Recombinant T-cell epitope conjugation: A new approach for Dermatophagoides hypoallergen design.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.

Novel Approaches for Inhibiting the Indoor Allergen Der f 2 Excreted from House Dust Mites by Todomatsu Oil Produced from Woodland Residues.

House dust mite (HDM) is a globally ubiquitous domestic cause of allergic diseases. There is a pressing demand to discover efficient, harmless, and eco-friendly natural extracts to inhibit HDM allergens that are more likely to trigger allergies and challenging to be prevented entirely. This study, therefore, is aimed at assessing the inhibition of the allergenicity of major HDM allergen Der f 2 by todomatsu oil extracted from residues of Abies Sachalinensis. The inhibition was investigated experimentally (using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) and in silico using molecular docking. The results showed that todomatsu oil inhibits the allergenicity of Der f 2 by reducing its amount instead of the IgG binding capacity of a single protein. Moreover, the compounds in todomatsu oil bind to Der f 2 via alkyl hydrophobic interactions. Notably, most compounds interact with the hydrophobic amino acids of Der f 2, and seven substances interact with CYS27. Contrarily, the principal compounds fail to attach to the amino acids forming the IgG epitope in Der f 2. Interestingly, chemical components with the lowest relative percentages in todomatsu oil show high-affinity values on Der f 2, especially ß-maaliene (-8.0 kcal/mol). In conclusion, todomatsu oil has been proven in vitro as a potential effective public health strategy to inhibit the allergenicity of Der f 2.

Regulation of IL (Interleukin)-33 Production in Endothelial Cells via Kinase Activation and Fas/CD95 Upregulation.

OBJECTIVE: The occurrence of new blood vessel formation in the lungs of asthmatic patients suggests a critical role for airway endothelial cells (ECs) in the disease. IL-33 (Interleukin-33)-a cytokine abundantly expressed in human lung ECs-recently emerged as a key factor in the development of allergic diseases, including asthma. In the present study, we evaluated whether mouse and human ECs exposed to the common Dermatophagoides farinae allergen produce IL-33 and characterized the activated signaling pathways. Approach and Results: Mouse primary lung ECs were exposed in vitro to D farinae extract or rmIL-33 (recombinant murine IL-33). Both D farinae and rmIL-33 induced Il-33 transcription without increasing the IL-33 production and upregulated the expression of its receptor, as well as genes involved in angiogenesis and the regulation of immune responses. In particular, D farinae and rmIL-33 upregulated Fas/Cd95 transcript level, yet without promoting apoptosis. Inhibition of caspases involved in the Fas signaling pathway, increased IL-33 protein level in ECs, suggesting that Fas may decrease IL-33 level through caspase-8-dependent mechanisms. Our data also showed that the NF-κB (nuclear factor-κB), PI3K/Akt, and Wnt/ß-catenin pathways regulate Il-33 transcription in both mouse and human primary ECs. CONCLUSIONS: Herein, we described a new mechanism involved in the control of IL-33 production in lung ECs exposed to allergens.

Temperature stress response: A novel important function of Dermatophagoides farinae allergens.

Dermatophagoides farinae, an important pathogen, has multiple allergens. However, their expression under physiological conditions are not understood. Our previous RNA-seq showed that allergens of D. farinae were up-regulated under temperature stress, implying that they may be involved in stress response. Here, we performed a comprehensive study. qRT-PCR detection indicated that 26 of the 34 allergens showed differential expression. Der f1 had the most abundant basic expression quantity. Der f 28.0201 (HSP70) and Der f3 had the same regulation pattern in 9 highly expressed transcripts, which only up-regulated at 41 °C and 43 °C, but Der f 28.0201 showed stronger regulation than Der f 3 (19.88-fold vs 6.02-fold). Whereas Der f 1, 2, 7, 21, 22, 27, and 30 were up-regulated under both heat and cold stress, and Der f 27 showed the strongest regulation ability among them. Der f 27 showed more significant up-regulation than Der f 28.0201 under heat stress (23.59-fold vs 19.88-fold), and Der f27 had more obvious up-regulation under cold than heat stress (30.70-fold vs 23.59-fold). The expression of Der f 27, 28.0201 and 1, and D. farinae survival rates significantly decreased following RNAi, indicating the upregulation of these allergens under temperature stress conferred thermo-tolerance or cold-tolerance to D. farinae. In this study, we described for the first time that these allergens have temperature-stress response functions. This new scientific discovery has important clinical value for revealing the more frequent and serious allergic diseases caused by D. farinae during the change of seasons.

