Evaluation of 'implications for research' statements in systematic reviews of interventions in advanced cancer patients - a meta-research study.

BACKGROUND: Implications for research (IfR) sections are an important part of systematic reviews (SRs) to inform health care researchers and policy makers. PRISMA 2020 recommends reporting IfR, while Cochrane Reviews require a separate chapter on IfR. However, it is unclear to what extent SRs discuss IfR. We aimed i) to assess whether SRs include an IfR statement and ii) to evaluate which elements informed IfR statements. METHODS: We conducted a meta-research study based on SRs of interventions in advanced cancer patients from a previous project (CRD42019134904). As suggested in the Cochrane Handbook, we assessed if the following predefined variables were referred to in IfR statements: patient, intervention, control, outcome (PICO) and study design; concepts underlying Grading of Recommendations, Assessment, Development and Evaluation (GRADE) domains: risk of bias, inconsistency, indirectness, imprecision, publication bias. Data were independently extracted by three reviewers after piloting the data extraction form. Discrepancies were resolved in weekly in-depth discussions. RESULTS: We included 261 SRs. The majority evaluated a pharmacological intervention (n = 244, 93.5%); twenty-nine were Cochrane Reviews (11.1%). Four out of five SRs included an IfR statement (n = 210, 80.5%). IfR statements commonly addressed 'intervention' (n = 121, 57.6%), 'patient ' (n = 113, 53.8%), and 'study design' (n = 107, 51.0%). The most frequent PICO and study design combinations were 'patient and intervention ' (n = 71, 33.8%) and 'patient, intervention and study design ' (n = 34, 16.2%). Concepts underlying GRADE domains were rarely used for informing IfR recommendations: 'risk of bias ' (n = 2, 1.0%), and 'imprecision ' (n = 1, 0.5%), 'inconsistency ' (n = 1, 0.5%). Additional elements informing IfR were considerations on cost effectiveness (n = 9, 4.3%), reporting standards (n = 4, 1.9%), and individual patient data meta-analysis (n = 4, 1.9%). CONCLUSION: Although about 80% of SRs included an IfR statement, the reporting of PICO elements varied across SRs. Concepts underlying GRADE domains were rarely used to derive IfR. Further work needs to assess the generalizability beyond SRs in advanced cancer patients. We suggest that more specific guidance on which and how IfR elements to report in SRs of interventions needs to be developed. Utilizing PICO elements and concepts underlying GRADE according to the Cochrane Handbook to state IfR seems to be a reasonable approach in the interim. REGISTRATION: CRD42019134904.

When and how do patients with cardiac amyloidosis die?

BACKGROUND: Cardiac amyloidosis (CA) is an underappreciated cause of morbidity and mortality. Light-chain (AL) and transthyretin (ATTR) amyloidosis have different disease trajectories. No data are available on subtype-specific modes of death (MOD) in patients with CA. METHODS AND RESULTS: We retrospectively investigated 66 with AL and 48 with wild-type ATTR amyloidosis (ATTRwt) from 2000 to 2018. ATTRwt differed from AL by age (74.6 ± 5.4 years vs. 63 ± 10.8 years), posterior wall thickness (16.8 ± 3.3 mm vs. 14.3 ± 2.2 mm), left ventricular mass index (180.7 ± 63.2 g/m2 vs. 133.5 ± 42.2 g/m2), and the proportions of male gender (91.7% vs. 59.1%), atrial enlargement (92% vs. 68.2%) and atrial fibrillation (50% vs. 12.1%). In AL NYHA Functional Class and proteinuria (72.7% vs. 39.6%) were greater; mean arterial pressure (84.4 ± 13.5 mmHg vs. 90.0 ± 11.3 mmHg) was lower. Unadjusted 5-year mortality rate was 65% in AL-CA vs. 44% in the ATTRwt group. Individuals with AL-CA were 2.28 times ([95%CI 1.27-4.10]; p = 0.006) more likely to die than were individuals with ATTRwt-CA. Information on MOD was available in 56 (94.9%) of 59 deceased patients. MOD was cardiovascular in 40 (66.8%) and non-cardiovascular in 16 (27.1%) patients. Cardiovascular [28 (68.3%) vs. 13 (80%)] death events were distributed equally between AL and ATTRwt (p = 0.51). CONCLUSION: Our data indicate no differences in MOD between patients with AL and ATTRwt cardiac amyloidosis despite significant differences in clinical presentation and disease progression. Cardiovascular events account for more than two-thirds of fatal casualties in both groups.

