Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential

AbstractPlant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries. Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen. The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass. To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E coli. The coding region of Zm13 was PCR amplified from a genomic clone and expressed as as a glutathione-S-transferase fusion protein. The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens

Component-Resolved Diagnosis (CRD) of Type I Allergy with Recombinant Grass and Tree Pollen Allergens by Skin Testing

The diagnosis of Type I allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens, most frequently conducted by skin testing. Both forms of diagnosis are currently performed with allergen extracts that are difficult to standardize regarding their allergen contents, and which contain additional undefined nonallergenic components. We report the expression in Escherichia coli and purification of some of the most relevant timothy grass- and birch pollen allergens. Recombinant timothy grass- (rPhl p 1, rPhl p 2, rPhl p 5) and birch pollen (rBet v 1, rBet v 2) allergens were purified and used for the measurement of allergen-specific IgE and IgG subclass responses as well as for skin prick testing in 55 pollen allergic patients and 10 nonatopic individuals. Results obtained showed that the recombinant allergens allowed in vivo allergy diagnosis in 52 of 54 of the grass pollen and in 35 of 36 of the birch pollen allergic patients. Positive skin reactions were observed almost exclusively in patients containing detectable allergen-specific IgE antibodies but not in the nonatopic group; however, sensitivity to a given allergen as measured by skin reactivity was weakly correlated with the levels of allergen-specific IgE. Our results demonstrate that recombinant allergens can be used for component-resolved skin test diagnosis (CRD) of the patients’ allergen sensitization profile, whereas allergen extracts at best allow to identify allergen-containing sources. CRD may thus represent the basis for novel forms of patient-tailored immunotherapy

Angiotensin-II-Evoked Ca2+ Entry in Murine Cardiac Fibroblasts Does Not Depend on TRPC Channels

TRPC proteins form cation conducting channels regulated by different stimuli and are regulators of the cellular calcium homeostasis. TRPC are expressed in cardiac cells including cardiac fibroblasts (CFs) and have been implicated in the development of pathological cardiac remodeling including fibrosis. Using Ca2+ imaging and several compound TRPC knockout mouse lines we analyzed the involvement of TRPC proteins for the angiotensin II (AngII)-induced changes in Ca2+ homeostasis in CFs isolated from adult mice. Using qPCR we detected transcripts of all Trpc genes in CFs; Trpc1, Trpc3 and Trpc4 being the most abundant ones. We show that the AngII-induced Ca2+ entry but also Ca2+ release from intracellular stores are critically dependent on the density of CFs in culture and are inversely correlated with the expression of the myofibroblast marker α-smooth muscle actin. Our Ca2+ measurements depict that the AngII- and thrombin-induced Ca2+ transients, and the AngII-induced Ca2+ entry and Ca2+ release are not affected in CFs isolated from mice lacking all seven TRPC proteins (TRPC-hepta KO) compared to control cells. However, pre-incubation with GSK7975A (10 µM), which sufficiently inhibits CRAC channels in other cells, abolished AngII-induced Ca2+ entry. Consequently, we conclude the dispensability of the TRPC channels for the acute neurohumoral Ca2+ signaling evoked by AngII in isolated CFs and suggest the contribution of members of the Orai channel family as molecular constituents responsible for this pathophysiologically important Ca2+ entry pathway

Salt-induced changes of colloidal interactions in critical mixtures

We report on salt-dependent interaction potentials of a single charged particle suspended in a binary liquid mixture above a charged wall. For symmetric boundary conditions (BC) we observe attractive particle-wall interaction forces which are similar to critical Casimir forces previously observed in salt-free mixtures. However, in case of antisymmetric BC we find a temperature-dependent crossover from attractive to repulsive forces which is in strong contrast to salt-free conditions. Additionally performed small-angle x-ray scattering experiments demonstrate that the bulk critical fluctuations are not affected by the addition of salt. This suggests that the observed crossover can not be attributed alone to critical Casimir forces. Instead our experiments point towards a possible coupling between the ionic distributions and the concentration profiles in the binary mixture which then affects the interaction potentials in such systems.Comment: 5 pages, 4 Figure

Genome-Wide Scan on Total Serum IgE Levels Identifies FCER1A as Novel Susceptibility Locus

High levels of serum IgE are considered markers of parasite and helminth exposure. In addition, they are associated with allergic disorders, play a key role in anti-tumoral defence, and are crucial mediators of autoimmune diseases. Total IgE is a strongly heritable trait. In a genome-wide association study (GWAS), we tested 353,569 SNPs for association with serum IgE levels in 1,530 individuals from the population-based KORA S3/F3 study. Replication was performed in four independent population-based study samples (total n = 9,769 individuals). Functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 (rs2251746 and rs2427837) were strongly associated with total IgE levels in all cohorts with P values of 1.85×10−20 and 7.08×10−19 in a combined analysis, and in a post-hoc analysis showed additional associations with allergic sensitization (P = 7.78×10−4 and P = 1.95×10−3). The “top” SNP significantly influenced the cell surface expression of FCER1A on basophils, and genome-wide expression profiles indicated an interesting novel regulatory mechanism of FCER1A expression via GATA-2. Polymorphisms within the RAD50 gene on chromosome 5q31 were consistently associated with IgE levels (P values 6.28×10−7−4.46×10−8) and increased the risk for atopic eczema and asthma. Furthermore, STAT6 was confirmed as susceptibility locus modulating IgE levels. In this first GWAS on total IgE FCER1A was identified and replicated as new susceptibility locus at which common genetic variation influences serum IgE levels. In addition, variants within the RAD50 gene might represent additional factors within cytokine gene cluster on chromosome 5q31, emphasizing the need for further investigations in this intriguing region. Our data furthermore confirm association of STAT6 variation with serum IgE levels

Cluster Cosmology Constraints from the 2500 deg2^2 SPT-SZ Survey: Inclusion of Weak Gravitational Lensing Data from Magellan and the Hubble Space Telescope

We derive cosmological constraints using a galaxy cluster sample selected from the 2500~deg$^2$ SPT-SZ survey. The sample spans the redshift range $0.25< z5$. The sample is supplemented with optical weak gravitational lensing measurements of 32 clusters with $0.29<z<1.13$ (from Magellan and HST) and X-ray measurements of 89 clusters with $0.25<z<1.75$ (from Chandra). We rely on minimal modeling assumptions: i) weak lensing provides an accurate means of measuring halo masses, ii) the mean SZ and X-ray observables are related to the true halo mass through power-law relations in mass and dimensionless Hubble parameter $E(z)$ with a-priori unknown parameters, iii) there is (correlated, lognormal) intrinsic scatter and measurement noise relating these observables to their mean relations. We simultaneously fit for these astrophysical modeling parameters and for cosmology. Assuming a flat $\nu\Lambda$CDM model, in which the sum of neutrino masses is a free parameter, we measure $\Omega_\mathrm{m}=0.276\pm0.047$, $\sigma_8=0.781\pm0.037$, and $\sigma_8(\Omega_\mathrm{m}/0.3)^{0.2}=0.766\pm0.025$. The redshift evolution of the X-ray $Y_\mathrm{X}$-mass and $M_\mathrm{gas}$-mass relations are both consistent with self-similar evolution to within $1\sigma$. The mass-slope of the $Y_\mathrm{X}$-mass relation shows a $2.3\sigma$ deviation from self-similarity. Similarly, the mass-slope of the $M_\mathrm{gas}$-mass relation is steeper than self-similarity at the $2.5\sigma$ level. In a $\nu w$CDM cosmology, we measure the dark energy equation of state parameter $w=-1.55\pm0.41$ from the cluster data. We perform a measurement of the growth of structure since redshift $z\sim1.7$ and find no evidence for tension with the prediction from General Relativity. We provide updated redshift and mass estimates for the SPT sample. (abridged)Comment: Accepted for publication in ApJ (v2 is accepted version), the catalog can be found at https://pole.uchicago.edu/public/data/sptsz-clusters