The structure of Cyn d 4, the group 4 allergen from Bermuda grass, is reported at 2.15 Å resolution and is the first crystal structure of a naturally isolated pollen allergen. A conserved N-terminal segment that is only present in the large isoallergens forms extensive interactions with surrounding residues and hence greatly enhances the structural stability of the protein. Cyn d 4 contains an FAD cofactor that is covalently linked to His88 and Cys152. To date, all identified bicovalent flavoproteins are oxidases and their substrates are either sugars or secondary metabolites. A deep large hydrophobic substrate-binding cleft is present. Thus, Cyn d 4 may be an oxidase that is involved in the biosynthesis of a pollen-specific metabolite. Cyn d 4 shares ~70% sequence identity with the Pooideae group 4 allergens. Various cross-reactivities between grass pollen group 4 allergens have previously been demonstrated using sera from allergic patients. The protein surface displays an unusually large number of positively charged clusters, reflecting the high pI of ~10. 38 decapeptides that cover the solvent-accessible sequences did not show any significant IgE-binding activity using sera with high Cyn d 4 reactivity from four patients, suggesting that the IgE epitopes of Cyn d 4 are predominantly conformational in nature. Several group 4 structures were then modelled and their potential cross-reactive and species-specific IgE epitopes were proposed.

Diagnosis of transplant rejection requires tissue biopsy and entails risks. Here, we describe a new 19F MRI approach for noninvasive visualization of organ rejection via the macrophage host response. For this, we employed biochemically inert emulsified perfluorocarbons (PFCs), known to be preferentially phagocytized by monocytes and macrophages. Isografts from C57BL/6 or allografts from C57B10.A mice were heterotopically transplanted into C57BL/6 recipients. PFCs were applied intravenously followed by 1H/19F MRI at 9.4 T 24 h after injection. 1H images showed a similar position and anatomy of the graft in the abdomen for both cases. However, corresponding 19F signals were only observed in allogenic tissue. 1H/19F MRI enabled us to detect the initial immune response not later than 3 days after surgery, when conventional parameters did not reveal any signs of rejection. In allografts, the observed 19F signal strongly increased with time and correlated with the extent of rejection. In separate experiments, rapamycin was used to demonstrate the ability of 19F MRI to monitor immunosuppressive therapy. Thus, PFCs can serve as positive contrast agent for the early detection of transplant rejection by 19F MRI with high spatial resolution and an excellent degree of specificity due to lack of any 19F background.

Background: Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG4 antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. Methods: Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG1–4 antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. Results: Four major IgE-binding epitopes (peptides 15–1, 21, 33–2 and 35+1, corresponding to amino acids 70–79, 101–110, 159–167 and 172–181) and 5 major IgG4-binding epitopes (peptides 15–1, 30–2, 33–2, 35+1 and 39, corresponding to amino acids 70–79, 144–153, 159–167, 172–181 and 192–200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG4. Their critical amino acids were all characterized. Major epitopes for human IgG1 to IgG4 are almost identical. Conclusions: This is the first study to map the sequential epitopes for human IgE and IgG4 subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis.

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC- specific ICAM3-grabbing non-integrin (DCSIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha 1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Lex). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC- SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.

Although cockroaches are known to produce allergens that can cause IgE‐mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2‐DE and IgE‐binding proteins were analyzed by nanoLC‐MS/MS or N‐terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE‐binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross‐reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE‐binding proteins varied between cockroach‐allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.

Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca2+ mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca2+-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca2+-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.

BACKGROUND:Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells.METHODS:A549 cells, 16HBE14o- cells and primary cultures of human bronchial epithelial cells (HBEpC) were exposed to purified Pen ch 13 and mediators released into culture supernatants were assayed with enzyme-linked immunosorbent assay (ELISA) kits. Cleavage of occludin in 16HBE14o- cells was analysed by immunofluorescent staining of whole cells and immunoblot analysis of whole cell extracts. Fragments generated by incubating Pen ch 13 and a synthetic peptide carrying the occludin sequence were analysed by mass spectrometry.RESULTS:Pen ch 13 induced productions of prostaglandin-E2 (PGE2), interleukin (IL)-8 and transforming growth factor (TGF)-beta1 by A549 cells, 16HBE14o- cells and primary cultures of HBEpC. The protease activity of Pen ch 13 is needed for the induction of PGE2 IL-8, TGF-beta1 and cyclo-oxygenase (COX)-2 expression. A tight junction protein occludin of 16HBE14o- cells can be cleaved by Pen ch 13 at Gln202 and Gln211 which are within the second extracellular domain of the protein.CONCLUSION:Pen ch 13 may contribute to asthma by damaging the barrier formed by the airway epithelium and stimulating the release of mediators that orchestrate local immune responses and inflammatory process from HBEpC.

