The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. SDS-PAGE of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5kD band and the identity of Ara h 6 was confirmed by LC-MS/MS. Ara h 6 showed a higher seroprevalence in chimeric-IgE ELISA (n=54), but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays, as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.
Peanut; allergen; IgE reactivity; histamine release
Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of ~100 amino acids, but the fold of the protein and the biological function are unknown.
To determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays.
Natural Bla g 1 and recombinant constructs were compared by ELISA using specific murine IgG and human IgE. The structure of Bla g 1 was determined by X-ray crystallography. Mass spectrometry and NMR were utilized to examine ligand-binding properties of the allergen.
The structure of a recombinant Bla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by X-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic and stearic acids were associated with nBla g 1 from cockroach frass. One Unit of Bla g 1 was equivalent to 104 ng of allergen.
Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with non-specific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.
Allergen; asthma; Bla g 1; cockroach; structure; ligand-binding proteins; exposure assessment
Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well.
A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC).
Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD=0.1 - 9.8ng/ml) to higher than 6800ng/ml for all allergens except Mus m 1, which was up to 1700ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (∼0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%.
The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.
Allergen measurement; asthma; indoor air quality; immunoassay; multiplex array; occupational health
Most studies of indoor allergens have focused on the home environment. However, schools may be an important site of allergen exposure for children with asthma. We compared school allergen exposure to home exposure in a cohort of children with asthma. Correlations between settled dust and airborne allergen levels in classrooms were examined.
Settled dust and airborne samples from 12 inner-city schools were analyzed for indoor allergens using multiplex array technology (MARIA). School samples were linked to students with asthma enrolled in the School Inner-City Asthma Study (SICAS). Settled dust samples from students’ bedrooms were analyzed similarly.
From schools, 229 settled dust and 197 airborne samples were obtained. From homes, 118 settled dust samples were obtained. Linear mixed regression models of log-transformed variables showed significantly higher settled dust levels of mouse, cat and dog allergens in schools than homes (545% higher for Mus m 1, estimated absolute difference 0.55 μg/g, p<0.0001; 198% higher for Fel d 1, estimated absolute difference 0.13 μg/g, p=0.0033; and 144% higher for Can f 1, estimated absolute difference 0.05 μg/g, p=0.0008). Airborne and settled dust Mus m 1 levels in classrooms were moderately correlated (r=0.48; p< 0.0001). There were undetectable to very low levels of cockroach and dust mite allergens in both homes and schools.
Mouse allergen levels in schools were substantial. In general, cat and dog allergen levels were low, but detectable, and were higher in schools. Aerosolization of mouse allergen in classrooms may be a significant exposure for students. Further studies are needed to evaluate the effect of indoor allergen exposure in schools on asthma morbidity in students with asthma.
indoor allergens; asthma; inner city; urban; mouse; Mus m 1; Can f 1; Fel d 1; SICAS; school
Alternaria is one of the most common molds associated with allergic diseases and 80% of Alternaria-sensitive patients produce IgE antibodies to a major protein allergen, Alt a 1. The structure and function of Alt a 1 is unknown.
To obtain a high resolution structure of Alt a 1 by X-ray crystallography and to investigate structural relationships between Alt a 1 and other allergens and proteins reported in the Protein Data Bank.
X-ray crystallography was used to determine the structure of Alt a 1 using a custom-designed set of crystallization conditions. An initial Alt a 1 model was determined by the application of a Ta6Br122+ cluster and Single-wavelength Anomalous Diffraction. Bioinformatic analyses were used to compare the Alt a 1 sequence and structure with other proteins.
Alt a 1 is a unique β-barrel comprising 11 β-strands and forms a ‘butterfly-like’ dimer linked by a single disulfide bond, with a large (1345Å2) dimer interface. Intramolecular disulfide bonds are conserved among Alt a 1 homologs. Currently, the Alt a 1 structure has no equivalent in the Protein Data Bank. Bioinformatics analyses suggest that the structure is found exclusively in fungi. Four previously reported putative IgE binding peptides have been located on the Alt a 1 structure.
Alt a 1 has a unique, dimeric β-barrel structure that appears to define a new protein family with unknown function found exclusively in fungi. The location of IgE antibody binding epitopes is in agreement with the structural analysis of Alt a 1.The Alt a 1 structure will allow mechanistic structure/function studies and immunologic studies directed towards new forms of immunotherapy for Alternaria-sensitive allergic patients.
Asthma; Allergens; Molds; Alt a 1; Alternaria; X-ray crystallography; Protein structure; Oligomeric structure
Bla g 2, one of the major cockroach allergens, induces a strong IgE response against conformational epitopes, and on reexposure, sensitized individuals often display symptoms of allergic rhinitis and asthma. The aim of the current study was to perform a test of the efficacy of a modified phage display screening, characterization of selected phages and an automated algorithm, EpiSearch, in locating an important conformational epitope.
The monoclonal antibody 7C11, which partially inhibits the binding of patient IgE antibodies to Bla g 2, was used to screen a random peptide phage library. After 3 rounds of panning, 32 phage clones were isolated and the amino acid sequences of their peptides were determined. The relative affinity and specificity of the binding of these peptides to 7C11 were tested in ELISAs. The amino acid composition of these peptides was then matched with clusters of residues on the surface of the 3-dimensional (3D) structure of Bla g 2, using our EpiSearch algorithm.
The amino acid sequences of the peptides on selected phages differed at only one position, occupied by 1 of 2 negatively charged residues. The two 12-mer sequences bound to 7C11 with similar avidity and specificity. There was good concordance between the residues in the 3D clusters identified from our phage display/computational method with the co-crystal structural analysis.
Conformational epitopes may be mapped through screening of clones from random peptide phage display libraries and EpiSearch.
Bla g 2; Cockroach allergen; Conformational epitope; EpiSearch; Monoclonal antibody; Random peptide phage display library
Climate change and environmental stewardship are phrases that have been defining the past few decades and promoting change in our societies. The sensitivities of intensive care as a specialty make the process of greening an intensive care unit a challenge, but not one that is insurmountable. This paper discusses opportunities for critical care to reduce its environmental impact and provide a framework change. The article includes suggestions of what can be done as an individual, as a unit and as a hospital. Generally, practices in critical care are accepted without questioning the environmental consequences. We believe it is time for change, and critical care should give environmental stewardship a higher priority.
TSLP is expressed at sites of allergic inflammation, including eczematous skin. This cytokine has been reported to exert its Th2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated Th2 cells could amplify Th2 responses at inflamed sites through the direct actions of TSLP.
To rigorously test whether Th2 cells induced by “pro-allergic” factors express TSLP receptor and characterize these cells using an experimental platform that combines flow cytometry with microscopic capabilities.
CD4+ T cells isolated from patients with atopic dermatitis or normal healthy controls were co-cultured with autologous dendritic cells in the presence of Th2-promoting stimuli (TSLP±allergen and staphylococcal enterotoxin B±TSLP). Surface expression of TSLP receptor was analyzed by image-based flow cytometry and responsiveness of purified T cells to TSLP was assessed by phosphorylation of STAT5 and cytokine secretion.
Th2-promoting stimuli induced a robust population of activated Th2 cells (CD25+IL-4+). Regardless of the nature of the stimulus, flow cytometry imaging confirmed that T cells expressing TSLP receptor were rare, constituting a minor fraction of the IL-4+ T cell pool; however, TSLP-responsiveness was nonetheless detectable. Analysis of cell size and nuclear morphology revealed preferential expression of TSLP receptor on IL-4-expressing cells undergoing mitosis. Analysis of lesional skin in atopic dermatitis supported the view that rare IL-4+ T cells expressing TSLP receptor are present at inflamed sites.
In a “pro-allergic” milieu, TSLP receptor is preferentially expressed on rare actively dividing Th2 cells. The direct action of TSLP on T cells could amplify Th2 responses at sites of allergic inflammation.
TSLP; TSLP receptor; atopic dermatitis; Th2 cells; flow cytometry imaging
Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2.
Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab′ fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients.
Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates.
The crystal structure of a murine monoclonal antibody, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2, has been solved at 1.8 Å resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared to the monoclonal antibody 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn268 and that a large number of antigen-antibody contacts are mediated by water molecules and ions, most likely zinc. Antibody binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant, showed that the carbohydrate contributes, without being essential, to the Bla g 2-4C3 mAb interaction. Inhibition of IgE antibody binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that amino acids Lys251, Glu233, and Ile199 are important for the recognition of Bla g 2 by the 4C3 mAb antibody. The results show the relevance of X-ray crystallographic studies of allergen-antibody complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.
Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in mite allergic patients. However, the structure and biological function of this important allergen are unknown. Allergen function may contribute to allergenicity as shown for the protease activity of Group 1 mite allergens and the interaction with the innate immune system by Group 2 mite allergens.
To determine the crystal structure of Der p 7 and to investigate its biological function.
X-ray crystallography was utilized to determine the Der p 7 structure. NMR analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands.
Der p 7 has an elongated structure with two 4-stranded anti-parallel β-sheets which wrap around a long C-terminal helix. The fold of Der p 7 is similar to lipopolysaccharide binding protein (LBP), which interacts with Toll-like receptors (TLRs) after binding lipopolysaccharide and other bacterially-derived lipid ligands. NMR and biochemical assays indicate that Der p 7 does not bind lipopolysaccharide but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7.
Der p 7 binds a bacterially-derived lipid product, a common feature of some allergens. The finding that the Group 7 as well as the Group 2 mite allergens are structurally similar to different proteins in the TLR pathway further strengthens the connections between dust mites, innate immunity, and allergy.
Asthma; allergens; dust mites; Der p 7; lipopolysaccharide binding protein; TLR4; lipopeptide; innate immunity
The molecule H22-Fel d 1, which targets cat allergen to FcγRI on dendritic cells, has the potential to treat cat allergy owing to its T-cell modulatory properties.
To investigate whether the T-cell response induced by H22-Fel d 1 is altered in the presence of the Th2-promoting cytokine, TSLP.
Studies were performed in cat-allergic subjects with and without atopic dermatitis. Monocyte-derived dendritic cells were primed with H22-Fel d 1 in the presence or absence of TSLP and the resulting T-cell cytokine repertoire was analyzed by flow cytometry. The capacity for H22-Fel d 1 to modulate TSLP receptor expression on dendritic cells was examined by flow cytometry in the presence or absence of inhibitors of Fc receptor signaling molecules.
Surprisingly, TSLP alone was a weak inducer of Th2 responses irrespective of atopic status; however, dendritic cells co-primed with TSLP and H22-Fel d 1 selectively and synergistically amplified Th2 responses in highly atopic subjects. This effect was OX40 ligand-independent pointing to an unconventional TSLP-mediated pathway. Expression of TSLP receptor was upregulated on atopic dendritic cells primed with H22-Fel d 1 through a pathway regulated by FcγRI-associated signaling components, including src related tyrosine kinases and Syk, as well as the downstream molecule, PI3-kinase. Inhibition of TSLP receptor upregulation triggered by H22-Fel d 1 blocked TSLP-mediated Th2 responses.
Discovery of a novel Th2 regulatory pathway linking FcγRI signaling to TSLP receptor upregulation and consequent TSLP-mediated effects questions the validity of receptor-targeted allergen vaccines.
This study establishes a pivotal role for Fc receptor ligation in promoting TSLP-mediated Th2 responses associated with allergic disease.
Atopic dendritic cells are equipped to efficiently upregulate TSLP receptor upon Fc receptor ligation by allergen. These findings suggest that dendritic cell-based vaccines that target Fc receptors could amplify Th2-driven inflammatory responses by potentiating the effects of TSLP.
H22-Fel d 1; TSLP; TSLP receptor; atopic dermatitis; blood dendritic cells; monocyte-derived dendritic cells; Th2 cells; FcγRI
In the 2003 Toronto SARS outbreak, SARS-CoV was transmitted in hospitals despite adherence to infection control procedures. Considerable controversy resulted regarding which procedures and behaviours were associated with the greatest risk of SARS-CoV transmission.
A retrospective cohort study was conducted to identify risk factors for transmission of SARS-CoV during intubation from laboratory confirmed SARS patients to HCWs involved in their care. All SARS patients requiring intubation during the Toronto outbreak were identified. All HCWs who provided care to intubated SARS patients during treatment or transportation and who entered a patient room or had direct patient contact from 24 hours before to 4 hours after intubation were eligible for this study. Data was collected on patients by chart review and on HCWs by interviewer-administered questionnaire. Generalized estimating equation (GEE) logistic regression models and classification and regression trees (CART) were used to identify risk factors for SARS transmission.
45 laboratory-confirmed intubated SARS patients were identified. Of the 697 HCWs involved in their care, 624 (90%) participated in the study. SARS-CoV was transmitted to 26 HCWs from 7 patients; 21 HCWs were infected by 3 patients. In multivariate GEE logistic regression models, presence in the room during fiberoptic intubation (OR = 2.79, p = .004) or ECG (OR = 3.52, p = .002), unprotected eye contact with secretions (OR = 7.34, p = .001), patient APACHE II score ≥20 (OR = 17.05, p = .009) and patient Pa02/Fi02 ratio ≤59 (OR = 8.65, p = .001) were associated with increased risk of transmission of SARS-CoV. In CART analyses, the four covariates which explained the greatest amount of variation in SARS-CoV transmission were covariates representing individual patients.
