Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. METHODS: The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. RESULTS: A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. CONCLUSIONS: Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.

Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen.

BACKGROUND: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. METHODS: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. RESULTS: The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αßα sandwich structure containing 4 α-helices and 6 antiparallel ß-sheets, encompassing 4 conserved CAP domain. CONCLUSION: The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.

Purification and characterization of enolase as a novel allergen in Platanus acerifolia pollen.

BACKGROUND: The pollen from Platanus acerifolia (P. acerifolia) is one of the main causes of allergic disorders. To date, only 4 allergens have been identified from this pollen. But previous studies showed that there still exist under-recognized allergens in it. The aim of this study was to comprehensively investigate the newly identified enolase (Pla a 6) as a novel allergen in the P. acerifolia pollen. METHODS: The natural (n) Pla a 6 was purified by combined chromatographic strategies. According to the identified internal peptides, the cDNA sequence encoding this allergen was matched from the mRNA-sequencing results of P. acerifolia pollen, which was further amplified and cloned. The recombinant (r) Pla a 6 was expressed and purified from E. coli. The allergenicity of this novel allergen was characterized by enzyme linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test (BAT). RESULTS: A novel allergen from P. acerifolia pollen, named as Pla a 6 was thoroughly studied, which contained an open reading frame of 1338 bp encoding 445 amino acids. The IgE-binding activity of nPla a 6 was initially proved by Western-blot, and a similar IgE-binding pattern to rPla a 6 was also exhibited. Moreover, the positivity for specific IgE against rPla a 6 was tested as 45.95% (17/37) by ELISA, and IgE binding to pollen extract could be inhibited up to 45.77% by 10 µg/ml of rPla a 6. The protein was also confirmed to activate patients' basophils. CONCLUSIONS: In this study, a novel allergen belonging to enolase family was comprehensively investigated and characterized through its natural and recombinant forms in P. acerifolia pollen. The study will contribute to the development of novel molecular-based diagnostic and therapeutic approaches for P. acerifolia pollen allergy.

A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms.

Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy.

Prediction of clinical efficacy of subcutaneous immunotherapy for Artemisia sieversiana pollen allergic rhinitis by serum metabolomics.

BACKGROUND/PURPOSE: Specific immunotherapy is the only effective etiological treatment for allergic rhinitis, but subcutaneous immunotherapy has a slow onset and poor compliance. Predicting the clinical efficacy of subcutaneous immunotherapy in advance can reduce unnecessary medical costs and resource waste. This study aimed to identify metabolites that could predict the efficacy of subcutaneous immunotherapy on seasonal allergic rhinitis by serum metabolomics. METHODS: Patients (n = 43) with Artemisia sieversiana pollen allergic rhinitis were enrolled and treated with subcutaneous immunotherapy for one year. Patients were divided into the ineffective group (n = 10) and effective group (n = 33) according to the therapeutic index. Serum samples were collected before treatment. Metabolomics was determined by liquid chromatography-mass spectrometry combined with gas chromatography-mass spectrometry and analyzed differential compounds and related metabolic pathways. RESULTS: A total of 129 differential metabolites (P < 0.05) were identified and 4 metabolic pathways, namely taurine and hypotaurine metabolism, pentose and glucuronate interconversions, pentose phosphate pathway, and alanine, aspartate, and glutamate metabolism, were involved. CONCLUSION: Some metabolites, such as hypotaurine, taurine, and l-alanine, have the potential to become predictive biomarkers for effective subcutaneous immunotherapy.

Molecular and immunochemical characterization of profilin as major allergen from Platanus acerifolia pollen.

BACKGROUND: The Platanus acerifolia (P. acerifolia) pollen is one of the most common causes of allergic respiratory symptoms in China. However, the allergenic components in P. acerifolia are not fully studied yet. The study aimed to determine the molecular and immunochemical characterization of the profilin from P. acerifolia pollen. METHODS: The coding sequence of profilin was amplified, cloned, and then expressed in Escherichia coli BL21 cells and purified by nickel affinity chromatography. Protein refolding was followed by structural characterization and homology 3D model building. The allergenicity and cross-reactivity were assessed by ELISA, immunoblotting, or basophil activation test (BAT) using the sera of P. acerifolia allergic patients. RESULTS: The cDNA sequence of profilin was cloned with a 396 bp open reading frame coding for 131 amino acids. The molecular weight of the profilin was approximately 14 kDa, and the predicted structure consisted of 3 α-helixes and 7 ß-sheets. Physicochemical analysis indicated the profilin was a stable, relatively thermostable, and relatively conserved protein. The allergenicity determined by ELISA, western blot, and BAT suggested 76.9% (30/39) of the P. acerifolia pollen allergic patients displayed specific IgE recognition of the profilin. The profilin shared > 80% sequence identity with Pop n 2, the profilin from Populus nigra, and observed a significant cross-reactivity with Pop n 2 in IgE-inhibition assay. CONCLUSION: Profilin, as one of the major component allergens in P. acerifolia pollen, was identified and characterized at molecular and immunochemical levels in this study. These findings would contribute to developing diagnostic and therapeutic strategies for P. acerifolia pollen allergic patients.