Validation of Strasbourg environmental exposure chamber (EEC) ALYATEC® in mite allergic subjects with asthma.

Objective: Environmental Exposure Chamber (EEC) should have standardized and controlled allergenic and non-allergenic exposures to perform reproducible clinical studies. The aim was to demonstrate that mite exposure in the Alyatec® EEC could induce early (EAR) and/or late asthmatic reactions (LAR) in at least 60% of subjects allergic to mite.Methods: The EEC has a volume of 147-m3 with 20 seats. The nebulized particle number, airborne Der p1, endotoxins, and volatile organic compound (VOC) concentrations were measured. Twenty-four asthmatics allergic to mite were randomly exposed to 15, 25, and 46 ng/m3 Der p1. Specificity was assessed in not mite-sensitized asthmatics.Results: No significant endotoxin or VOC contamination was measured. The mean inter-assay CVs were 12.5% for the airborne particle number and 28.7% for airborne Der p1 concentrations. For the three Der p1 concentrations, at least 88% of the subjects developed EAR and/or LAR, and at least 46% developed a dual response. No reaction occurred with placebo or in the control group. No severe bronchial reaction occurred.Conclusions: The Alyatec® EEC demonstrated a tight control of allergenic and non-allergenic exposures. The EEC was clinically validated, with airborne Der p1 levels close to levels found in natural settings.

Synergistic effect of Dermatophagoides pteronyssinus allergen and Escherichia coli lipopolysaccharide on human blood cells.

PURPOSE: House dust mites Dermatophagoides pteronyssinus are the main source of major inhalatory allergens inducing inflammatory response. Mite extract contain both allergenic proteins and lipopolysaccharides (LPS). The main allergenic protein, Der p 2, is a functional homolog of sMD-2, a protein providing blood cell response on LPS. Der p 2 may restore the response to LPS in absence of MD-2, but its interaction with LPS in whole blood is unknown. We studied the effect of Der p 2 on LPS-mediated activation of human whole blood cells. METHODS: Interaction of Der p 2 and LPS was studied on eight healthy donors. The whole blood was incubated with extract of house dust mite Dermatophagoides pteronyssinus (DP-e), recombinant antigenic protein Der p 2 variant 5 (rDep 2), Escherichia coli lipopolysaccharide and their combination. Supernatants were collected for ELISA analysis of protein content. Activation degree was determined by change in concentration of TNF-α, IL-8, IL-1Ra cytokines and sMD-2 protein. RESULTS: extract of mite Dermatophagoides pteronyssinus (DP-e) possessed weak inherent activity and did not cause significant increase of cytokine production. Simultaneous activation of blood cells by LPS and DP-e led to considerable increase of pro-inflammatory cytokine production. We have shown the intrinsic inducing activity of Der p 2 allergen on sMD-2 protein and TNF-α cytokine expression. CONCLUSIONS: Der p 2 allergen enhances the response of human whole blood cells to external LPS by inducing additional expression of LPS-transporting protein sMD-2. The obtained data show an important role of LPS contamination of allegrens in the progress of allergic inflammatory response.

Annexin A5 Protein as a Potential Biomarker for the Diagnosis of Asthma.

