Ferroptotic alveolar epithelial type II cells drive TH2 and TH17 mixed asthma triggered by birch pollen allergen Bet v 1.

Asthma is a common allergic disease characterized by airway hypersensitivity and airway remodeling. Ferroptosis is a regulated death marked by iron accumulation and lipid peroxidation. Several environmental pollutants and allergens have been shown to cause ferroptosis in epithelial cells, but the relationship between birch pollinosis and ferroptosis in asthma is poorly defined. Here, for the first time, we have identified ferroptosis of type II alveolar epithelial cells in mice with Bet v 1-induced asthma. Further analysis revealed that treatment with ferrostatin-1 reduced TH2/TH17-related inflammation and alleviated epithelial damage in mice with Bet v 1-induced asthma. In addition, ACSL4-knocked-down A549 cells are more resistant to Bet v 1-induced ferroptosis. Analysis of clinical samples verified higher serum MDA and 4-HNE concentrations compared to healthy individuals. We demonstrate that birch pollen allergen Bet v 1 induces ferroptosis underlaid TH2 and TH17 hybrid asthma. Lipid peroxidation levels can be considered as a biomarker of asthma severity, and treatment with a specific ferroptosis inhibitor could be a novel therapeutic strategy.

Efficacy and safety of intratonsillar immunotherapy for allergic rhinitis: A randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: A lower adherence rate existed in patients receiving allergen-specific immunotherapy due to its lengthy period and adverse effects even though it is the only curative treatment for IgE-mediated allergies. Therefore, exploring innovative allergen-specific immunotherapy routes is necessary. OBJECTIVE: To explore the efficacy and safety of the intratonsillar injection of house dust mite (HDM) extract in patients with HDM-induced allergic rhinitis (AR). METHODS: A randomized, double-blind, placebo-controlled clinical trial was conducted. A total of 80 patients with HDM-induced AR were randomized to receive 6 intratonsillar injections with HDM extract or placebo in 3 months. The total nasal symptom score (TNSS), visual analogue scale of nasal symptoms, combined symptom and medication score, mini rhinoconjunctivitis quality of life questionnaire, and serum allergen-specific IgG4 to Dermatophagoides pteronyssinus were all monitored at baseline and 3 months, 6 months, and 12 months after the treatment was finished. The intent-to-treat and per-protocol set (PPS) are both analyzed. RESULTS: The primary end points TNSS and ΔTNSS were improved significantly at 3 months after the patients with AR finished a 3-month 6-injection intratonsillar immunotherapy compared with those in the placebo treatment in both intent-to-treat and PPS. Results of visual analogue scale, combined symptom and medication score, and mini rhinoconjunctivitis quality of life questionnaire were also improved significantly at 3 months after the treatment in PPS. However, the improvement effect of intratonsillar immunotherapy at 6 and 12 months was limited and uncertain based on the data. The increase of serum Der p IgG4 in the active group was significantly higher than that in the placebo group at 3, 6, and 12 months after the treatment was finished. Adverse events were monitored, and no systemic adverse reactions were observed. CONCLUSION: The clinical trial revealed that intratonsillar injection with HDM extract was safe and effective in patients with AR. Optimizing the protocol and allergen formulations is expected to increase and maintain the efficacy of this novel approach. TRIAL REGISTRATION: https://www.chictr.org.cn/index.html, identifier: ChiCTR-TRC-13003600.

Toll-like Receptor 4 Deficiency Aggravates Airway Hyperresponsiveness and Inflammation by Impairing Neutrophil Apoptosis in a Toluene Diisocyanate-Induced Murine Asthma Model.

PURPOSE: Accumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation. METHODS: TLR4-/- and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4-/- mice after each challenge. RESULTS: TDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4-/- mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4-/- mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis. CONCLUSIONS: These data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.

Synergistic activation of Src, ERK and STAT pathways in PBMCs for Staphylococcal enterotoxin A induced production of cytokines and chemokines.

