Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface

Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma

IntroductionEquine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF).MethodsSerum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays.ResultsMEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig.DiscussionA. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis

Allergen-specific immunoglobulin E in sera of horses affected with insect bite hypersensitivity, severe equine asthma or both conditions

Background Genetic, epidemiologic, and clinical evidence suggests that, in horses, there are manifestations of hypersensitivity that can occur together. Objectives To investigate whether concurrent insect bite hypersensitivity (IBH) and severe equine asthma (EA) is associated with higher allergen‐specific and total serum immunoglobulin E (IgE) concentrations than only EA or IBH. Animals Healthy control horses (C, n = 40), horses with IBH (IBH, n = 24), severe EA (EA, n = 18), and both conditions (IBH/EA, n = 23) were included. Methods In our retrospective comparative study, sera from horses with signs of severe EA, IBH, and control animals were used. IgE specific for 15 recombinant (r) allergens as well as total serum IgE concentrations were measured by enzyme‐linked immunosorbent assay. Results Group IBH (median sum r‐Culicoides IgE: optical density at 405 nm [OD405] = 3.54 [0.48‐15.07]) and group IBH/EA (OD405 = 4.55 [0.46‐17.15]) had significantly (P  .05) differences between group IBH and group IBH/EA. No significant differences among the groups were found for the other r‐allergens or total serum IgE concentration. Compared to controls, horses with severe IBH had significantly increased IgE concentration to 5 Culicoides r‐allergens (P < .05), whereas horses with moderate IBH had significantly increased IgE concentration to only 3 Culicoides r‐allergens (P < .05). Conclusions and Clinical Importance Susceptibility of IBH‐affected horses to develop EA is likely not associated with IgE‐mediated immune reactions but with other immunopathological mechanisms

IL‐13, periostin and dipeptidyl‐peptidase‐4 reveal endotype‐phenotype associations in atopic dermatitis

Background: The heterogeneous (endo)phenotypes of atopic dermatitis (AD) require precision medicine. Currently, systemic therapy is recommended to patients with an Eczema Area and Severity Index (EASI) ≥ 16. Previous studies have demonstrated an improved treatment response to the anti‐interleukin (IL)‐13 antibody tralokinumab in AD subgroups with elevated levels of the IL‐13‐related biomarkers dipeptidyl‐peptidase (DPP)‐4 and periostin. Methods: Herein, 373 AD patients aged ≥12 years were stratified by IL‐13$^{high}$, periostin$^{high}$ and DPP‐4$^{high}$ endotypes using cross‐sectional data from the ProRaD cohort Bonn. “High” was defined as >80th quantile of 47 non‐atopic controls. We analyzed endotype‐phenotype associations using machine‐learning gradient boosting compared to logistic regression. Results: Atopic dermatitis severity and eosinophils correlated with IL‐13 and periostin levels. Correlations of IL‐13 with EASI were stronger in patients with increased (rs = 0.482) than with normal (rs = 0.342) periostin levels. We identified eosinophilia >6% and an EASI range of 5.5–17 dependent on the biomarker combination to be associated with increasing probabilities of biomarker$^{high}$ endotypes. Also patients with mild‐to‐low‐moderate severity (EASI < 16) featured increased biomarkers (IL‐13$^{high}$: 41%, periostin$^{high}$: 48.4%, DPP‐4$^{high}$: 22.3%). Herthoge sign (adjusted Odds Ratio (aOR) = 1.89, 95% Confidence Interval (CI) [1.14–3.14]) and maternal allergic rhinitis (aOR = 2.79–4.47) increased the probability of an IL‐13$^{high}$‐endotype, “dirty neck” (aOR = 2.83 [1.32–6.07]), orbital darkening (aOR = 2.43 [1.08–5.50]), keratosis pilaris (aOR = 2.21 [1.1–4.42]) and perleche (aOR = 3.44 [1.72–6.86]) of a DPP‐4$^{high}$‐endotype. Conclusions: A substantial proportion of patients with EASI < 16 featured high biomarker levels suggesting systemic impact of skin inflammation already below the current cut‐off for systemic therapy. Our findings facilitate the identification of patients with distinct endotypes potentially linked to response to IL‐13‐targeted therapy