PURPOSE: Annexin A5 (ANXA5) has a potential role in cellular signal transduction, inflammation, and fibrosis. However, the exact role of ANXA5 in asthma remains to be clarified. The aims of the present study were to investigate ANXA5 protein expression in a mouse model of asthma and pollutant exposure and to elucidate the relationships between clinical variables and plasma ANXA5 levels in patients with asthma. METHODS: A murine model of asthma induced by ovalbumin (OVA) and titanium dioxide (TiO2) nanoparticles has been established using BALB/c mice, and we examined ANXA5 expression and lung fibrosis using this model. Moreover, we also compared ANXA5 plasma levels in patients with controlled vs. exacerbated asthma. RESULTS: ANXA5 protein levels were lower in lung tissue from OVA + OVA mice than in control mice. Lung ANXA5, connective tissue growth factor (CTGF), and transforming growth factor ß1 (TGF-ß1) protein levels were higher in OVA + TiO2-exposed mice than in control or OVA + OVA mice. Although Dermatophagoides pteronyssinus (Derp1) treatment increased lung ANXA5 protein levels in MRC-5 cells and A549 epithelial cells, it decreased lung ANXA5 levels in NHBE cells. Treatment with TiO2 nanoparticles increased lung ANXA5, CTGF, and TGF-ß1 protein levels in MRC-5 cells, A549 epithelial cells, and NHBE cells. Plasma ANXA5 levels were lower in asthmatic patients than in healthy controls, and they were significantly enriched in patients with exacerbated asthma compared with those with controlled asthma (P < 0.05). ANXA5 levels were correlated with pulmonary function as assessed by spirometry. CONCLUSION: Our results imply that ANXA5 plays a potential role in asthma pathogenesis and may be a promising marker for exacerbated bronchial asthma and exposure to air pollutants.

Heparinoid suppresses Der p-induced IL-1ß production by inhibiting ERK and p38 MAPK pathways in keratinocytes.

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro-inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen-induced interleukin (IL)-1ß release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1ß, but also suppressed IL-1ß messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal-regulated kinase and p38 pathways, which are required for IL-1ß expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL-1ß mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte-mediated skin inflammation.

House dust mite allergens Der f and Der p induce IL-31 production by blood-derived T cells from atopic dermatitis patients.

Aero-allergens, such as house dust mite (HDM), have been suggested to play a role in the initiation of atopic dermatitis (AD)-related skin inflammation. Here, we analysed the proliferation and the cytokine expression of blood-derived T cells from AD and healthy individuals upon HDM-allergen stimulation. The proliferating cells from healthy individuals and AD patients had a significantly different, distinct cytokine profile: in AD blood, we found increased frequencies of HDM-reactive IL-31-producing T cells, as well as a decreased Th1/Th2 and Tc1/Tc2 ratio, suggesting that allergen-specific T cells in blood of chronic AD patients are subject to pre-existent Th2-Tc2 and "Th31-Tc31" programming.

Dust mite allergen-specific immunotherapy increases IL4 DNA methylation and induces Der p-specific T cell tolerance in children with allergic asthma.

Allergen-specific immunotherapy (allergen-SIT) is a highly effective treatment for children with allergic asthma (AA), an immune-mediated chronic disease leading to bronchial muscle hypertrophy and airway obstruction in response to specific allergens. T helper cells and secreted cytokines play important roles in the pathogenesis of asthma, and epigenetic modulation controls genes important for T cell development and cytokine expression. This study evaluated T helper cell-secreted cytokines and DNA methylation patterns in children treated with Dermatophagoides pteronyssinus (Der p) allergen-SIT. Our results showed that after Der p challenge, peripheral blood mononuclear cells (PBMCs) from the SIT group, compared with the non-SIT AA group, produced lower levels of IL-4, IL-5 and IL-2. The SIT group, compared with the AA group, exhibited decreased sensitivity to the Der p allergen, concurrent with IL-4 down-modulation due to increased promoter DNA methylation, as estimated in PBMCs. Our results showed that SIT decreased IL-4 and IL-5, and inhibited T cell proliferation, by inhibiting IL-2 production after the specific allergen challenge. These results suggest that decreased IL-2 production and increased IL-4 cytokine promoter methylation is a potential mechanism of Der p-specific allergen desensitization immunotherapy.

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation.