BACKGROUND: Staphylococcal enterotoxin A (SEA) is a well-known superantigen and stimulates human peripheral blood mononuclear cells (PBMCs) involving in the pathogenesis of inflammatory disorders and cancer. OBJECTIVE: To better understand the biological activities of SEA and the possible intracellular mechanisms by which SEA plays its roles in conditions like staphylococcal inflammatory and/or autoimmune disorders and immunotherapy. METHODS: Recombinant SEA (rSEA) was expressed in a prokaryotic expression system and its effects on the cytokine and chemokine production was examined by Enzyme-linked Immunospot (ELISpot) Assay and ELISA analysis. RESULTS: In vitro experiments showed rSEA could significantly enhance secretion of a broad spectrum of cytokines and chemokines from PBMCs dose-dependently. Increased secretion of cytokines and chemokines from rSEA stimulated PBMCs was barely affected by C-C motif chemokine receptor 2 (CCR2) antagonist INCB3344. However, Src, ERK and STAT pathway inhibitors were able to successfully block the enhanced secretion of most of cytokines and chemokines produced by rSEA stimulated PBMCs. CONCLUSIONS: Our work suggested that rSEA serves as a potent stimulant of PBMCs, and induces the release of cytokines and chemokines through Src, ERK and STAT pathways upon a relatively independent network. Our work also strongly supported that Src, ERK and STAT signaling inhibitors could be effective therapeutic agents against diseases like toxic shock syndrome or infection by microbes resistant to antibiotics.

Blockade of the NLRP3/Caspase-1 Axis Ameliorates Airway Neutrophilic Inflammation in a Toluene Diisocyanate-Induced Murine Asthma Model.

Multiple studies have addressed the vital role of Nod-like receptor protein 3(NLRP3)/caspase-1/IL-1ß signaling in asthma. Yet, the role of NLRP3/caspase-1 in toluene diisocyanate (TDI)-induced asthma is still obscure. The aim of this study is to investigate the role of the NLRP3/caspase-1 axis in TDI-induced asthma. Using an established murine model of TDI-induced asthma as described previously, we gave the asthmatic mice a highly selective NLRP3 inhibitor, MCC950, as well as the specific caspase-1 inhibitors VX-765 and Ac-YVAD-CHO for therapeutic purposes. Airway resistance was measured and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology, immunohistochemistry, Western blotting, and flow cytometry. TDI exposure elevated the expression of NLRP3 and caspase-1 that was coupled with increased airway hyperresponsiveness (AHR), neutrophil-dominated cell infiltration, pronounced goblet cell metaplasia, extensive collagen deposition, and increased TH2/TH17 responses. Both VX-765 and Ac-YVAD-CHO effectively inhibited the activation of caspase-1 in TDI-asthmatic mice that was accompanied by dramatic attenuation of AHR, airway inflammation, and airway remodeling, in addition to a decreased TH2 response and lower levels of IL-18 and IL-1ß. MCC950 blocked the activation of NLRP3 and downregulated protein expression of caspase-1, IL-1ß, and IL-18 in TDI-exposed mice. Furthermore, MCC950 remarkably alleviated AHR, airway inflammation, airway remodeling, and significantly suppressed TH2/TH17 responses. These findings suggested that blockade of the NLRP3/caspase-1 axis effectively prevents the progression of TDI-induced asthma and could be used as therapeutic targets for asthmatics.

Dust mite-derived Der f 3 activates a pro-inflammatory program in airway epithelial cells via PAR-1 and PAR-2.

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.

Activation profile of THP-1 derived dendritic cells stimulated by allergen Mal f 1 beyond its IgE-binding ability.