Close contact with the airway of severely ill patients and failure of infection control practices to prevent exposure to respiratory secretions were associated with transmission of SARS-CoV. Rates of transmission of SARS-CoV varied widely among patients.
The Group 1 mite allergens, Der f 1 and Der p 1, are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human IgE antibody responses to the Group 1 allergens show more cross-reactivity than the murine IgG antibody responses which are largely species-specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new, high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding is observed in the structure of Der f 1, despite the fact that all amino acids involved in Ca2+ binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features which could explain the differences in murine and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 which are unevenly distributed on the allergens’ surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1.
mite allergy; Der f 1; Der p 1; antibody; asthma
A number of environmental factors can affect the development and severity of allergy and asthma; however, it can be argued that the most significant inhaled agents that modulate the development of these conditions are biologics. Sensitization to environmental allergens is an important risk factor for the development of asthma. Innate immune responses are often mediated by receptors on mononuclear cells whose primary ligands arise from microorganisms. Many pathogens, especially viruses, target epithelial cells and affect the host immune response to those pathogens. The acquired immune response to an allergen is influenced by the nature of the innate immune system. Products of innate immune responses to microbes promote TH1-acquired responses. In the absence of TH1 responses, TH2 responses can dominate. Central to TH1/TH2 balance is the composition of contaminants that derive from microbes. In this review we examine the biology of the response to allergens, viruses, and bacterial products in the context of the development of allergy and asthma.
asthma; allergy; allergens; endotoxin; respiratory virus; immunoglobulins; tolerance; leukotrienes; neurotrophins
This commentary represents a selective survey of developments relevant to critical care. Selected themes include advances in point-of-care diagnostic testing, glucose control, novel microbiological diagnostics and infection control measures, and developments in information technology that have implications for intensive care. The latter encompasses an early example of an artificially intelligent clinical decision support mechanism, the introduction of a national health care information technology programme (UK NPfIT) and its implications, and exotic threats to patient safety due to emergent behaviour in complex information systems.
glucose; health technology assessment; information technology; intensive care; point-of-care
Carbon dioxide has been monitored in the body using a variety of technologies with a multitude of applications. The monitoring of this common physiologic variable in medicine is an illustrative example of the different levels of evidence that are required before any new health technology should establish itself in clinical practice. End-tidal capnography and sublingual capnometry are two examples of carbon dioxide monitoring that require very different levels of evidence before being disseminated widely. The former deserves its status as a basic standard based on observational data. The latter should be considered investigational until prospective controlled data supporting its use become available. Other applications of carbon dioxide monitoring are also discussed.
biomedical technology assessment; capnography; critical care; evidence-based medicine; physiologic monitoring
We assessed the accuracy of questionnaire reports of cat and dog ownership and presence of cockroaches in predicting measured allergen concentrations in house dust. We collected dust samples in the homes of 932 newborns living in New England. Dust samples were taken from the main living area and the infant's bedding. Allergen content of house dust was measured by enzyme-linked immunosorbent assays (ELISA) and related to questionnaire information on past and current cat and dog ownership and presence of cockroaches. Allergen levels were dichotomized using the limit of detection and the following cut points: 1.0 microg/g and 8.0 microg/g for cat, 2.0 microg/g and 10.0 microg/g for dog, and 2 U/g and 8 U/g for cockroach allergen. For the upper cut point, both specificity and sensitivity of questionnaire-reported cat and dog ownership and presence of cockroaches were high. For the limit of detection and lower cut point, specificity was high (> 80%), whereas sensitivity was low, particularly for current cat and dog ownership (21-60%). Taking pet ownership during the preceding 2 years into account increased the sensitivity by 10%, but it remained relatively poor. In conclusion, questionnaire-reported pet ownership and presence of cockroaches predicts allergen levels above the upper cut point but is a relatively poor measure of allergen exposure above the limit of detection and the lower cut point. Knowledge of past pet ownership can improve pet allergen exposure assessment by means of questionnaire. However, for epidemiologic purposes, measured concentrations of allergens are necessary.
This new section in Critical Care presents a selection of clinically important examples of advances in critical care health technology. This article is divided into two main areas: diagnostics and monitoring. Attention is given to how bedside echocardiography can alter the cardiovascular physical examination, and to novel imaging techniques such as virtual bronchoscopy. The monitoring section discusses recent claims of improved efficiency with telemedicine for intensive care units.
echocardiography; health technology; telemedicine; telemetry; virtual imaging
antibiotics; communicable diseases; guidelines; infection