The evolution of allergen and non-specific immunotherapy: past achievements,current applications and future outlook

Recent epidemiological studies estimated that more than 30% of European suffer from allergic rhinitis or conjunctivitis, while up to 20% suffer from asthma and 15% from allergic skin conditions, while for many other regions the prevalence is increasing. Allergen immunotherapy represents the only available treatment that can modify the allergic disease process, and thus is worth considering as a treatment in affected individuals. A beneficial effect of allergen immunotherapy has been shown in both adults and children affected by allergic rhinitis, allergic conjunctivitis, allergic asthma and hymenoptera venom allergy. The present study represents an overview on allergen immunotherapy, focusing on the principal aspects of the use of immunotherapy in the past, its recent clinical applications and future outlook.     

Risk factors for renal disease in systemic lupus erythematosus and their clinical implications

Lupus nephritis is one of the most common severe manifestations of systemic lupus erythematosus and is associated with significant morbidity and mortality. Genetic, ethnic and hormonal factors may influence the presence and severity of renal involvement and therefore affect the outcome and overall prognosis of patients. In this review, we will discuss the association of known lupus risk factors in developing renal disease and explore the recent literature to identify potential risk factors and their clinical implications in terms of diagnostic vigilance, management and prognosis.     

Complement deficiency and systemic lupus erythematosus: consensus and dilemma

The involvement of the complement system in the pathogenesis of autoimmune diseases is a matter of debate. However, the link between complement abnormalities and systemic lupus erythematosus (SLE) is well established and widely described. Homozygous and/or heterozygous complement-component deficiencies of the classical pathway (C1q, C1r, C1s, C4A, C4B and C2) are causally associated with susceptibility to the development of SLE. Although the severity of the disease and the strength of the association are heterogeneous for deficiencies of these proteins, they commonly cause peculiar SLE syndromes with an early age of onset, a susceptibility to bacterial infections and negative anti-dsDNA antibodies. In this review, we highlight the available data on complement deficiency and SLE with a focus on deficiencies in classical complement pathway components. We also discuss the paradox of the link between complement deficiency and lupus. The complement system acts as a ‘friend’ through the clearance of immune complexes and apoptotic cells, which explains the close association between complement deficiency and lupus. It also acts as an ‘enemy’ by participating in the effector inflammatory phase of the autoimmune response. Understanding the importance of complement deficiencies should provide novel targets for therapeutic interventions in the modulation of the immune response.     

Hashimoto’s thyroiditis: TGAb,TPOAb, TRAb and recovery from hypothyroidism

Hashimoto described four patients with goiter. The histology of the goiter was characterized by diffuse lymphocytic infiltration, fibrosis and epithelial cell destruction. Thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb) have been used to diagnose Hashimoto’s thyroiditis. Patients with positive TGAb and/or TPOAb have been assumed to have Hashimoto’s thyroiditis. Approximately 10% of those with positive TGAb and/or TPOAb have hypothyroidism. There are two types of autoimmune thyroiditis: goitrous Hashimoto’s thyroiditis and atrophic thyroiditis. The latter patients have blocking antibody (thyroid-stimulating hormone [TSH]-stimulation blocking antibody [TSBAb]). TSBAb is a TSH-receptor antibody (TRAb). TSBAb causes thyroid atrophy and hypothyroidism. TGAb and/or TPOAb do not necessarily cause hypothyroidism. Hypothyroid patients with Hashimoto’s thyroiditis usually receive life-long l-thyroxine therapy. However, spontaneous recovery from hypothyroidism has been reported. Patients who had Hashimoto’s hypothyroidism and then Graves’ hyperthyroidism (and vice versa), have also been reported. Hashimoto’s hypothyroidism and Graves’ hyperthyroidism could be the opposite spectrums of one disease.     

A Quantitative Nitrocellulose Enzyme Immunoassay and its Application to the Screening of Hybrbidomas for the Detection of Uncharacterized Tumor-Associated Antigens in Sera of Cancer Patients

Abstract

An enzyme-linked immunosorbent assay on nitrocellulose based microtiter plates for the detection of uncharacterized tumor associated antigens in squamous cell carcinoma and adenocarcinoma cancer patients' sera is described. Nitrocellulose microtiter plates are more sensitive than the plastic plates of polystyrene and polyvinyl chloride for the detection of antigens in serum. Monoclonal antibodies were selected for their net reactivities toward cancer patients' sera as compared to nomal sera. Sera from benign liver and kidney disease patients and activated human peripheral blood leukocyte supernatant were used to reduce potential false positives toward inflammatory and benign diseases. Using this system, fourteen antibodies were selected out of over eight hundred antibodies for their potential serodiagnostic application.     

Development of an Enzyme‐Linked Immunoassay for Sensitive Detection of Native and Recombinant Human Interferon‐γ Using Whole IgG Fraction as Polyclonal Tracer

Abstract

Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against human interferon gamma (IFN‐γ) were produced and used for development of a sensitive enzyme‐linked immunosorbent assay (ELISA) for the detection and quantitation of native and recombinant human IFN‐γ in tissue culture fluid and human sera. The human IFN‐γ ELISA was constructed using mAb CAy‐IFNg111 as the capture antibody (Ab) and biotinylated polyclonal mouse immunoglobulin G (IgG) as the tracer Ab. The assay is completed within 4?hr at room temperature (RT). The human IFN‐γ ELISA worked in tissue culture medium and human serum and was capable of detecting both recombinant and native human IFN‐γ. The assay dynamic range extended from 16 to 1000?pg/mL and the sensitivity level was less than 3?pg/mL of human IFN‐γ with averaged intra‐ and inter‐assay variation coefficients less than 8% for both. The results demonstrated that without the need of an antigen‐affinity purification, biotinylation of protein G‐purified pAb, obtained from 1?mL of mouse blood, was sufficient for constructing the tracer reagent for the establishment of a highly sensitive ELISA (40,000 test) for the quantitative detection of native and recombinant human IFN‐γ in culture supernatant and human sera.     

Development of a PAN‐Specific,Affinity‐Purified Anti‐acetylated Lysine Antibody for Detection,Identification, Isolation,and Intracellular Localization of Acetylated Protein

Abstract

Acetylation on the lysine residue is an important event of posttranslational modification of proteins. In this study, we developed a simple method to produce and to affinity purify the specific anti‐acetylated lysine polyclonal antibody, which is useful for the detection, identification, isolation, and intracellular localization of acetylated proteins on the lysine residues. We utilized the chemically acetylated hemocyanin of keyhole limpets (KLH) as an immunogen to raise the immune serum and to isolate the population of the acetylated lysine specific antibody using the immobilized acetylated lysine as immunoaffinity‐ligand. The isolated antibody was tested to be useful for ELISA, immunoblotting detection, immunofluorescent localization, and affinity isolation of the acetylated proteins.     

Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein–Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.     

Effects of Interleukin-9 Blockade on Chronic Airway Inflammation in Murine Asthma Models

Purpose

Asthma is a chronic inflammatory disease of the airways associated with structural changes and airway remodeling. Interleukin (IL)-9 has pleiotropic effects on both inflammatory cells and airway structural cells, which are involved in asthma pathogenesis. We evaluated the effects of IL-9 blockade on chronic airway inflammation.

Methods

Acute airway inflammation was induced in Balb/c mice using aerosolized ovalbumin (OVA), whereas chronic asthma was induced by OVA exposure for 5 weeks with anti-IL-9 or isotype-matched antibody (Ab) treatment during the OVA challenge. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and lung tissues were stained to detect cellular infiltration, mucus deposition, and collagen accumulation. The levels of interferon (IFN)-γ, IL-4, IL-5, IL-9, IL-17, and immunoglobulin E (IgE) in BALF were measured using enzyme linked immunosorbent assays, and profiles of inflammatory cells and subsets of T helper (Th) cells were analyzed using flow cytometry.