BACKGROUND: Mal f 1, the first allergen cloned from Malassezia furfur, has positive IgE reactivity in sera from atopic dermatitis (AD) patients. The mechanism by which Mal f 1 induces the maturation of human dendritic cells (DCs) and maintains the symptoms of AD is not well understood. OBJECTIVE: The present study aims to explore the activation profile of THP-1 derived dendritic cells (TDDCs) stimulated by recombinant Mal f 1, as well as to explore the IgE-binding ability of rMal f 1 and its correlation with IgE-binding activity of complete allergens of M. furfur. METHODS: rMal f 1 was produced by expression in E. coli and purification with affinity chromatography. The ability of rMal f 1 and ImmunoCAP complete allergens of M. furfur to bind to serum specific IgE was assayed in parallel by ELISA and immunoblotting. Immature TDDCs were stimulated with rMal f 1 or an enzyme-digested product of rMal f 1. The expression levels of markers, CD83, CD80, CD86, and HLA-DR, were investigated by flow cytometry. The levels of interleukin (IL)-6, IL-10, IL-12p70 and tumor necrosis factor (TNF)-α in culture supernatants were determined by ELISA. RESULTS: Eighteen patient sera were identified that reacted positively to the complete allergens of M. furfur as determined by ImmunoCAP and also showed positive responses to rMal f 1. Five patient sera were identified that had no reaction to ImmunoCAP complete allergens of M. furfur and also exhibited negative response to rMal f 1. All sera, except for one, had no reaction to the unrelated allergen Bet v 1. rMal f 1 upregulated the maturation surface marker CD83 on TDDCs. In addition, rMal f 1 also induced high levels of CD80 and CD86. Increased expression of HLA-DR, a first signal for T cell activation, was observed. Secretion of IL-6, TNF-α and IL-10 by TDDCs increased significantly (P < 0.0001 for IL-6, P < 0.01 for TNF-α and P < 0.05 for IL-10) after stimulation by rMal f 1, while the IL-12p70 level was unaltered. CONCLUSION: We have shown that rMal f 1 has ideal IgE binding ability and good correlation with binding activity to M. furfur. Moreover, we have revealed a hitherto unknown DC activation profile after rMal f 1 stimulation whereby TNF-α, IL-6, and IL-10 were significantly increased and IL-12 was unaltered, suggesting that rMal f 1 can predispose a DC bias toward the TH22/TH17 pathway beyond the routine IgE-dependent TH2 pathway, thus providing intriguing clues for clinical treatment involving both pathways.

A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies.

Peanut (Arachis hypogaea) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10 µg/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

Construction, Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of its Antitumor Activity Against Nasopharyngeal Cancer.

Staphylococcal enterotoxin A is well known as a superantigen and able to be used for cancer immunotherapy. In this study, recombinant Staphylococcal enterotoxin A was genetically conjugated to epidermal growth factor to produce a chimeric protein recombinant Staphylococcal enterotoxin A-epidermal growth factor expressed in Escherichia coli. The recombinant Staphylococcal enterotoxin A-epidermal growth factor protein was purified using Strep-Tactin affinity chromatography and Endotoxin Removal Resin and identified by sodium dodecyl sulfate-polyacrylamide gel electropheresis and liquid chromatography-tandem mass spectrometry analysis. Furthermore, in vitro experiments showed purified recombinant Staphylococcal enterotoxin A-epidermal growth factor could successfully bind to the human nasopharyngeal carcinoma cell line CNE2, significantly promote the proliferation of human peripheral blood mononuclear cells, and enhance the secretion of several cytokines that have broad antitumor activities, such as interferon-γ, tumor necrosis factor-α, and interleukin-2 . Importantly, recombinant Staphylococcal enterotoxin A-epidermal growth factor significantly inhibited proliferation of CNE2 cells and promoted apoptosis in CNE2 cells when cocultured with peripheral blood mononuclear cells. Finally, both the binding of recombinant Staphylococcal enterotoxin A-epidermal growth factor and the toxicity of recombinant Staphylococcal enterotoxin A-epidermal growth factor-activated peripheral blood mononuclear cells were demonstrated as specific and only effective on high epidermal growth factor receptor-expressing cell lines. In all, our work suggests that recombinant Staphylococcal enterotoxin A-epidermal growth factor serves as a promising novel immunotherapeutic agent. More in vivo and in vitro studies are needed to verify its antitumor potency, as well as investigate the underlying mechanisms in cancer immunotherapy.

PBMC activation via the ERK and STAT signaling pathways enhances the anti-tumor activity of Staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is well known as a superantigen and is highly potent in activating T lymphocytes. And it has been used clinically as an immunomodifier in the treatment of a number of tumors for years. However, the mechanism of its action remains largely unclear. In this study, SEA was found to significantly inhibit the proliferation and induce the death of human lung carcinoma A549 cells when co-cultured with human peripheral blood mononuclear cells (PBMCs). SEA could also induce the proliferation of human PBMCs and stimulate human PBMCs to release a wide range of cytokines that have broad anti-tumor activities such as IFN-γ, TNF-α, IL-2. Furthermore, SEA was found in PBMCs to induce a rapid and long-lasting phosphorylation of extracellular signal-regulated kinases (ERKs) which was significantly inhibited by MEK/ERK pathway inhibitors U0126 and PD0325901, and a late onset of phosphorylation of signal transducers and activators of transcription (STATs) which was significantly inhibited by a pan-JAK inhibitor Pyridone 6 (P6). Unexpectedly constitutive ERK or STATs phosphorylation was also significantly inhibited by P6 or U0126 in a dose-dependent manner, respectively. Summing up, our data reveal SEA may function as a novel protein drug used for cancer immunotherapy via inducing activation of PBMCs, immune cell crosstalk-dependent activation of ERK and STATs, and production of tumor-suppressive cytokines.

Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells.

PURPOSE: Cell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3. METHODS: We investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines. RESULTS: Our results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis. CONCLUSION: The occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death.

Generation and purification of monoclonal antibodies against Der f 2, a major allergen from Dermatophagoides farinae.

Monoclonal antibodies (mAbs) are needed for the quantitation of environmental allergens for precise diagnosis and immunotherapy. In this study, we produced and purified monoclonal antibodies against Der f 2, one of the major allergens of the house dust mite Dermatophagoides farina, in order to develop an assay for the detection of this allergen. BALB/c mice were immunized four times with the protein Der f 2 together with an adjuvant after which splenocytes were collected and fused with SP2/0 (myeloma cells) in the presence of polyethylene glycol (PEG). The fused cells were selected in the presence of Hypoxanthine-Aminopterin-Thymidine (HAT) and then Hypoxanthine-Thymidine (HT) medium. Positive cells were screened with ELISA and subcloned by limited dilution at least three times to achieve stable mAb-producing clones. Four stable mAb-producing clones were obtained. One clone with IgG1 isotype and another with IgG2b isotype were chosen to produce large amounts of mAb by inoculation of the cells into the abdominal cavity of mice. Ascites were collected and the mAbs were purified using protein A affinity chromatography. Testing of the ascites by ELISA showed the titration of IgG1 and IgG2b to be higher than 1/10(6) dilution. The specificity of both antibodies was confirmed by immunoblotting. Thus, we produced two mAb clones against Der f 2 that can be used to create a precise quantitative method to identify allergen components in dust samples and facilitate further study in Der f 2 component-resolved diagnosis (CRD).

Mast Cell Targeted Chimeric Toxin Can Be Developed as an Adjunctive Therapy in Colon Cancer Treatment.

The association of colitis with colorectal cancer has become increasingly clear with mast cells being identified as important inflammatory cells in the process. In view of the relationship between mast cells and cancer, we studied the effect and mechanisms of mast cells in the development of colon cancer. Functional and mechanistic insights were gained from ex vivo and in vivo studies of cell interactions between mast cells and CT26 cells. Further evidence was reversely obtained in studies of mast cell targeted Fcε-PE40 chimeric toxin. Experiments revealed mast cells could induce colon tumor cell proliferation and invasion. Cancer progression was found to be related to the density of mast cells in colonic submucosa. The activation of MAPK, Rho-GTPase, and STAT pathways in colon cancer cells was triggered by mast cells during cell-to-cell interaction. Lastly, using an Fcε-PE40 chimeric toxin we constructed, we confirmed the promoting effect of mast cells in development of colon cancer. Mast cells are a promoting factor of colon cancer and thus also a potential therapeutic target. The Fcε-PE40 chimeric toxin targeting mast cells could effectively prevent colon cancer in vitro and in vivo. Consequently, these data may demonstrate a novel immunotherapeutic approach for the treatment of tumors.

[In vitro differentiation of human umbilical cord blood mononuclear cells into mature mast cells induced by cytokines].

OBJECTIVE: To isolate and induce human umbilical cord blood mononuclear cells to differentiate into mature mast cells of high purity. METHODS: Human umbilical cord blood mononuclear cells were differentiated into mature mast cells by the treatment of recombinant human stem cell factor (rhSCF) and recombinant human interleukin 6 (rhIL-6). The cultured cells at different time points were stained with toluidine blue for the detection of anti-FcepsilonRI and the maturity of mast cells was detected by flow cytometry. After the mature mast cells were stimulated with allergen, the levels of histamine and tryptase in the supernatant were determined. RESULTS: The cells started to express FcepsilonRI receptor after 2-week treatment of rhSCF and rhIL-6. After 3 weeks, the amount of FcepsilonRI receptor reached its peak accompanied by increased intracellular basophilic granules. The mature mast cells released tryptase and histamine effectively after allergen challenge. CONCLUSION: Human umbilical cord blood mononuclear cells can be differentiated into mature mast cells by the treatment of rhSCF and rhIL-6. The mature mast cells may be used for the study of allergenicity in vitro.

Efficacy of Sublingual Immunotherapy with Dermatophagoides farinae Extract in Monosensitized and Polysensitized Patients with Allergic Rhinitis: Clinical Observation and Analysis.

AIM: To investigate differences in the efficacy of sublingual immunotherapy with Dermatophagoides farinae drops in monosensitized and polysensitized allergic rhinitis patients. METHODS: The patients enrolled in the study were treated for more than one year by sublingual immunotherapy (SLIT) using Dermatophagoides farinae drops and were divided into a monosensitized group (n = 20) and a polysensitized group (n = 30). Total nasal symptom scores of patients before and after SLIT were analyzed to evaluate the curative effect. The phylogenetic tree of dust mite allergens as well as other allergens that were tested by skin prick test was constructed to help the analysis. RESULTS: There was no significant difference in the efficacy of SLIT between dust mite monosensitized and polysensitized patients. CONCLUSIONS: Both dust mite monosensitized and polysensitized patients could be cured by SLIT using Dermatophagoides farinae drops. This study provides a reference for the selection of allergens to be used in immunotherapy for polysensitized AR patients.

Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli.

House dust mite (Dermatophagoides farinae) allergen Der f1 is one of the most important indoor allergens associated with asthma, eczema and allergic rhinitis in humans. Therefore, sufficient quantities of Der f1 cysteine protease to be used for both experimental and therapeutic purposes are very much needed. Using recombinant DNA technology, high expression rates of cysteine proteases were obtained. The cDNA sequence encoding pro-Der f1 was cloned and expressed in Escherichia coli using the T7 based expression vector pET-44a and induced by isopropyl-ß-d-thiogalactoside at a final concentration of 0.2mM. Recombinant pro-Der f1 (pro-rDer f1) was expressed as an inclusion body and the isolated protease was solubilized, refolded and purified. The protease activities and IgE reactivities of pro-rDer f1 that were refolded by size-exclusion chromatography (SEC) were higher than those obtained by dilution. The pair of pro-rDer f1 polypeptides produced by this method could be used for more effective and safer allergen-specific immunotherapy or to produce enzymatically and immunologically active Der f1 for diagnostic testing and deciphering of immunotherapy mechanisms.

Reduction of the number of major representative allergens: from clinical testing to 3-dimensional structures.

Vast amounts of allergen sequence data have been accumulated, thus complicating the identification of specific allergenic proteins when performing diagnostic allergy tests and immunotherapy. This study aims to rank the importance/potency of the allergens so as to logically reduce the number of allergens and/or allergenic sources. Meta-analysis of 62 allergenic sources used for intradermal testing on 3,335 allergic patients demonstrated that in southern China, mite, sesame, spiny amaranth, Pseudomonas aeruginosa, and house dust account for 88.0% to 100% of the observed positive reactions to the 62 types of allergenic sources tested. The Kolmogorov-Smironov Test results of the website-obtained allergen data and allergen family featured peptides suggested that allergen research in laboratories worldwide has been conducted in parallel on many of the same species. The major allergens were reduced to 21 representative allergens, which were further divided into seven structural classes, each of which contains similar structural components. This study therefore has condensed numerous allergenic sources and major allergens into fewer major representative ones, thus allowing for the use of a smaller number of allergens when conducting comprehensive allergen testing and immunotherapy treatments.

SORTALLER: predicting allergens using substantially optimized algorithm on allergen family featured peptides.