Distinct epitope structures of defensin-like proteins linked to proline-rich regions give rise to differences in their allergenic activity.

BACKGROUND: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens. METHODS: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro. RESULTS: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 125-36 was observed. CONCLUSIONS: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis.

Amb a 1 isoforms: Unequal siblings with distinct immunological features.

BACKGROUND: Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunological characteristics of Amb a 1 isoforms are not fully investigated. Here, we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms. METHODS: After purification, the isoforms were physicochemically characterized, tested for antibody binding and induction of human T-cell proliferative responses. Their immunological properties were further evaluated in vitro and in vivo in a mouse model. RESULTS: Amb a 1 isoforms exhibited distinct patterns of IgE binding and immunogenicity. Compared to Amb a 1.02 or 03 isoforms, Amb a 1.01 showed higher IgE-binding activity. Isoforms 01 and 03 were the most potent stimulators of patients' T cells. In a mouse model of immunization, Amb a 1.01 induced higher levels of IgG and IgE antibodies when compared to isoforms 02 and 03. Interestingly, ragweed-sensitized patients also displayed an IgG response to Amb a 1 isoforms. However, unlike therapy-induced antibodies, sensitization-induced IgG did not show IgE-blocking activity. CONCLUSION: The present study showed that naturally occurring isoforms of Amb a 1 possess different immunogenic and sensitizing properties. These findings should be considered when selecting sequences for molecule-based diagnosis and therapy for ragweed allergy. Due to its high IgE-binding activity, isoform Amb a 1.01 should be included in diagnostic tests. In contrast, due to their limited B- and T-cell cross-reactivity patterns, a combination of different isoforms might be a more attractive strategy for ragweed immunotherapy.

Influence of the Ag concentration on the medium-range order in a CuZrAlAg bulk metallic glass.

Fluctuation electron microscopy of bulk metallic glasses of CuZrAl(Ag) demonstrates that medium-range order is sensitive to minor compositional changes. By analyzing nanodiffraction patterns medium-range order is detected with crystal-like motifs based on the B2 CuZr structure and its distorted structures resembling the martensitic ones. This result demonstrates some structural homology between the metallic glass and its high temperature crystalline phase. The amount of medium-range order seems slightly affected with increasing Ag concentration (0, 2, 5 at.%) but the structural motifs of the medium-range ordered clusters become more diverse at the highest Ag concentration. The decrease of dominant clusters is consistent with the destabilization of the B2 structure measured by calorimetry and accounts for the increased glass-forming ability.

The quantity and quality of α-gal-specific antibodies differ in individuals with and without delayed red meat allergy.

BACKGROUND: IgG to galactose-α-1,3-galactose (α-gal) are highly abundant natural antibodies (Ab) in humans. α-Gal-specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3-7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their α-gal-specific IgE and IgG responses in more detail. METHODS: α-Gal-specific IgE was determined by ImmunoCAP. IgE reactivity to meat extract and bovine gamma globulin (BGG) was assessed by immunoblotting and ELISA, respectively. In some experiments, sera were pre-incubated with α-gal or protein G to deplete IgG Ab. α-Gal-specific IgG1-4 Ab in individuals with and without meat allergy were assessed by ELISA. RESULTS: In immunoblots, BGG was the most frequently recognized meat protein. Binding of IgE and IgG to BGG was confirmed by ELISA and completely abolished after pre-incubation with α-gal. Neither the depletion of autologous α-gal-specific IgG Ab nor the addition of α-gal-specific IgG Ab from nonallergic individuals changed the IgE recognition of BGG of meat-allergic patients. Meat-allergic patients showed significantly higher α-gal-specific IgG1 and IgG3 Ab than nonallergic individuals, whereas the latter showed significantly higher levels of α-gal-specific IgG4 Ab. CONCLUSION: Patients with delayed meat allergy display IgE and IgG Ab that selectively recognize the α-gal epitope on BGG. Their enhanced α-gal-specific IgE levels are accompanied by high levels of α-gal-specific IgG1 devoid of IgE-blocking activity. This subclass distribution is atypical for food allergies and distinct from natural α-gal IgG responses in nonallergic individuals.

Pilot Study of Endothelin Receptor Blockade in Heart Failure with Diastolic Dysfunction and Pulmonary Hypertension (BADDHY-Trial).

BACKGROUND: In this multi-centre, randomised, placebo-controlled pilot trial, we investigated the clinical and haemodynamic effects of the endothelin-receptor blocker Bosentan in patients with heart failure, preserved ejection fraction and pulmonary hypertension (PH-HFpEF). MATERIALS AND METHODS: Eligible patients received either 12 weeks of Bosentan therapy, or a placebo drug. Patients were thereafter followed for a further period of 12 weeks without the study medication. At three points during the study (study Commencement, Week 12 and Week 24), a six-minute walk test (6MWT), echocardiographic and laboratory assessments were performed, as well as a quality of life survey. Right heart catheterisation (RHC) was undertaken at commencement only. The study was aborted early, after an interim analysis favoured the placebo. RESULTS: Six-minute walk distance (6MWD) did not change in the Bosentan group (309.7±96.3m (Commencement), 317.0±126.1m (Week 12), 307.0±84.4m (Week 24); p=0.86), but almost reached statistical significance in the placebo group from 328.8±79.6m, to 361.6±98.2m and 384.0±74.9m (Week 24); p=0.075. In the placebo group, estimated systolic pulmonary artery pressure (measured via echocardiography) significantly decreased (from 62.3±16.7mmHg [Commencement], 45.3±13.9mmHg [Week 12], to 44.6±14.5mmHg [Week 24]; p=0.014) as did right atrial pressure (13.1±5.3 [Commencement], 10.0±3.8 [Week 12], to 9.4±3.2 [Week 24]; p=0.046). CONCLUSION: Despite this study's limited sample size and premature cessation, it nevertheless suggests that endothelin receptor blockade in patients with PH-HFpEF may have no beneficial effects and could even be detrimental in comparison to a placebo.

Local, atomic-level elastic strain measurements of metallic glass thin films by electron diffraction.

A novel technique is used to measure the atomic-level elastic strain tensor of amorphous materials by tracking geometric changes of the first diffuse ring of selected area electron diffraction patterns (SAD). An automatic procedure, which includes locating the centre and fitting an ellipse to the diffuse ring with sub-pixel precision is developed for extracting the 2-dimensional strain tensor from the SAD patterns. Using this technique, atomic-level principal strains from micrometre-sized regions of freestanding amorphous Ti0.45Al0.55 thin films were measured during in-situ TEM tensile deformation. The thin films were deformed using MEMS based testing stages that allow simultaneous measurement of the macroscopic stress and strain. The calculated atomic-level principal strains show a linear dependence on the applied stress, and good correspondence with the measured macroscopic strains. The calculated Poisson's ratio of 0.23 is reasonable for brittle metallic glasses. The technique yields a strain accuracy of about 1×10(-4) and shows the potential to obtain localized strain profiles/maps of amorphous thin film samples.

HLA class II peptide tetramers vs allergen-induced proliferation for identification of allergen-specific CD4 T cells.

BACKGROUND: Fluorescence-labeled MHC class II/peptide tetramer complexes are considered as optimal tools to characterize allergen-specific CD4(+) T cells, but this technique is restricted to frequently expressed HLA class II molecules and knowledge of immunodominant epitopes. In contrast, allergen-stimulated proliferation assessed by CFSE dilution is less sophisticated and widely applicable. The major mugwort allergen, Art v 1, contains only one single, immunodominant, HLA-DR1-restricted epitope (Art v 125-36 ). Thus, essentially all Art v 1-reactive cells should be identified by a HLA-DRB1*01:01/Art v 119-36 tetramer. METHODS: We compared specificity and sensitivity of tetramer(+) and allergen-induced proliferating (CFSE(lo) ) CD4(+) T cells by flow cytometry. RESULTS: The frequency of tetramer(+) CD4(+) T cells determined ex vivo in PBMC of mugwort-allergic individuals ranged from 0 to 0.029%. After 2-3 weeks of in vitro expansion, sufficient tetramer(+) T cells for phenotyping were detected in 83% of Art v 125-36 -reactive T-cell lines (TCL) from mugwort-allergic individuals, but not in TCL from healthy individuals. The tetramers defined bona fide Th2 cells. Notably, Art v 125-36 -reactive TCL depleted of tetramer(+) T cells still reacted to the peptide, and only 44% of Art v 125-36 -specific T-cell clones were detected by the tetramer. CFSE(lo) CD4(+) T cells contained only 0.3-10.7% of tetramer(+) T cells and very low proportions of Th2 cells. CONCLUSION: Allergen-specific T cells can be identified by HLA class II tetramers with high specificity, but unexpected low sensitivity. In contrast, allergen-stimulated CFSE(lo) CD4(+) T cells contain extremely high fractions of bystander cells. Therefore, for T-cell monitoring, either method should be interpreted with caution.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

BACKGROUND: Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. METHODS: Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. RESULTS: Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. CONCLUSION: Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.

Bet v 1--a Trojan horse for small ligands boosting allergic sensitization?

BACKGROUND: Birch pollen allergy represents the main cause of winter and spring pollinosis in the temperate climate zone of the northern hemisphere and sensitization towards Bet v 1, the major birch pollen allergen, affects over 100 million allergic patients. The major birch pollen allergen Bet v 1 has been described as promiscuous acceptor for a wide variety of hydrophobic ligands. OBJECTIVE: In search of intrinsic properties of Bet v 1, which account responsible for the high allergenic potential of the protein, we thought to investigate the effects of ligand-binding on immunogenic as well as allergenic properties. METHODS: As surrogate ligand of Bet v 1 sodium deoxycholate (DOC) was selected. Recombinant and natural Bet v 1 were characterised physico-chemically as well as immunologically in the presence or absence of DOC, and an animal model of allergic sensitization was established. Moreover, human IgE binding to Bet v 1 was analysed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Ligand-binding had an overall stabilizing effect on Bet v 1. This translated in a Th2 skewing of the immune response in a mouse model. Analyses of human IgE binding on Bet v 1 in mediator release assays revealed that ligand-bound allergen-induced degranulation at lower concentrations; however, in basophil activation tests with human basophils ligand-binding did not show this effect. For the first time, human IgE epitopes on Bet v 1 were determined using antibodies isolated from patients' sera. The IgE epitope mapping of Bet v 1 demonstrated the presence of multiple binding regions. CONCLUSIONS AND CLINICAL RELEVANCE: Deoxycholate binding stabilizes conformational IgE epitopes on Bet v 1; however, the epitopes themselves remain unaltered. Therefore, we speculate that humans are exposed to both ligand-bound and free Bet v 1 during sensitization, disclosing the ligand-binding cavity of the allergen as key structural element.

The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes.

BACKGROUND: Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes. METHODS: A cDNA expression library representing the wheat transcriptome was constructed in phage lambda gt11 and screened with IgE antibodies from wheat food allergic patients. IgE-reactive cDNA clones coding for portions of high molecular weight (HMW) glutenin subunits were identified by sequence analysis of positive clones. IgE epitopes were characterized using recombinant fragments from the HMW Bx7 and synthetic peptides thereof for testing of allergic patients' sera and in basophil degranulation assays. RESULTS: We found that the major IgE-reactive areas of HMW glutenins are located in the repetitive regions of the protein and could show that two independent IgE-reactive fragments from HMW Bx7 contained repetitive IgE epitopes. CONCLUSIONS: Our results demonstrate that IgE antibodies from wheat food allergic patients can recognize repetitive epitopes in one of the important wheat food allergens. Recombinant HMW Bx7 may be included into the panel of allergens for component-resolved diagnosis of wheat food allergy.

Dose matters! Optimisation of guideline adherence is associated with lower mortality in stable patients with chronic heart failure.