Results

IL-9, IL-17, and IFN-γ levels were significantly increased in the chronic group compared to the acute asthma group. However, the number of IL-9-positive cells was not affected, with a decrease in Th17 cells in OVA-challenged caspase-1 knockout mice. Numbers of eosinophils, neutrophils, B cells, mast cells, and Th17 cells decreased after administration of anti-IL-9 Ab. Total IgE, IL-5, IL-9, and IL-17 levels were also lower in the anti-IL-9 group.

Conclusions

Our results suggest that anti-IL-9 Ab treatment inhibits pulmonary infiltration of inflammatory cells and cytokine production, especially IL-17. These results provide a basis for the use of an anti-IL-9 Ab to combat IL-17-mediated airway inflammation.     

应用蛋白质芯片技术筛选人舌鳞癌细胞株差异性炎症因子的初步研究

目的研究应用蛋白质芯片(抗体芯片)技术筛选、分析人舌鳞状细胞癌细胞株中差异性炎症因子的效果,为进一步探讨口腔癌与炎症的关系奠定基础。方法体外培养人舌鳞癌细胞株UM-1、CAL-27、Tca-8113和人正常口腔黏膜上皮细胞,消化后分别提取胞内、胞外蛋白,上芯片孵育,封闭,清洗。采用激光扫描仪扫描芯片,Axon GenePix Pro6.0软件提取芯片数据,对获得的80种炎症因子表达数据采用AAH-CYT-G5软件分析,上调因子信号值以大于200、比值大于2.0为纳入标准;下调因子信号值以大于200、比值小于0.66为纳入标准,分别对3种人舌鳞癌细胞株(UM-1、CAL-27、Tca-8113)与人正常口腔黏膜上皮细胞,以及高侵袭性细胞株(UM-1、CAL-27)与低侵袭性细胞株(Tca-8113)进行两两比较,筛选出差异性炎症因子。挑选3种显著差异性炎症因子,用酶联免疫吸附法再次检测其蛋白表达,所得结果用单因素方差分析进行统计学分析,进一步验证芯片法所获得的结果。结果根据纳入标准,从检测的80种因子中筛选出有表达的炎症因子9个,包括干扰素诱导蛋白10(inter-feron inducible protein10,IP-10)、巨噬细胞炎症蛋白-1β(macrophage inflammatory protein1β,MIP-1β)、巨噬细胞炎症蛋白-3α(macrophage inflammatory protein-3α,MIP-3α)、骨保护素蛋白(osteoprotegerin,OPG)、调节活化蛋白(regulated upon activation normal T-cell expressed and secreted,RANTES)、白细胞介素1β(interleukin1β,IL-1β)6种舌鳞癌细胞株中高表达的细胞因子,及单核细胞趋化因子4(monocyte chemoattractant protein4,MCP-4)、胰岛素样生长因子结合蛋白4(insulin-like growth factor binding protein-4,IGFBP-4)、转化生长因子-β3(transforminggrowth factor-β3,TGF-β3)3种舌鳞癌细胞株中低表达的细胞因子。鳞状细胞癌细胞和正常口腔鳞状上皮细胞之间比较,胞内IL-1β呈高表达,MCP-4呈低表达,胞外OPG呈高表达;高、低侵袭性细胞株之间比较,胞内RANTES呈高表达,IGFBP-4、TGF-β3呈低表达,胞外IP-10、MIP-1β、MIP-3α呈高表达。酶联免疫吸附法检测发现UM-1和CAL-27胞外IP-10、MIP-1β、MIP-3α的表达量和Tca-8113相比呈高表达,差异有统计学意义(P<0.05)。其结果与芯片筛选结果一致。结论蛋白质芯片技术能够较为准确地筛选出不同舌鳞癌细胞株之间差异性炎症因子,这些差异性炎症因子可能与肿瘤细胞的生物学行为有一定的联系。