Japanese Cedar Pollen Allergens in Japan.

Pollen from members of the Cupressaceae tree family is one of the most important causes of allergic disease in the world. Cryptomeria japonica (Japanese cedar) and Chamaecyparis obtusa (Japanese cypress) are Japan's most common tree species. The pollen dispersal season is mainly from February to May. The major allergens of Japanese cedar and Japanese cypress exhibit high amino acid sequence similarity due to the phylogenetic relationship between the two species. An epidemiological study has shown that the prevalence of Japanese cedar pollinosis is approximately 40%. Younger children (5 to 9 years old) showed a high prevalence of Japanese cedar pollinosis as 30% in 2019, indicating that season pollinosis is getting worse. Pharmacotherapy is the most common treatment for pollinosis induced by Japanese cedar and Japanese cypress. Patients' satisfaction with pharmacotherapy is low due to insufficient experienced effect and daytime somnolence. Unlike pharmacotherapy, allergy immunotherapy (AIT) addresses the basic immunological mechanisms of allergic disease and activates protective allergen-reactive pathways of the immune system. AIT is now recognized as the only treatment option with the potential to provide long-term post-treatment benefits and alter the natural course of the allergic disease, including Japanese cedar pollinosis.

Polymorphisms in Cha o 1 and Cha o 2, major allergens of Japanese cypress (Chamaecyparis obtusa) pollen from a restricted region in Japan.

Japanese cedar pollinosis is a major seasonal allergy in Japan, and Japanese cypress pollinosis is a growing concern because the cypress pollen season follows the cedar pollen season and cross-reactivity among allergens occurs between these closely related species. Allergens purified from pollen under unspecified collecting conditions can potentially heterogenous allergens profiles and batch to batch variability, and amino acid sequence variants in allergens possibly exist among trees. Polymorphisms have not been investigated for the cypress pollen major allergens, Cha o 1 and Cha o 2. Our aim was to examine the homogeneity of allergen amino acid sequences. DNA sequences of Cha o 1 and Cha o 2 from pollen collected from Chiba and Ibaraki prefectures and from needles of 47 plus trees located at seed orchards in Chiba Prefecture were examined by amplicon sequencing and amino acid substitutions were deduced. Sequence analysis of the pollen samples revealed that eight and seven residues of Cha o 2 were polymorphic, respectively. Thirteen residues in Cha o 2, including those residues identified in pollen, were deduced to be polymorphic for the plus trees. Cha o 2 expressed by the 47 plus trees included amino acid differences when compared with that of isoallergen Cha o 2.0101. No substitution was deduced in Cha o 1 for pollen taken from the two prefectures. One conservative amino acid substitution was deduced in Cha o 1 for the plus trees. Of the 47 plus trees examined, 38 were deduced to express only the isoallergen Cha o 1.0101 isoform, whereas eight trees were heterozygous and a single tree was homozygous for the non-synonymous mutation, which indicates relative uniformity of Cha o 1. Cha o 2 was found to be a heterogeneous allergen which suggests that studies using pollen from different trees may not give the same results.

Reduced anaphylactic potential of denatured pullulan-conjugated Cry j 1.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.

Recombinant Fusion Allergens, Cry j 1 and Cry j 2 from Japanese Cedar Pollen, Conjugated with Polyethylene Glycol Potentiate the Attenuation of Cry j 1-Specific IgE Production in Cry j 1-Sensitized Mice and Japanese Cedar Pollen Allergen-Sensitized Monkeys.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent seasonal rhinitis in Japan. A standardized Japanese cedar pollen extract (CPE) containing 1.5-4.2 µg of Cry j 1 is currently the highest-concentration extract available for allergen-specific immunotherapy (SIT) against this pollinosis. Therefore, we developed a PEGylated fusion protein as a more effective SIT vaccine against Japanese cedar pollinosis. METHODS: The fusion protein of major allergens for Japanese cedar pollen, Cry j 1 and Cry j 2, was expressed in Escherichia coli and conjugated with polyethylene glycol (PEG). The purified PEGylated Cry j 1/2 fusion protein (PEG-fusion) was subcutaneously injected four times into Cry j 1- sensitized mice and CPE-sensitized monkeys. The mice were then subcutaneously challenged with Cry j 1 and serum levels of Cry j 1-specific immunoglobulin, and the proliferation and cytokine production of splenocytes were analyzed. The monkeys were intranasally challenged with CPE and analyzed for Cry j 1-specific immunoglobulin levels in plasma. RESULTS: Cry j 1-specific IgE was significantly attenuated in the PEG-fusion-treated group after Cry j 1-challenge and Cry j 1-specific IgG was significantly increased following PEG-fusion treatment in mice and monkeys. Proliferation and Th2-type cytokine production in splenocytes stimulated with Cry j 1 were also reduced in PEG-fusion-treated mice. IL10 and IL2 production were reduced, but not significantly, while IFN-x03B3; was significantly increased in the PEG-fusion-treated group. CONCLUSIONS: A high-dose injection of PEG-fusion appears to be a valid candidate for a safer and more effective vaccine than the conventional SIT extract for Japanese cedar pollinosis.

Hepatic de novo lipogenesis is present in liver-specific ACC1-deficient mice.

Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes carboxylation of acetyl-CoA to form malonyl-CoA. In mammals, two isozymes exist with distinct physiological roles: cytosolic ACC1 participates in de novo lipogenesis (DNL), and mitochondrial ACC2 is involved in negative regulation of mitochondrial beta-oxidation. Since systemic ACC1 null mice were embryonic lethal, to clarify the physiological role of ACC1 in hepatic DNL, we generated the liver-specific ACC1 null mouse by crossbreeding of an Acc1(lox(ex46)) mouse, in which exon 46 of Acc1 was flanked by two loxP sequences and the liver-specific Cre transgenic mouse. In liver-specific ACC1 null mice, neither hepatic Acc1 mRNA nor protein was detected. However, to compensate for ACC1 function, hepatic ACC2 protein and activity were induced 1.4 and 2.2 times, respectively. Surprisingly, hepatic DNL and malonyl-CoA were maintained at the same physiological levels as in wild-type mice. Furthermore, hepatic DNL was completely inhibited by an ACC1/2 dual inhibitor, 5-tetradecyloxyl-2-furancarboxylic acid. These results strongly demonstrate that malonyl-CoA from ACC2 can access fatty acid synthase and become the substrate for the DNL pathway under the unphysiological circumstances that result with ACC1 disruption. Therefore, there does not appear to be strict compartmentalization of malonyl-CoA from either of the ACC isozymes in the liver.