UNLABELLED: SORTALLER is an online allergen classifier based on allergen family featured peptide (AFFP) dataset and normalized BLAST E-values, which establish the featured vectors for support vector machine (SVM). AFFPs are allergen-specific peptides panned from irredundant allergens and harbor perfect information with noise fragments eliminated because of their similarity to non-allergens. SORTALLER performed significantly better than other existing software and reached a perfect balance with high specificity (98.4%) and sensitivity (98.6%) for discriminating allergenic proteins from several independent datasets of protein sequences of diverse sources, also highlighting with the Matthews correlation coefficient (MCC) as high as 0.970, fast running speed and rapidly predicting a batch of amino acid sequences with a single click. AVAILABILITY AND IMPLEMENTATION: http://sortaller.gzhmc.edu.cn/.

Strengths and weaknesses of immunotherapy for advanced non-small-cell lung cancer: a meta-analysis of 12 randomized controlled trials.

BACKGROUND: Lung cancer is one of the leading causes of cancer death worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Immunotherapy has yielded no consistent benefit to date for those patients. Assessing the objective efficacy and safety of immunotherapy for advanced NSCLC patients will help to instruct the future development of immunotherapeutic drugs. METHODOLOGY AND PRINCIPAL FINDINGS: We performed a meta-analysis of 12 randomized controlled trials including 3134 patients (1570 patients in the immunotherapy group and 1564 patients in the control group) with histologically confirmed stage IIIA, IIIB, or IV NSCLC. The analysis was executed with efficacy end points regarding overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and total effective rate. Overall unstratified OS, PFS, PR, and total effective rate were significantly improved in advanced NSCLC patients in the immunotherapy group (P = 0.0007, 0.0004, 0.002, 0.003, respectively), whereas CR was not improved (P = 0.97). Subgroup analysis showed that monoclonal antibody (mAb) immunotherapy significantly improved the PFS, PR, and total effective rate and showed a trend of improving OS of advanced NSCLC patients compared with the control group, with one kind of adverse event being significantly dominant. Compared with the control group, the vaccine subgroup showed no significant difference with regard to serious adverse events, whereas cytokine immunotherapy significantly induced three kinds of serious adverse events. CONCLUSIONS: Immunotherapy works efficiently on advanced NSCLC patients. Of several immunotherapies, mAb therapy may be a potential immunotherapy for advanced NSCLC patients, and become a standard complementary therapeutic approach in the future if the issues concerning toxicity and allergenicity of mAbs have been overcome.

[Detection of hearing threshold and polymorphic molecular marker analysis of guinea pigs of mimetic aging].

OBJECTIVE: To explore the establishment of the mimetic aging effect in guinea pigs induced by D-galactose, and to detect the biological indicatrix associated with hearing loss and provide a new tool for molecular pathogenesis of hearing loss. METHODS: Total of 51 guinea pigs were randomly divided into three groups: group A (model aging group, n = 25), which were injected with D-galactose (200 mgxkg(-1)xd(-1)) by intra peritoneum for 6 weeks, group B (model control group, n = 18), which were given the same amount of saline only, and group C (vacant group, n = 15) were not treated. Then, The guinea pigs in group A and B were exposed in noise for 8 days, 8 hours once a day. Auditory brainstem response (ABR) was used to test the hearing threshold of guinea pigs thrice, first before the drug administered, then after 6 weeks the drug used, third after noise exposure. And colorimetry was used to analyze the activity of superoxide dismutase (SOD) and malon dialdehyde (MDA) in brain and liver tissue. The DNA of inner ear tissue was harvested and amplified fragment length polymorphism (AFLP) was used to detect the differential polymorphic markers. RESULTS: After injection, there was no significant difference in elevation of ABR threshold between the group A and group B (t = 1.14, P > 0.05). However, exposure of noise later, elevation in ABR threshold of (22.97 +/- 10.56) dB peSPL was observed in group A, and (14.16 +/- 7.36) dB peSPL in group B. The was significant difference in variation of hearing threshold between group A and group B (t = 2.78, P < 0.05). The activity of SOD in brain and liver tissue in group A was lower than that in group B. the level of MDA was opposite between group A and group B. The difference between group A and group B was significant (P < 0.01). A differential polymorphic marker was observed by AFLP. CONCLUSIONS: The mimetic aging effect of the guinea pigs can be induced by D-galactose, and this model can not directly induce the hearing loss. The differential polymorphic marker possibly act as a predisposing factor which can greatly enhance the sensitivity of the ear to the noise.