Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

The Role of the E3 Ligase Cbl-B in Murine Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb−/− bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb−/− BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb−/− BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb−/− BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8⁺ OT-I or CD4⁺ OT-II transgenic T cells. However, cblb−/− BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb−/− peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.     

3-Deoxyglucosone: metabolism,analysis, biological activity,and clinical implication

3-Deoxyglucosone (3-DG) is synthesized via the Maillard reaction and the polyol pathway, and is detoxified to 3-deoxyfructose and 2-keto-3-deoxygluconic acid. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, N^ε-(carboxymethyl)1ysine and pentosidine, among which imidazolone is the AGE most specific for 3-DG. As demonstrated by using gas chromatography–mass spectrometry or high-performance liquid chromatography, plasma 3-DG levels are markedly increased in diabetes and uremia. Although the plasma 3-DG levels had been controversial, it was clearly demonstrated that its plasma level depends on the deproteinization method by which either free or total 3-DG, presumably bound to proteins, is measured. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway, and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG, which may be due to the loss of 3-DG reductase activity in the end-stage kidneys, may lead to high plasma 3-DG level. The elevated 3-DG levels in plasma and erythrocytes may promote the formation of AGEs such as imidazolone, as demonstrated by immunohistochemistry and immunochemistry using an anti-imidazolone antibody. Although AGE-modified proteins prepared in vitro exhibit a variety of biological activities, known AGE structures have not yet been demonstrated to show any biological activities. Because 3-DG is potent in the formation of AGEs and has some biological activities, such as cellular toxicity, it may be more important in the development of diabetic and uremic complications than the known AGE structures. By demonstrating that treatment with an aldose reductase inhibitor reduces the erythrocyte levels of 3-DG and AGEs, such as imidazolone, light is shed on the mystery of how aldose reductase inhibitors may prove beneficial in diabetic complications. These evidences suggest that 3-DG plays a principal role in the development of diabetic and uremic complications.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

Candida soluble cell wall β-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells

Background

Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro.

Methods

In vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0–12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity.

Results

In vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs.

Conclusion

CSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.     

NMR Structure and IgE Epitopes of Blo t 21, a Major Dust Mite Allergen from Blomia tropicalis

Blo t 21 is a paralogue of the group 5 allergen, Blo t 5, a major allergen from the dust mite Blomia tropicalis. Blo t 21 has moderate sequence identity (40.7%) to Blo t 5 and low to moderate cross-reactivity to Blo t 5. In B. tropicalis, the most prevalent and allergenic allergens are in the order of Blo t 21, Blo t 5, and Blo t 7. Here, we determined the NMR solution structure of Blo t 21, which represents the first structure of the group 21 dust mite allergen. The structure of Blo t 21 closely resembles the structures of Blo t 5 and Der p 5, comprising three anti-parallel α-helices arranged in a helical bundle. Using site-directed mutagenesis and specific IgE binding ELISA, Blo t 21 was found to contain both conserved and unique charged IgE epitope residues at the L2 loop region and on helix α3. Cross-inhibition assays confirmed that Blo t 21 has a low to moderate cross-reactivity with Blo t 5 and Der p 5 and represents a novel group of major allergen in B. tropicalis. In addition to group 5 allergens, Blo t 21 has also a low to moderate cross-reactivity with group 21 allergens from Dermatophagoides mites, confirming that B. tropicalis is a major and distinct source of dust mite allergens.     

3-Deoxyglucosone: A Potential Glycating Agent Accountable for Structural Alteration in H3 Histone Protein through Generation of Different AGEs

Advanced glycation end-products (AGEs) are heterogeneous group of compounds, known to be implicated in diabetic complications. One of the consequences of the Maillard reaction is attributed to the production of reactive intermediate products such as α-oxoaldehydes. 3-deoxyglucosone (3-DG), an α-oxoaldehyde has been found to be involved in accelerating vascular damage during diabetes. In the present study, calf thymus histone H3 was treated with 3-deoxyglucosone to investigate the generation of AGEs (N^ε-carboxymethyllysine, pentosidine), by examining the degree of side chain modifications and formation of different intermediates and employing various physicochemical techniques. The results clearly indicate the formation of AGEs and structural changes upon glycation of H3 by 3-deoxyglucosone, which may hamper the normal functioning of H3 histone, that may compromise the veracity of chromatin structures and function in secondary complications of diabetes.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Background

Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.

Methodology/Principal Findings

A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals'' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.

Conclusion/Significance

This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.     

FEEL-1 and FEEL-2 are endocytic receptors for advanced glycation end products

Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate in vascular tissues in aging and diabetes. Receptors for AGEs include scavenger receptors, which recognize acetylated low density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SR-BI), and lectin-like oxidized low density lipoprotein receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs have prompted us to examine whether AGEs are also recognized by the novel scavenger receptors, which we have recently isolated from a cDNA library prepared from human umbilical vein endothelial cells, such as the scavenger receptor expressed by endothelial cells-I (SREC-I); the fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At 4 degrees C, (125)I-AGE-bovine serum albumin (BSA) exhibited high affinity specific binding to Chinese hamster ovary (CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) with K(d) of 2.55 and 1.68 microg/ml, respectively, but not to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At 37 degrees C, (125)I-AGE-BSA was taken up and degraded by CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The (125)I-AGE-BSA binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids, and dextran sulfate but not by native LDL, oxidized LDL, or HDL. FEEL-1, which is expressed by the liver and vascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis.     

Scavenger receptor-A functions in phagocytosis of E. coli by bone marrow dendritic cells

Class-A scavenger receptors (SR-A) are cellular pattern recognition receptors that bind and traffic a variety of endogenous and microbial ligands. However, despite an emerging role for SR-A as a contributor to the innate immune system, little is known of the regulation or function of SR-A on dendritic cells (DCs). Here we show that SR-A expression is upregulated during murine DC differentiation and that SR-A expression levels correlate with the expression of the murine DC marker CD11c. Using bone marrow-derived DCs (BMDCs) from SR-A knockout (SR-A(-/-)) mice, we investigated the contribution of SR-A to BMDC particulate phagocytosis. Functional analyses demonstrated that SR-A is a critical phagocytic receptor for BMDC internalization of the gram-negative bacteria E. coli. SR-A(-/-) BMDCs were impaired in their ability to phagocytose bacteria, and this deficit varied with the bacteria:BMDC cell ratio. Microscopic and biochemical analyses revealed that SR-A is broadly distributed on the surface of BMDCs and is not physically associated with lipid rafts. However, cholesterol depletion demonstrated dependence of SR-A-mediated phagocytosis upon lipid rafts. These data demonstrate a functional contribution for SR-A in the BMDC phagocytic pathway.     

A Protein Allergen Microarray Detects Specific IgE to Pollen Surface,Cytoplasmic, and Commercial Allergen Extracts

Background

Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.

Methodology/Principal Findings

We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.

Conclusions/Significance

These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line ¹²⁵I-LDL, and on a murine macrophage cell line (J774) ¹²⁵I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.     

Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

BackgroundGenetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release.ObjectiveTo compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize.MethodsAn integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed.ResultsIn silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.ConclusionCry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

The Transaldolase,a Novel Allergen of Fusarium proliferatum,Demonstrates IgE Cross-Reactivity with Its Human Analogue

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.     

Recognition of lipoproteins by scavenger receptor class A members

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca²⁺-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca²⁺-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.