The abundance of Ruminococcus bromii is associated with faecal butyrate levels and atopic dermatitis in infancy

Background: Impaired microbial development and decreased levels of short chain fatty acids, particularly butyrate, is suggested to have a role in the development of atopic dermatitis (AD). Methods: Faecal microbiota composition, abundance of selected bacterial groups and fermentation metabolites were compared at 90, 180 and 360 days of life between 27 children who developed AD by age one (AD group), and 39 controls (non-AD group) among the CARE (Childhood AlleRgy, nutrition and Environment) study cohort. Results: Diversity within the Firmicutes and Bacteroidetes phylum in the faecal microbiota was lower in the AD group compared to the non-AD group. Longitudinal analysis showed multiple amplicon sequence variants (ASV) within the same bacterial family to be differentially abundant. Namely, Ruminococcus bromii, a keystone primary starch degrader, and Akkermansia muciniphila, a mucin-utilizer, had lower abundance among the AD group. Children with AD were less likely to have high levels of faecal butyrate at 360 days compared to those without AD (11.5% vs 34.2%). At 360 days, children with high abundance of R. bromii had higher level of butyrate as well as lower proportion of children with AD compared to children with low abundance of R. bromii (11.1-12.5% vs 44.4-52.5%), which was independent of the abundance of the major butyrate producers. Conclusion: Our results suggested that R. bromii and other primary degraders might play an important role in the differences in microbial cross-feeding and metabolite formation between children with and without AD, which may influence the risk of developing the disease. Keywords: atopic dermatitis; butyrate; microbiota; resistant starch; short chain fatty acid

Atopic dermatitis: Correlation of distinct risk factors with age of onset in adulthood compared to childhood

Background: Atopic dermatitis (AD) has long been regarded as a primarily pediatric disease. However, there is growing evidence for a high rate of adult-onset AD. We aimed to characterize factors associated with adult-onset versus childhood-onset AD and controls. Methods: We analyzed cross-sectional data of the CK-CARE-ProRaD cohorts Bonn, Augsburg, Davos, Zürich of 736 adult patients stratified by age of AD onset (childhood-onset <18 years: 76.4% (subsets: 0 to 2; ≥2 to 6; ≥7 to 11; ≥12 to 18); adult-onset ≥18 years: 23.6% (subsets: ≥18 to 40; ≥41 to 60; ≥61) and 167 controls (91 atopic, 76 non-atopic)). Results: We identified active smoking to be associated with adult-onset AD versus controls (adjusted Odds Ratio (aOR) = 5.54 [95% Confidence Interval: 1.06-29.01] vs. controls$^{non-atopic}$ , aOR = 4.03 [1.20-13.45] vs. controls$^{atopic}$ ). Conjunctivitis showed a negative association versus controls$^{atopic}$ (aOR = 0.36 [0.14-0.91]). Food allergy (aOR = 2.93 [1.44-5.96]), maternal food allergy (aOR = 9.43 [1.10-80.95]), palmar hyperlinearity (aOR = 2.11 [1.05-4.25]), and academic background (aOR = 2.14 [1.00-4.54]) increased the odds of childhood-onset AD versus controls$^{atopic}$. Shared AD-associated factors were maternal AD (4-34x), increased IgE (2-20x), atopic stigmata (2-3x) with varying effect sizes depending on AD onset and control group. Patients with adult-compared to childhood-onset had doubled odds of allergic rhinitis (aOR = 2.15 [1.12-4.13]), but reduced odds to feature multiple (3-4) atopic comorbidities (aOR = 0.34 [0.14-0.84]). Adult-onset AD, particularly onset ≥61 years, grouped mainly in clusters with low contributions of personal and familial atopy and high frequencies of physical inactivity, childhood-onset AD, particularly infant-onset, mainly in "high-atopic"-clusters. Conclusions: The identified associated factors suggest partly varying endo- and exogeneous mechanisms underlying adult-onset versus childhood-onset AD. Our findings might contribute to better assessment of the individual risk to develop AD throughout life and encourage prevention by non-smoking and physical activity as modifiable lifestyle factors

In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients