Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.     

Mite-allergen exposure and spontaneous histamine release in monosensitive and polysensitive mite-allergic patients

Forty-three patients allergic to mites and suffering mainly from asthma were recruited: 25 were mite-monosensitive and 18 were polysensitive, as determined by skin tests and specific serum IgE determinations with various allergens. In vitro spontaneous histamine release (SHR) by washed blood basophils was measured once or several times for each patient. Throughout this study, the mean periods of high and low mite-allergen exposure were defined on the basis of relative indoor humidity and temperature data. For the mite-monosensitive patients, there was a significant increase in mean SHR during the season of high mite-allergen exposure as compared to the months of lower mite-allergen presence ( P < 0.002). No significant difference between mean SHR values was observed when comparing the monosensitive group during the season of high mite-allergen exposure with polysensitive patients (allergic to mite and pollen) during the period of exposure to both allergens. Differences in mean SHR reported here emphasize the positive relationship between intense allergen exposure and the in vitro SHR increase in blood basophils of mite-allergic patients.     

A 300 IR sublingual tablet is an effective,safe treatment for house dust mite–induced allergic rhinitis: An international,double-blind,placebo-controlled,randomized phase III clinical trial

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Hyposensitization in asthmatics with mPEG-modified and unmodified house dust mite extract

Forty-six adult asthmatics allergic to D. pteronyssinus (Dp) participated in a 2-year study. Thirty-one underwent hyposensitization (HS-group). Fifteen were treated with Dp-extract (Dp-group), and 16 with a similar extract modified by monomethoxypolyethylene glycol with reduced allergenicity (mPEG-Dp-group). Fifteen patients served as controls. Dp-specific antibodies and histamine release from blood basophils were determined and compared with Dp-sensitivity in lungs and skin. In addition, IgG and IgE against the major allergen Der p I were followed in a subgroup. Dp-specific IgG, IgG1, and IgG4 increased significantly in both HS-treated groups after 1 and 2 years (median: 2.5- to 11.6-fold). IgG4 was not induced if maintenance dose during the first year was less than 20,000 BU. Median skin sensitivity decreased 4.4- to 8.2-fold after 1 year and 7.4- to 21.4-fold after 2 years. Der p I specific IgG response was unrelated to the occurrence or change in IgE with the same specificity. The mPEG-Dp-extract tended to have less effect on skin sensitivity and immunological parameters, differences reaching statistical significance for skin sensitivity only. In the HS-group, the decrease in bronchial sensitivity was significantly correlated to a decrease in IgE (r = 0.36), IgG1/IgG4 (r = 0.49), Dp-specific histamine release (r = 0.58), and to an increase in Dp-specific IgG4 (r = -0.36) and IgG4/IgE (r = -0.48). In patients improving clinically, Dp-specific IgG4/IgE increased, and median Dp-specific IgE was reduced to 80% compared with an increase to 150-160% seen in the unchanged or deteriorated group (P less than 0.05). Findings indicate an improvement of effect, if the allergen dose is sufficient to reduce specific IgE and/or induce an IgG and especially IgG4 response.     

Importance of including Blomia tropicalis in the routine diagnosis of Venezuelan patients with persistent allergic symptoms

Background:  Blomia tropicalis is a common mite found in the house dust of many tropical countries including Venezuela. The prevalence of skin test and specific serum immunoglobulin (Ig)E reactivity to B. tropicalis in Venezuela has not been previously evaluated.
Methods:  In the present study we evaluated the skin reactivity by skin prick test and specific IgE by a multiple antigen blot assay, against B. tropicalis and Dermatophagoides pteronyssinus , in a group of 115 subjects who attended the Allergy Clinic of the Institute of Biomedicine, Caracas, Venezuela, and we studied possible cross reactions between similar proteins of these two mites.
Results:  One hundred and six patients with persistent allergic respiratory symptoms showed a positive skin prick test to at least one of the mite extracts, with the frequency of positive reactions to B. tropicalis being as high as to D. pteronyssinus . Twelve patients reacted only to D. pteronyssinus and 13 different patients only to B. tropicalis . Specific IgE to each of the mite extracts was found with similar frequency, and the results coincided with the skin test reactivity.
Conclusions:  The study indicated the importance of including B. tropicalis in routine diagnostic testing in tropical and sub-tropical situations.     

粉尘螨滴剂舌下特异性免疫治疗儿童哮喘的疗效及其对T淋巴细胞亚群的影响

目的:观察粉尘螨滴剂舌下特异性免疫治疗(SLIT)对支气管哮喘患儿T淋巴细胞亚群的影响,分析影响疗效的因素。方法:采用前瞻性随机对照研究方法,选取对粉尘螨过敏的轻度支气管哮喘患儿120例,按随机数表法分为观察组和对照组各60例。对照组给予常规药物治疗,观察组在对照组治疗基础上加用粉尘螨滴剂SLIT,疗程1年。分别于治疗前后采用流式细胞仪检测患儿的T淋巴细胞亚群,比较两组临床疗效并探讨影响疗效的因素。结果:治疗后两组患儿CD3+、CD4+、CD4+/CD8+均升高,且观察组均高于对照组(P均<0.05),CD8+均降低,且观察组低于对照组(P均<0.05)。观察组总有效率为78.33%,高于对照组的58.33%(P<0.05)。观察组患儿中,治疗有效的患儿(n=47)和治疗无效的患儿(n=13)在年龄、病程、气道高反应、特异性免疫球蛋白E(sIgE)水平、痰液嗜酸粒细胞(EOS)水平方面比较差异有统计学意义(P均<0.05)。结论:粉尘螨滴剂SLIT能有效纠正哮喘患儿T淋巴细胞亚群紊乱,提高疗效,可能影响其疗效的因素包括年龄、病程、气道高反应、sIgE水平、痰液EOS水平等。     

Analysis of basophil activation by flow cytometry in pediatric house dust mite allergy

Detection of allergen‐induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non‐allergic children (n = 9). Allergen‐induced basophil activation was detected as a CD63‐upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut‐off value of activated basophils discriminating mite allergic and non‐allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite‐sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97–1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 μg/ml mite extract. Analysis of the data obtained with 1.6 μg/ml mite extract defined a cut‐off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91–1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non‐allergic children produced a cut‐off of 8% activated basophils (AUC = 1.0) with 16 μg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 μg/ml extract (AUC = 97%, 95% CI = 0.92–1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1‐specific IgE (>100 kU/l). Cross‐reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA‐CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen‐induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.     

舌下含服粉尘螨滴剂治疗支气管哮喘伴变应性鼻炎的疗效

目的 探讨舌下含服粉尘螨滴剂特异性免疫治疗支气管哮喘(哮喘)伴变应性鼻炎的疗效及安全性.方法 选取516例年龄4～13岁哮喘伴变应性鼻炎患儿.其中291例完成1 a舌下免疫治疗(免疫治疗组),非免疫治疗对照组225例.患儿均完成10种常见变应原皮肤点刺试验,点刺试验结果呈阳性反应.免疫治疗组根据皮肤点刺试验结果分尘螨过敏组80例、尘螨及蟑螂过敏组71例、尘螨及花粉过敏组74例、尘螨及狗毛过敏组66例.应用粉尘螨滴剂进行临床免疫治疗,记录治疗前后哮喘控制问卷(ACQ)评分、鼻炎症状评分、用药情况和不良反应.结果 1.治疗12个月后,免疫治疗组和非免疫治疗对照组ACQ评分分别为(0.28±0.33)分和(1.07±0.68)分,与治疗前[(1.76±0.75)分和(1.55±0.62)分]比较,分别下降了(74.03±37.66)%和(29.32±44.53)%,2组ACQ评分比较差异有统计学意义(Z=-154.109,P<0.000 1).2.治疗12个月,免疫治疗组和非免疫治疗对照组鼻炎症状评分分别为(0.337±0.479)分和(0.560±0.634)分,较治疗前[(0.899±0.667)分和(0.892±0.688)分]分别有70.8%和39.1%的患儿评分级别降低,2组比较差异有统计学意义(χ2=51.949,P<0.000 1).3.治疗12个月,免疫治疗组和非免疫治疗对照组治疗哮喘月均用药评分分别为(20.91±18.03)分 和(85.22±47.84)分,与治疗初始月均用药评分[(113.41±35.02)分和(108.86±35.24)分]比较,分别下降了(75.10±28.80)%和(20.60±39.52)%.4.治疗12个月,免疫治疗组肺功能呼气峰流速(预计值百分比)[(91.38±8.82)%]较治疗前上升(8.84±9.64)%.5.免疫治疗结束,免疫治疗组粉尘螨试验阳性级别降低,与非免疫治疗对照组比较差异有统计学意义(χ2=70.850,P<0.000 1).6.各免疫治疗亚组ACQ评分、月均用药评分和粉尘螨皮试阳性级别降低的差异均无统计学意义.7.与用药相关的皮疹、鼻咽痒和哮喘发作不良反应发生率为24.7%,未出现过敏性休克等严重不良反应.结论 特应性舌下免疫治疗方法安全有效,是治疗儿童哮喘伴变应性鼻炎的重要措施之一.