AIMS: Guidelines have been published for improving management of chronic heart failure (CHF). We examined the association between improved guideline adherence and risk for all-cause death in patients with stable systolic HF. METHODS: Data on ambulatory patients (2006-2010) with CHF and reduced ejection fraction (HF-REF) from the Austrian Heart Failure Registry (HIR Austria) were analysed. One-year clinical data and long-term follow-up data until all-cause death or data censoring were available for 1014 patients (age 65 [55-73], male 75%, NYHA class I 14%, NYHA II 56%, NYHA III/IV 30%). A guideline adherence indicator (GAI [0-100%]) was calculated for each patient at baseline and after 12 ± 3 months that considered indications and contraindications for ACE-I/ARB, beta blockers, and MRA. Patients were considered ΔGAI-positive if GAI improved to or remained at high levels (≥ 80%). ΔGAI50+ positivity was ascribed to patients achieving a dose of ≥ 50% of suggested target dose. RESULTS: Improvements in GAI and GAI50+ were associated with significant improvements in NYHA class and NT-proBNP (1728 [740-3636] to 970 [405-2348]) (p<0.001). Improvements in GAI50+, but not GAI, were independently predictive of lower mortality risk (HR 0.55 [95% CI 0.34-0.87; p=0.01]) after adjustment for a large variety of baseline parameters and hospitalisation for heart failure during follow-up. CONCLUSIONS: Improvement in guideline adherence with particular emphasis on dose escalation is associated with a decrease in long-term mortality in ambulatory HF-REF subjects surviving one year after registration.

Allergen hybrids - next generation vaccines for Fagales pollen immunotherapy.

BACKGROUND: Trees belonging to the order of Fagales show a distinct geographical distribution. While alder and birch are endemic in the temperate zones of the Northern Hemisphere, hazel, hornbeam and oak prefer a warmer climate. However, specific immunotherapy of Fagales pollen-allergic patients is mainly performed using birch pollen extracts, thus limiting the success of this intervention in birch-free areas. OBJECTIVES: T cells are considered key players in the modification of an allergic immune response during specific immunotherapy (SIT), therefore we thought to combine linear T cell epitope-containing stretches of the five most important Fagales allergens from birch, hazel, alder, oak and hornbeam resulting in a Fagales pollen hybrid (FPH) molecule applicable for SIT. METHODS: A Fagales pollen hybrid was generated by PCR-based recombination of low IgE-binding allergen epitopes. Moreover, a structural-variant FPH4 was calculated by in silico mutagenesis, rendering the protein unable to adopt the Bet v 1-like fold. Both molecules were produced in Escherichia coli, characterized physico-chemically as well as immunologically, and tested in mouse models of allergic sensitization as well as allergy prophylaxis. RESULTS: Using spectroscopic analyses, both proteins were monomeric, and the secondary structure elements of FPH resemble the ones typical for Bet v 1-like proteins, whereas FPH4 showed increased amounts of unordered structure. Both molecules displayed reduced binding capacities of Bet v 1-specific IgE antibodies. However, in a mouse model, the proteins were able to induce high IgG titres cross-reactive with all parental allergens. Moreover, prophylactic treatment with the hybrid proteins prevented pollen extract-induced allergic lung inflammation in vivo. CONCLUSION: The hybrid molecules showed a more efficient uptake and processing by dendritic cells resulting in a modified T cell response. The proteins had a lower IgE-binding capacity compared with the parental allergens, thus the high safety profile and increased efficacy emphasize clinical application for the treatment of Fagales multi-sensitization.

Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis.

BACKGROUND: Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen-food syndrome (PFS) and caused by cross-reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies. METHODS: As vaccine candidates, apple Mal d 1 as well as hazelnut Cor a 1 derivatives were designed by in silico backbone analyses of the respective allergens. The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens. Because Mal d 1 and Cor a 1 form cysteine-mediated aggregates, nonaggregative cysteine to serine mutants were also generated. The proteins were characterized physicochemically, immunologically, and in in vivo models with or without adjuvant. RESULTS: The structurally modified proteins showed significantly decreased IgE binding capacity. Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants. When formulated with alum, the monomeric cysteine mutants induced a similar immune response as the aggregated parental allergens, which is in contrast with data published on Bet v 1. CONCLUSION: These findings lead to the suggestion that the Bet v 1 structure has unique intrinsic properties, which could account for its high allergenicity. Obviously, these characteristics are not entirely shared with its food homologues from apple and hazelnut. Thus, it is important to tackle pollen-related food allergies from different angles for the generation of effective vaccine candidates to treat birch PFS.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.

BACKGROUND: IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. METHODS: IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. RESULTS: BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. CONCLUSION: Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Carrier-bound Alt a 1 peptides without allergenic activity for vaccination against Alternaria alternata allergy.

BACKGROUND: The mould Alternaria alternata is a major elicitor of allergic asthma. Diagnosis and specific immunotherapy (SIT) of Alternaria allergy are often limited by the insufficient quality of natural mould extracts. OBJECTIVE: To investigate whether recombinant Alt a 1 can be used for reliable diagnosis of Alternaria alternata allergy and to develop a safe, non-allergenic vaccine for SIT of Alternaria allergy. METHODS: The qualitative sensitization profile of 80 Alternaria-allergic patients from Austria and Italy was investigated using an allergen micro-array and the amount of Alternaria-specific IgE directed to rAlt a 1 was quantified by ImmunoCAP measurements. Peptides spanning regions of predicted high surface accessibility of Alt a 1 were synthesized and tested for IgE reactivity and allergenic activity, using sera and basophils from allergic patients. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Alt a 1 and inhibit allergic patients' IgE reactivity to Alt a 1. RESULTS: rAlt a 1 allowed diagnosis of Alternaria allergy in all tested patients, bound the vast majority (i.e. >95%) of Alternaria-specific IgE and elicited basophil activation already at a concentration of 0.1 ng/mL. Four non-allergenic peptides were synthesized which, after coupling to the carrier protein keyhole limpet hemocyanin, induced Alt a 1-specific IgG and inhibited allergic patients' IgE binding to Alt a 1. CONCLUSIONS AND CLINICAL RELEVANCE: rAlt a 1 is a highly allergenic molecule allowing sensitive diagnosis of Alternaria allergy. Carrier-bound non-allergenic Alt a 1 peptides are candidates for safe SIT of Alternaria allergy.

Molecular characterization of wheat allergens specifically recognized by patients suffering from wheat-induced respiratory allergy.

BACKGROUND: Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). OBJECTIVE: The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. METHODS: A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. RESULTS: We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. CONCLUSION AND CLINICAL RELEVANCE: The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.

Patients suffering from non-IgE-mediated cow's milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens.

BACKGROUND: Cow's milk is one of the most common causes of food allergy. In two-thirds of patients, adverse symptoms following milk ingestion are caused by IgE-mediated allergic reactions, whereas for one-third, the mechanisms are unknown. Aim of this study was to investigate whether patients suffering from non-IgE-mediated cow's milk protein intolerance can be distinguished from persons without cow's milk protein intolerance based on serological measurement of IgG and IgA specific for purified cow's milk antigens. METHODS: We determined IgG(1-4) subclass and IgA antibody levels to purified recombinant αS1-casein, αS2-casein, ß-casein, κ-casein, α-lactalbumin, and ß-lactoglobulin in four patient groups by ELISA: Patients with IgE-mediated cow's milk allergy (CMA, n=25), patients with non-IgE-mediated cow's milk protein intolerance (CMPI, n=19), patients with gastrointestinal symptoms not associated with cow's milk ingestion (GI, n=15) and control persons without gastrointestinal problems (C, n=26). Cow's milk-specific IgE levels were determined by ImmunoCAP. RESULTS: Only CMA patients had IgE antibodies to cow's milk. Cow's milk allergic patients mounted the highest IgG(1) and IgG(4) antibody levels to αS1-casein, αS2-casein, ß-casein, κ-casein, and α-lactalbumin. No elevated levels of IgG(4) , IgA, and complement-binding IgG subclasses (IgG(1) , IgG(2) , IgG(3) ) to purified cow's milk allergens were found within the CMPI patients compared to persons without cow's milk protein intolerance (GI and C groups). CONCLUSION: Cow's milk protein intolerant patients cannot be distinguished from persons without cow's milk protein intolerance on the basis of IgG subclass or IgA reactivity to cow's milk allergens.

The performance of a component-based allergen microarray for the diagnosis of kiwifruit allergy.

BACKGROUND: Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. OBJECTIVE: To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. METHODS: With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. RESULTS: The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. CONCLUSIONS: Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests.