Insecticide resistance in mosquitoes has become a severe impediment to global vector control and manifests as decreased insecticide effectiveness. The role of target site mutations and detoxification enzymes as resistance markers has been documented in mosquitoes; however, the emergence of complex resistant phenotypes suggest the occurrence of additional mechanisms. Cuticular proteins (CPs) are key constituents of the insect cuticle, and play critical roles in insect development and insecticide resistance. In this study, via electron microscopy we observed that the leg cuticle thickness in deltamethrin-resistant (DR) Anopheles sinensis mosquitoes was significantly greater than that measured in deltamethrin-susceptible (DS) An. sinensis. Transcription analysis revealed that cuticle proteins were enriched in the legs, including members of the CPR, CPAP, and CPF families. Further comparisons revealed the specific overexpression of four CP genes in the legs of DR An. sinensis; whose expression levels increased after treatment with deltamethrin. The RNAi-mediated silencing of one CP gene, AsCPF1, resulted in a significant decrease in the leg cuticle thickness of DR mosquitoes and significantly elevated the mortality rate when exposed to deltamethrin. These findings suggest that alterations in the An. sinensis leg cuticle contribute to the insecticide resistance phenotype. AsCPF1 is thereby a target study molecule for investigation of its mode of action, and broader attention should be paid to the role of mosquito legs in the development of insecticide resistance.

PURPOSE:Pet-derived allergens are the common indoor inhalant allergens. Among them, cat and dog allergens constitute more than 80% of animal allergic patients, which greatly affect the quality-of-life of patients and increase the burden of social health care. The aim of this study was to identify Cat-Niemann pick type C2 (NPC2) protein, a homologue of Can f 7, as a new allergen.METHODS:Cat-NPC2 complementary DNA (cDNA) was cloned and optimized for amplification and expression in Escherichia coli. Then, recombinant Cat-NPC2 (rCat-NPC2) was purified by Ni2+ affinity chromatography. The allergenicity was assessed by enzyme-linked immunosorbent assay (ELISA), western blot and basophil activation test (BAT). Based on the sequence similarity, the cross-reactivity between Cat-NPC2 and Can f 7 was investigated by inhibition ELISA. Circular dichroism spectroscopy and homology modeling were used to characterize the structure of Cat-NPC2.RESULTS:The cDNA sequence of Cat-NPC2 was cloned with a 450-bp open reading frame coding for 149 amino acids (GenBank MN_737596). The condon-optimized NPC2 gene was subcloned and expressed in E. coli with a molecular weight of 18.9 kDa. The native Cat-NPC2 was detected in cat dander extracts. The allergenicity determined by ELISA, western blot and BAT suggested at least 14.5% cat-allergic patients displayed high specific immunoglobulin E (IgE) recognition of Cat-NPC2. The predicted structure of Cat-NPC2 was found to consist of 7 β-strands arranged in 2 β-sheets. An ELISA based assay showed that rCat-NPC2 bound to cholesterol in a dose dependent manner. Based on the structure and sequence similarities, IgE cross-reactivity was demonstrated between Cat-NPC2 and Can f 7/Der f 2.CONCLUSIONS:In the study, a novel cat allergen, belonging to the NPC2 protein family, was identified and characterized at both molecular and immunological levels. The study will offer a deeper understanding of cat allergens and improve a component-resolved diagnosis in pet allergy.

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 β-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infancy, which is a major risk factor for recurrent wheezing and asthma. Orosomucoid 1-like protein 3 (ORMDL3) has been reported to associate with virus-triggered recurrent wheezing and asthma in children. However, little is known about how ORMDL3 is involved into RSV infection. In this study, we showed that the mRNA expression of ORMDL3 is significantly increased in the peripheral blood lymphocytes of infants with RSV-induced bronchiolitis compared with uninfected controls, also increased in bronchial epithelial cells and lung fibroblasts following RSV infection in vitro. To investigate the underlying mechanisms of RSV-induced ORMDL3 expression, we performed in silico analysis of the binding sites of several transcription factors in the ORMDL3 promoter. The proximal interferon-regulatory factor-3 (IRF-3) binding site positively regulated ORMDL3 transcription following exposure to RSV, as determined through mutational analysis. Overexpression and RNA interference experiments targeting IRF-3 showed that it regulates the expression of ORMDL3 following RSV exposure. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay showed that IRF-3 binds directly to the promoter of the ORMDL3 gene. Furthermore, we confirmed that expression of IRF-3 is significantly increased and shows a strong linear correlation with increased ORMDL3 in the peripheral blood lymphocytes from infants with RSV-induced bronchiolitis. Our results indicate that IRF-3 is an important regulator of ORMDL3 induction following RSV infection by binding directly to the promoter of ORMDL3, which may be implicated in the inflammatory and immune reactions involved in bronchiolitis and wheezing diseases.

The pathophysiology of allergic disease such as asthma and allergic rhinitis tell the similar story: when the endogenous and exogenous inflammatory mechanisms occur disorder, the body may begin with inflammatory cell activation, namely through the release of cytokine and inflammatory mediator role in the corresponding target cells, activate the sensory nerve fiber, acting on the cell organ specificity effect, clinical symptoms. This article is divided into the following five parts focused on the research progress of allergic inflammatory diseases: (1) inflammatory cells; (2) staphylococcus aureus superantigen; (3) small molecules (cytokines, inflammatory mediators, lipid classes medium); (4) nerve fibers and effect cells; (5) genetic and epigenetic factors.

第30届欧洲变态反应和临床免疫学会（European Academy of Allergyand Clinical Immunology，EAACI）会议2011年6月在土耳其伊斯坦布尔举行，又适逢免疫治疗提出百年，故而本届大会的主题为：“Bridging science and culture”。作者荣幸获得本届会议旅行奖，亲历本届会议盛况。本文结合会议论文汇编^[1]仅对变应性鼻炎（AR）的相关研究进行简略报道。

Objective To express the excellular domain of glycoprotein D(gD) of herpes simplex virus type 2(HSV-2) in mammalian cells and analyze the immunologic activity of expressed product.Methods The gDt gene sequence encoding excellular domain of gD of HSV-2 G strain was chemically synthesized and inserted into vector pCEP4.The constructed recombinant plasmid pCEP4-gDt,with a His label at N-terminus,was transfected to HEK293 cells for expression.The expressed protein was purified by nickel ion column affinity chromatography and tested for antigenicity by ELISA.Polyclonal antisera were prepared by immunizing mice with the purified recombinant protein and determined for titer by ELISA.Results PCR,restriction analysis and DNA sequencing proved that recombinant plasmid pCEP4-gDt was constructed correctly.Western blot showed a target protein band with relative molecular mass of about 46 000.The purified recombinant protein,at a concentration of about 45 μg/ml,showed good antigenicity as proved by ELISA,which induced specific antibody titer of 5 × 103 in the sera of mice 5 weeks after immunization.Conclusion The excellular domain of gD of HSV-2 was expressed in mammalian cells,and showed good antigenicity and immunogenicity,which laid a foundation of preparation of recombinant subunit vaccine against HSV.

Background: Programmed death 1 (PD-1) on T cells is expressed persistently at a high level in chronic hepatitis B virus (HBV) infection, however, there are few studies focus on the change of PD-1 expression on T cells in patients with chronic hepatitis B (CHB) during antiviral therapy and its relationship with viral load. Aims: To observe longitudinally the PD-1 expression on CD4 + and CD8+ T cells in peripheral blood of CHB patients at early phase of antiviral therapy, and to explore the relationship between PD-1 expression and serum levels of HBV DNA and alanine aminotransferase (ALT). Methods: Levels of PD-1 expression on peripheral blood CD4+ and CD8+ T cells in 31 CHB patients undergoing antiviral therapy were assessed by flow cytometry prior to the treatment or at baseline (T1), and 4-8 weeks (T2) and 12-16 weeks (T3) during treatment course, respectively. Serum HBV DNA load was determined by real-time fluorescent quantitative polymerase chain reaction (PCR). Serum ALT level was also measured at the same time points. Results: A significant reduction in PD-1 expression level on both peripheral CD4+ and CD8+ T cells in CHB patients was observed at early phase of antiviral therapy (P<0.05). Serum HBV DNA and ALT levels were also markedly reduced from T1 to T3 (P<0.01). Furthermore, significant positive correlations between PD-1 expression on CD4+ and CD8 + T cells and HBV DNA load (P<0.01), and ALT level (P<0.05) were observed. Conclusions: Effective antiviral treatment induced reduction of viral load results in down-regulation of PD-1 expression on T cells in CHB patients. Furthermore, PD-1 expression on T cells is closely correlated with disease status.