House Dust Mites Induce Production of Endothelin-1 and Matrix Metalloproteinase-9 in Keratinocytes via Proteinase-Activated Receptor-2 Activation.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction and abnormal immune response. House dust mites (HDM) are a major source of allergens, some of which have cysteine and serine protease activities. Keratinocytes stimulated by HDM-derived proteases have been suggested to contribute to the pathogenesis of AD by producing various cytokines. However, whether keratinocytes contribute to the induction of pruritus in AD, especially by producing pruritus-related mediators upon stimulation with HDM-derived proteases, has not been fully elucidated. METHODS: We examined whether the production of endothelin-1 (ET-1), matrix metalloproteinase (MMP)-2, and MMP-9 in keratinocytes can be induced by stimulation with Dermatophagoides farinae extracts, and if so, whether pretreatment with a protease inhibitor or proteinase-activated receptor-2 (PAR-2) antagonist affects the production of these mediators in keratinocytes. RESULTS: Although MMP-2 levels were undetectable in the culture supernatants, the production of ET-1 and MMP-9 was increased upon stimulation with HDM extracts in a concentration- and time-dependent manner and suppressed by pretreatment of HDM extracts with serine protease inhibitor, but not with cysteine protease inhibitor. Mite-derived serine proteases also induced ET-1 and MMP-9 production in a concentration- and time-dependent manner. Moreover, pretreatment with a PAR-2 antagonist inhibited the production of ET-1 and MMP-9 in keratinocytes. CONCLUSION: These results suggest that the activation of PAR-2 on keratinocytes by HDM-derived serine proteases induces the production of ET-1 and MMP-9, and may contribute to the induction of pruritus in AD.

Aeroallergen Der p 2 promotes motility of human non-small cell lung cancer cells via toll-like receptor-mediated up-regulation of urokinase-type plasminogen activator and integrin/focal adhesion kinase signaling.

House dust mite (HDM) allergens are one of the major causes leading to respiratory hypersensitiveness and airway remodeling. Here we hypothesized that a major HDM allergen Der p 2 could increase cell motility and invasiveness of non-small cell lung cancer (NSCLC) cells. Our results showed that low dose (1 and 3 µg/mL) recombinant Der p 2 protein (DP2) enhanced the migration and invasiveness of human NSCLC cell A549, H1299 and CL1-5, but nonsignificantly altered their growth. Further investigation revealed that integrin αV level was increased and its downstream signaling including focal adhesion kinase (FAK) and paxillin were activated in A549 cells exposed to DP2. In parallel, DP2 also activated the FAK-associated signaling effectors such as Src, phosphatidyl inositol 3-kinase (PI3K), AKT, p38 mitogen-activated protein kinase (P38), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Our findings also revealed that DP2 increased expression level of urokinase type plasminogen-activated kinase (uPA) and uPA receptor (uPAR), and subsequently enhanced the binding of uPAR to integrin αV. Moreover, the involvement of toll-like receptor 2/4 (TLR2/4)-triggered ERK1/2 activation in the increased expression of uPA and uPAR was also demonstrated. Collectively, these findings indicate that DP2 can enhance cell motility and invasiveness of NSCLC cells, attributing to TLR2/4-ERK1/2 activation, increased uPA and uPAR expression, enhanced binding of uPAR to integrin αV, and the consequent FAK signaling cascades. Thus, we suggest that DP2 may exacerbate NSCLC via promoting metastatic ability of carcinoma cell.

CB2 receptors regulate natural killer cells that limit allergic airway inflammation in a murine model of asthma.

BACKGROUND: Allergic asthma is a chronic airway inflammatory disease involving the complementary actions of innate and adaptive immune responses. Endogenously generated cannabinoids acting via CB2 receptors play important roles in both homeostatic and inflammatory processes. However, the contribution of CB2-acting eicosanoids to the innate events preceding sensitization to the common house dust mite (HDM) allergen remains to be elucidated. We investigated the role of CB2 activation during allergen-induced pulmonary inflammation and natural killer (NK) cell effector function. METHODS: Lung mucosal responses in CB2-deficient (CB2-/- ) mice were examined and compared with wild-type (WT) littermates following intranasal exposure to HDM allergen. RESULTS: Mice lacking CB2 receptors exhibited elevated numbers of pulmonary NK cells yet were resistant to the induction of allergic inflammation exemplified by diminished airway eosinophilia, type 2 cytokine production and mucus secretion after allergen inhalation. This phenomenon was corroborated when WT mice were treated with a CB2-specific antagonist that caused a pronounced inhibition of HDM-induced airway inflammation and goblet cell hyperplasia. Unexpectedly, the preponderance of NK cells in the lungs of CB2-/- mice correlated with reduced numbers of group 2 innate lymphoid cells (ILC2s). Depletion of NK cells restored the allergen responsiveness in the lungs and was associated with elevated ILC2 numbers. CONCLUSIONS: Collectively, these results reveal that CB2 activation is crucial in regulating pulmonary NK cell function, and suggest that NK cells serve to limit ILC2 activation and subsequent allergic airway inflammation. CB2 inhibition may present an important target to modulate NK cell response during pulmonary inflammation.

Dermatophagoides pteronyssinus group 2 allergen bound to 8-OH modified adenine reduces the Th2-mediated airway inflammation without inducing a Th17 response and autoimmunity.

8-OH modified adenine bound to Dermatophagoides pteronyssinus group 2 (nDer p2-Conj), a novel allergen-TLR7 agonist conjugate, improves murine airway inflammation in priming and therapeutic settings, however no data are known on the activity of this construct on Th17 cells. The aim of the study was to evaluate if nDer p2-Conj elicited in vivo Th17 cells and Th17-driven autoimmune responses, by using both short- and long-term priming and therapeutic protocols in a nDer p2-driven model of murine airway inflammation. The conjugate induced the in vitro production of cytokines favouring the Th17 polarization by bone marrow-derived dendritic cells. In short-term protocols, the priming or treatment with the conjugate ameliorated the airway inflammation by shifting Th2 allergen-specific cells into T cells producing IFN-γ, IL-10, but not IL-17A. Similar results were found in long-term protocol where the conjugate down-regulated airway inflammation without any evidence of autoimmune response and B cell compartment expansion. nDer p2-Conj also failed to shorten the spontaneous onset of diabetes on conjugates-primed NOD/LtJ mice. We found that neutrophils in BALF, ROR-γt and IL-17A expression in lungs were increased in conjugate-treated IL-10KO mice. These data emphasize the role of conjugate-driven IL-10 production, which can regulate the activity of memory Th17 cells and prevent the onset of autoimmune response.

Effect of the house dust mite allergen Der p 1 on tryptase release from human mast cells.

This study aimed to investigate the effects of the house dust mite allergen Der p 1 on the secretion of tryptase from the human mast cell line HMC-1. Flow cytometry was used to determine the expression levels of protease-activated receptor-2 (PAR2) on the surface of HMC-1 cells. HMC-1 cells were treated with Der p 1, SLIGRL-NH2 (PAR2 agonist), LRGILS-NH2 (control peptide for PAR2), or Der p 1 + FSLLRY (PAR2 antagonist), and the tryptase levels were measured using enzyme-linked immunosorbent assay. The biological functions of PAR2 were determined using the calcium green indicator, and intracellular calcium fluorescence intensity in the different groups (Der p 1, SLIGRL-NH2, LRGILS- NH2, Der p 1 + FSLLRY, tryptase, tryptase + FSLLRY, or cell culture medium) was detected by laser scanning confocal microscopy. The mast cells expressed PAR2 receptor on their surfaces. Der p 1 alone induced a significant release of intracellular calcium and tryptase in HMC-1 cells compared with the SLIGRL- NH2 treatment group and the control group. The combination of Der p 1 and FSLLRY partly inhibited intracellular calcium and tryptase release in HMC-1 cells compared with the Der p 1 treatment group. Moreover, tryptase induced a significant release of intracellular calcium in the HMC-1 cells. Der p 1 induced HMC-1 cell degranulation and the release of tryptase by activating the PAR2 receptor on the cell surfaces. Tryptase activated the PAR2 receptor and induced intracellular calcium release from the HMC-1 cells in a positive feedback loop.

Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation.

A strategy to improve allergen-specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8-OH-modified adenine. Humoral and cellular responses were analysed in allergen-sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co-culture system was also investigated. The nDer p 2-Conj treatment in nDer p 2-primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen-driven interleukin-13 (IL-13) production in lung mononuclear cells and IgE, in comparison with nDer p 2-treated mice. The increase of IgG2a paralleled that of interferon-γ (IFN-γ) and IL-10 in allergen-stimulated spleen cells. Similar effects were elicited by treatment with OVA-Conj in an OVA-driven BALB/c model. The nDer p 2-Conj or OVA-Conj redirected memory T helper type 2 cells towards the production of IL-10 and IFN-γ also in C57BL/6 mice and when subcutaneously administered. Interleukin-10, IL-12 and IL-27 were produced in vitro by Conj-stimulated bone marrow dendritic cells, whereas IL-10 and IFN-γ were up-regulated in co-cultures of CD11c(+) and CD4(+) T cells from Conj-treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN-γ- and IL-10- producing memory T cells. The Conj effects on IL-10(-/-) and IL-12(-/-) mice confirmed the role of IL-10 and IFN-γ in inducing a protective and balanced redirection the T helper type 2-mediated airway inflammation.

Zingiber cassumunar ROXb. and its active constituent inhibit MMP-9 direct activation by house dust mite allergens and MMP-9 expression in PMA-stimulated human airway epithelial cells.

BACKGROUND: House dust mite (HDM) induced matrix metalloproteinase (MMP)-9 plays a role in asthma. Zingiber cassumunar Roxb. (Phlai in Thai) has been used in folk medicine for asthma treatment. OBJECTIVE: We investigated effects of Phlai and its constituent (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D) on the cleavage of pro- MMP-9 by HDM. The effects of these compounds on phorbol 12-myristate 13-acetate (PMA)- induced MMP-9 gene and protein expression in airway epithelial cells (NCI-H292) were also investigated. METHODS: Pro-MMP-9 was directly activated in vitro with HDM in the presence or absence of the ethanolic extracts of Phlai or compound D for 1 hour. The amount of activated MMP-9 was determined using gelatin zymography. To study the cellular response of Phlai, NCI-H292 cells were pretreated with crude Phlai extracts or compound D for 2 hours, and then the cells were stimulated with PMA for 48 hours. The mRNA RT-PCR and Western blotting, respectively. MMP-9 activity was determined by gelatin zymography. RESULTS: Crude Phlai extracts (0.25 - 2.0 mg/ml) and compound D (0.5 - 4.0 mg/ml) inhibited pro- MMP-9 cleavage by HDM. Furthermore, crude Phlai extracts (100 mg/ml) and compound D, at concentrations of 50 and 100 mg/ml, attenuated the PMA-induced MMP-9 gene and expression in NCI-H292 cells. These compound also suppressed MMP-9 release from PMA-induced NCI-H292 cells. CONCLUSION: The crude ethanolic extract of Z. cassumunar and its active constituent compound D inhibited the cleavage of pro-MMP-9 by HDM. They also inhibited PMA-induced MMP-9 gene and protein synthesis in human airway epithelial cells.

Development of a poly (lactic-co-glycolic acid) particle vaccine to protect against house dust mite induced allergy.

Poly(lactic-co-glycolic acid) (PLGA) particles carrying antigen and adjuvant is a promising vaccine system which has been shown to stimulate systemic antigen-specific immune responses. In this study, we investigated the relationship of (i) the sizes of PLGA particle and (ii) the presence of cytosine-phosphate-guanine motifs (CpG), with the extent and type of immune response stimulated against Dermatophagoides pteronyssinus-2 (Der p2) antigen. Different sizes of PLGA particles encapsulating CpG were prepared using a double emulsion solvent evaporation method. Mice were vaccinated with Der p2 and different sizes of empty or CpG-loaded PLGA particles. Vaccinated mice were exposed to daily intranasal instillation of Der p2 for 10 days followed by euthanization to estimate leukocyte accumulation in bronchoalveolar lavage (BAL) fluids, antibody profiles, and airway hyperresponsiveness. PLGA particles showed a size-dependent decrease in the proportion of eosinophils found in BAL fluids. Mice vaccinated with the Der p2 coated on 9-µm-sized empty PLGA particles showed increased levels of IgE and IgG1 antibodies as well as increased airway hyperresponsiveness. All sizes of PLGA particles encapsulating CpG prevented airway hyperresponsiveness after Der p2 exposures. Inflammatory responses to Der p2 exposure were significantly reduced when smaller PLGA particles were used for vaccination. In addition, encapsulating CpG in PLGA particles increased IgG2a secretion. This study shows that the size of PLGA particles used for vaccination plays a major role in the prevention of house dust mite-induced allergy and that incorporation of CpG into the PLGA particles preferentially develops a Th1-type immune response.