ABSTRACTObjectives:Cow's milk protein hydrolysate formulas are widely used for genetically predisposed atopic infants. Whether hydrolysate formulas can induce oral tolerance to α‐casein was studied for the first time in naive and sensitized mice.Methods:Using immunoblotting, residual antigenicity to α‐casein was examined for in animals fed hydrolysate formulas. Naïve mice fed hydrolysate formulas for 1 to 4 weeks were later sensitized with α‐casein. Another group of mice sensitized first with α‐casein were then fed hydrolysate formulas continually for 12 weeks.Results:Oral tolerance measured by immunoglobulin (Ig)E and IgG1 antibody responses to α‐casein was induced in naïve mice fed NAN for 1 week or NAN‐HA for 4 weeks. IgE responses to α‐casein were suppressed in mice fed NAN‐HA for 1 week or Neoangelac FL for 4 weeks. In contrast, mice fed Alfare, Pepti‐Junior, or Pregestimil for 1 to 4 weeks did not develop tolerance to α‐casein. Antibody responses to α‐casein were not significantly suppressed in sensitized mice fed NAN or hydrolysate formulas for 12 weeks.Conclusions:Primary IgE responses to α‐casein are readily suppressed in naïve mice firts fed cow's milk formula or partially hydrolyzed formula for 1 week. Conversely, ongoing IgE, IgG1, and IgG antibody responses to α‐casein are poorly supressed in previously sensitized mice even after prolonged feeding of cow's milk formula or hydrolysate formulas.

RATIONALE: Bermuda grass (Cynodon dactylon) pollen contains a very complexity of allergens. One of the allergens with molecular mass of 21 kDa has been shown to bind serum IgE from 30% patients with BGP allergy. The aim of this study was to purify, characterize and clone the 21 kDa allergen and investigate its glycan structure in IgE binding.METHODS: Bermuda grass pollen extract was fractionated by using ion-exchange chromatography and reverse phase HPLC. Immunoblotting and ELISA were performed for investigating its allergenicity, and the endoproteinase Lys-C digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR based cloning. Putative glycan structures were elucidated using MALDI-TOF mass spectrometry.RESULTS: Cyn d 24allergen was purified from Bermuda grass pollen with an observed molecular mass of 21 kDa and pI of 5.7. The cDNA of Cyn d 24 was predicted as a 153-aa mature protein with an N terminus at Ser23. Sequence comparison revealed that Cyn d 24 possesses several features in common with the pathogen-related protein family. After removed the N-glycan, Cyn d 24 reduced > 80% and >90% of binding activities to human IgE. Six oligosaccharides fractions were isolated and the most abundant oligosaccharide (83.75%) was found to be Man3GlcNAc2Fuc.CONCLUSIONS: Cyn d 24, a novel glycoprotein allergen of Bermuda grass pollen has been isolated, characterized, and cloned. Its N-glycan structure play an important role in the immune response, especially in the IgE binding. RATIONALE: Bermuda grass (Cynodon dactylon) pollen contains a very complexity of allergens. One of the allergens with molecular mass of 21 kDa has been shown to bind serum IgE from 30% patients with BGP allergy. The aim of this study was to purify, characterize and clone the 21 kDa allergen and investigate its glycan structure in IgE binding. METHODS: Bermuda grass pollen extract was fractionated by using ion-exchange chromatography and reverse phase HPLC. Immunoblotting and ELISA were performed for investigating its allergenicity, and the endoproteinase Lys-C digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR based cloning. Putative glycan structures were elucidated using MALDI-TOF mass spectrometry. RESULTS: Cyn d 24allergen was purified from Bermuda grass pollen with an observed molecular mass of 21 kDa and pI of 5.7. The cDNA of Cyn d 24 was predicted as a 153-aa mature protein with an N terminus at Ser23. Sequence comparison revealed that Cyn d 24 possesses several features in common with the pathogen-related protein family. After removed the N-glycan, Cyn d 24 reduced > 80% and >90% of binding activities to human IgE. Six oligosaccharides fractions were isolated and the most abundant oligosaccharide (83.75%) was found to be Man3GlcNAc2Fuc. CONCLUSIONS: Cyn d 24, a novel glycoprotein allergen of Bermuda grass pollen has been isolated, characterized, and cloned. Its N-glycan structure play an important role in the immune response, especially in the IgE binding.

Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglobulin E (IgE) immunoblots. However, only a few allergens have been identified and characterized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE-reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI-TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identifying six novel allergens.

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18 411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man(3)GlcNAc(2)) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.

SummaryBackground For genetically predisposed atopic infants, cow's milk protein hydrolysed formulas have been widely used.Objective Whether hydrolysed formulas can induce oral tolerance to whey proteins will be extensively studied in naïve and sensitized mice.Methods Antigenicity of hydrolysed formulas was first studied using immunoblotting. Naïve mice fed hydrolysed formulas for 1–4 weeks were sensitized with whey allergens. In contrast, mice sensitized with whey allergens were fed hydrolysed formulas continually for 12 weeks.Results Whey allergens were found in Nan and Neoangelac FL. Large whey peptides with antigenicity were found in Nan‐HA. Profound suppression of IgE, IgG1 and IgG responses to whey allergens were induced in those fed Nan for 1 week, or Nan‐HA for 4 weeks. IgE responses to whey allergens were suppressed in those fed Neoangelac FL for 4 weeks, or Nan‐HA for 1–2 weeks. In contrast, those fed extensively hydrolysed formulas for 1–4 weeks failed to show decreased responses. On the other hand, IgE responses to β‐lactoglobulin, but not to bovine serum albumin or α‐lactalbumin, were decreased in sensitized mice fed Nan for 12 weeks. There was no suppression in sensitized mice fed hydrolysed formulas.Conclusion Suppression of IgE responses to whey proteins was readily induced in naïve mice fed Nan or Nan‐HA for 1 week. In contrast, it was hardly induced in sensitized mice even after prolonged feeding of Nan for 12 weeks, let alone hydrolysed formulas.

BACKGROUND:Garlic (Allium sativum) is one of the most common relishes used in cooking worldwide. Very few garlic allergens have been reported, and garlic allergy has been rarely studied.OBJECTIVE:The aim of the study was to identify allergenic proteins in garlic and to investigate their importance in allergies to other Allium species (leek, shallot, and onion).METHODS:A crude extract of garlic proteins was separated by SDS-PAGE and 2-dimensional electrophoresis; immunoblotting was then performed with the use of individual and pooled sera from patients with garlic allergy, and the major IgE-binding proteins were analyzed by amino acid sequencing and mass spectrometry. The putative allergens were further purified by chromatography; the antigenicity, allergenicity, and IgE-binding cross-reactivity of the purified protein were then studied by immunoblotting, periodate oxidation, skin tests, and IgE-binding inhibition assays.RESULTS:A major allergen, alliin lyase, was identified by mass spectrometry and Edman sequencing and purified to homogeneity through the use of a simple 2-step chromatographic method. Skin tests showed that the purified protein elicited IgE-mediated hypersensitive responses in patients with garlic allergy. Periodate oxidation showed that carbohydrate groups were involved in the antigenicity, allergenicity, and cross-reactivity. Garlic alliin lyase showed strong cross-reactivity with alliin lyases from other Allium species, namely leek, shallot, and onion.CONCLUSIONS:Alliin lyase was found to be a major garlic allergen in a garlic-allergic group of patients in Taiwan. The wide distribution of alliin lyase in Allium suggests it may be a new cross-reactive allergen.

A high-molecular-weight mite allergen Der f11 that was hardly purified for immunotherapy was used to develop the DNA vaccine pDf11. We have shown that vaccination of mice with pDf11 induces Th1 responses characterized by suppression of IgE responses. In the present study, effects of different adjuvants on pDf11 were first studied. Mice receiving pDf11 +/- CpG, bestatin, and bupivacaine had better suppression of IgE responses than those receiving pDf11 +/- lipofectin or alum. Bestatin could greatly boost IgG2a responses. Immunomodulating effects of different adjuvants between protein and DNA vaccines were further elucidated. CpG was the best for both protein and DNA vaccines to profoundly suppress IgE responses, but alum, bestatin and lipofectin were useless for rDf11 to induce IgE inhibition. Neither did the combination of rDf11 and pDf11 have further IgE suppression. In conclusion, CpG is the unique adjuvant for the protein vaccine rDf11 to inhibit IgE responses. In contrast, the DNA vaccine pDf11 +/- CpG, bestatin, or bupivacaine induces profound suppression of IgE responses.

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-γ. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4+ or CD8+ T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8+ T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulation of IgE responses by pDf11.

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization—time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni2+-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20°C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (∼180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS–PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.