The Diagnosis and Management of Allergic Reactions Caused by Chinese Materia Medica.

Traditional Chinese medicines (TCM) have been used in China for thousands of years. Although TCM has been generally perceived to be safe, adverse reactions to Chinese materia medica (CMM) have been reported. Most of the adverse reactions are allergic in nature, but other mechanisms may play a role. This review focuses on the mechanism and clinical presentation of these allergic reactions. Allergic reactions can occur as a result of the active and inactive ingredients of CMM. Impurities and chemicals generated during the production process can also lead to allergic or adverse reactions. Environmental factors such as temperature, humidity, and light can cause changes in the allergenicity of drugs. Human error in formulating CMM drugs also contributes to adverse drug reactions. The management of allergic reactions to CMM includes taking a good history, avoidance of medications in the same class as those which caused prior reactions, the proper training of staff, adherence to manufacturer guidelines and expiration dates, evaluation of benefit and risk balance, and the formulation of a risk management strategy for the use of CMM. A small test dose of a considered drug before using, improvements in drug purification technology, and proper storage and clinical administration help reduce allergic reactions due to CMM.

Identification of a Novel Major Allergen in Buckwheat Seeds: Fag t 6.

Buckwheat is one of the five main allergenic foods (eggs, milk, wheat, buckwheat, and peanuts). Oleosin is an important type of allergen in some allergic foods. However, although most diagnostic nut and seed extracts are defatted, some patients with food allergies may have false negative diagnostic results of oleosin in vitro. Recently, we found that the serum of buckwheat allergic patients responded strongly to an 18 kDa protein. Mass spectrometry analysis showed it is the oleosin protein family. We further purified and evaluated the allergenicity of this buckwheat oleosin-type allergen, which is involved in the formation of buckwheat oil bodies. The tartary buckwheat oleosin allergen was named Fag t 6, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria. The DNA sequence of tartary buckwheat oleosin was cloned. Dot blot and enzyme-linked immunosorbent assay (ELISA) showed half of the 20 buckwheat allergic patients' serum had strong reactivity with purified buckwheat Fag t 6. Circular dichroism experiment analysis of its thermal stability showed a Tm of 64.65 ± 0.65 °C. A buckwheat allergy showed possible cross-reaction with a wheat allergy. In summary, this study not only increases our understanding of buckwheat allergies and oil-soluble allergens in general, it may also be used to improve diagnostic tests for buckwheat allergies in the future.

Principles of Allergen Immunotherapy and Its Clinical Application in China: Contrasts and Comparisons with the USA.

Allergen immunotherapy (AIT) for allergic rhinitis (AR), asthma, and other allergic diseases has developed quickly. House dust mite (HDM), Artemisia (wormwood), Humulus japonicus (Japanese hop), Alternaria alternata, and Cladosporium herbarum are the five most common inhalant allergens in China. AIT has been performed in China for over 60 years. With the support of the Chinese Medical Association (CMA) and the Chinese Medical Doctors Association (CMDA), the Chinese College of Allergy and Asthma (CCAA) was established in 2016 as a specialized branch of CDMA and is the main certification authority for AIT. Chinese allergists and scientists have made tremendous progress in the development of AIT. There have been many publications by Chinese allergists and scientists worldwide encompassing original research studies, systematic reviews, case studies, and clinical trials. Currently, conventional subcutaneous immunotherapy (SCIT) is the preferred AIT in China, but sublingual immunotherapy (SLIT) is beginning to gain recognition. An increasing number of clinical trials have been conducted to investigate the clinical efficacy and side effects of SLIT and SCIT. In China, HDM is the only commercial standardized allergen extracts in clinical use, whereas the others are crude allergen extracts. Besides standardized allergen extracts, other forms of hypoallergenic extracts are still being investigated and developed in China. Immunotherapy in China is similar to that in the USA in which allergen extracts can be mixed for SCIT. However, allergen extracts cannot be mixed for SCIT in Europe.

Regional Differences in Food Allergies.

The prevalence of food allergies is increasing worldwide. To understand the regional specificities of food allergies and develop effective therapeutic interventions, extensive regional epidemiological studies are necessary. While data regarding incidence, prevalence, regional variation, and treatment in food allergies are available for western countries, such studies may not be available in many Asian countries. China accounts for almost 20% of the world's population and has a vast ethnic diversity, but large-scale meta-analyses of epidemiological studies of food allergy in China are lacking. A literature search revealed 22 publications on the prevalence of food allergy in Chinese populations. A review of these studies showed that the prevalence of food allergies in China is comparable to that in western countries, even though the Chinese diet is vastly different from that of the West and may vary even greatly within China, and finally, specific antigenic triggers of food allergy vary between China and the West and also within China. Current clinical management of food allergy in China includes allergen-specific immunotherapy, Chinese herbal medicine, acupuncture, and Western medicine. This study demonstrates an unmet need in China for a thorough investigation of the prevalence of food allergies in China, the specific foods involved, and characterization of the specific antigenic triggers of food allergy with respect to ethnicity, age, and diet in China.