozygosity are associated with the presence of bronchiectasis. Analysis of the relationship between HLA-C and KIR genes suggests a shift to activatory NK cell function. CONCLUSION: This is the first demonstration of genetic susceptibility in idiopathic bronchiectasis. The association with HLA-C group 1 homozygosity, and the interplay between HLA-C and KIR genes, argue for a role for NK cells in the progressive lung damage seen in this disease. This will require further investigation using functional studies.

HLA associations with occupational sensitization to rat lipocalin allergens: a model for other animal allergies?

BACKGROUND: Laboratory animal allergy is a common occupational health problem affecting between 11% and 44% of exposed researchers. Allergy to rats and mice is most common, probably because these are the animals most frequently used. OBJECTIVE: We hypothesized that HLA class II molecules, involved in the presentation of allergen to the T cell and likely candidates for controlling the immune response, might be associated with sensitization to rat urinary proteins among laboratory animal handlers. METHODS: We undertook a cross-sectional study of 741 employees at 6 pharmaceutical sites across the United Kingdom who had contact at work with laboratory rats. In all, 109 cases with specific sensitization to rat proteins and 397 referents were HLA-typed for DRB1 and DQB1 loci. Amino acid analyses of significantly associated HLA molecules were carried out. RESULTS: HLA-DR7 was associated with sensitization (odds ratio [OR], 1.82; CI, 1.12-2.97), respiratory symptoms at work (OR, 2.96; CI, 1.64-5.37) and, most strongly, sensitization with symptoms (OR, 3.81; CI, 1.90-7.65). HLA-DR3 was protective against sensitization (OR, 0.55; CI, 0.31-0.97). Amino acid analyses of these 2 molecules indicated a biologically plausible explanation for the associations. CONCLUSION: HLA phenotype is an important determinant of individual susceptibility to sensitization and asthma among laboratory animal workers. Similar mechanisms might apply in other animal allergies.

Domestic aeroallergen levels in Barcelona and Menorca (Spain).

Exposure to common indoor allergens is known to be associated with sensitization and triggers of asthma. Levels of allergens have been barely described in Mediterranean countries. This study reports domestic allergen levels among the general population of two regions of Spain. Dust samples were collected from living rooms and mattresses in homes of infants in Barcelona (n = 366) and Menorca (n = 475) and assayed for house dust mite (Der p 1) and cat allergen (Fel d 1) concentrations by enzyme-linked immunoabsorbent assay (ELISA). Geometric mean values (95% CI) of Der p 1 were 0.77 micro g/g (0.65, 0.92) in living rooms and 0.68 (0.56, 0.82) in children's mattresses in Barcelona, and 9.06 (7.93-10.34) and 3.12 (2.71-3.59) in Menorca, respectively. Fel d 1 levels were 0.37 micro g/g (0.31, 0.45) and 0.14 (0.12, 0.18) in Barcelona, and 0.42 (0.35, 0.50) and 0.20 (0.18, 0.24) in Menorca. Home characteristics were not consistently related to levels of aeroallergens in either location. Differences in Der p 1 levels in the two locations indicate that levels cannot be extrapolated from one part of a country to another with any certainty. Additionally, allergen reduction measures related to indoor sources must be specific to each location.