Dermatophagoides Pteronyssinus Extract Research Articles

In vivo and in vitro allergenicity of the domestic mite Chortoglyphus arcuatus

Chortoglyphus arcuatus is frequently identified in mattress dust samples in coastal areas of northern Spain. To establish the prevalence of positive skin test reactions to C. arcuatus and to analyze its allergenicity and cross-reactivity. One hundred thirty-eight consecutive patients entered the study. The main referred symptoms were rhinoconjunctivitis and asthma. Skin tests were performed with extracts of Dermatophagoides pteronyssinus, Lepidoglyphus destructor, Tyrophagus putrescentiae, C. arcuatus, and Glycyphagus domesticus. Enzyme-linked immunosorbent assay inhibition experiments were conducted with the different mite species and immunoblots with serum samples from 31 sensitized patients. Conjunctival provocation tests were performed in 10 patients with C. arcuatus-positive skin test reactions and in 9 with negative results; all the patients had positive skin test reactions to D. pteronyssinus. The prevalence of positive skin test reactions to D. pteronyssinus was 94.2% and to C. arcuatus was 58%. There was a significant correlation between the number of mites to which patients were sensitized and the time of disease evolution (P = .02). Results of enzyme-linked immunosorbent assay inhibition experiments suggested minimal cross-reactivity between C. arcuatus and D. pteronyssinus. Immunoblot results confirmed specific IgE binding to several bands. Conjunctival test results were positive in 9 of 10 C. arcuatus-positive individuals and slightly positive in 2 of 9 C. arcuatus-negative, D. pteronyssinus-positive individuals. There is a high prevalence of sensitization to C. arcuatus in northern Spain. Sensitization to this species should be considered of clinical significance. There is minimal cross-reactivity between C. arcuatus and D. pteronyssinus.

Induction of atopic eczema/dermatitis syndrome-like skin lesions by repeated topical application of a crude extract of Dermatophagoides pteronyssinus in NC/Nga mice

Mite antigen has been considered to play important roles in the development of atopic eczema/dermatitis syndrome (AEDS). In the present study, we attempted to induce an AEDS-like skin lesion in mice using Dermatophagoides pteronyssinus crude extract (DPE) as an antigen and performed pathophysiological evaluations. Ears of mice were tape-stripped and DPE was painted 3 times a week. Eczematous skin lesion and ear swelling were apparent in NC/Nga mice treated with DPE after 2 weeks, whereas neither skin lesion nor ear swelling were observed in BALB/c mice even after 30 days. Histological evaluation demonstrated that edema, epidermal hyperplasia and the accumulation of inflammatory cells were apparent in the ears of DPE-treated NC/Nga mice. In contrast to skin lesion and ear swelling, total serum IgE levels were increased in both NC/Nga and BALB/c mice. Treatment with DPE also increased auricular lymph node weight in both NC/Nga mice and BALB/c mice. To further characterize, we analyzed cytokine mRNA expression in ears and lymph nodes of DPE-treated NC/Nga mice. Increased expression of IL-4 and TNF-α mRNA was observed in both ears and lymph nodes of NC/Nga mice treated with DPE. Additionally, there was no change in the responsiveness of BALB/c mice to DPE treatment by adaptive transfer of serum from DPE-treated NC/Nga mice to BALB/c mice. Taken together, our results indicate that eczematous skin lesion and ear swelling caused by repeated application of DPE in NC/Nga mice has a Th2-dominant background and that inflammation is involved in this process. The animal model of AEDS established in this report may be used to investigate the pathogenesis of AEDS and evaluate the potential therapeutic agents for AEDS.

Platelet activation in allergic asthma patients during allergen challenge with Dermatophagoides pteronyssinus

Animal models of allergic asthma indicate that intravascular platelet activation is necessary for the development of allergen-induced chronic airway inflammation. To evaluate whether the development of a late asthmatic response (LAR) in allergic asthma patients challenged with a relevant allergen is consequent to platelet activation. Thirty-three house dust mite sensitive asthmatic patients were challenged intrabronchially with Dermatophagoides pteronyssinus (Dp) extract. Twelve non-atopic healthy subjects (HC) were used as controls. Platelet count and plasma levels of beta-thromboglobulin (beta-TG), platelet factor-4 (PF-4) and soluble P-selectin (sP-selectin) were assessed before the challenge (T(0)) and 30 min (T(EAR)), 6 h (T(LAR)) and 24 h (T(24)) after the challenge. Eleven patients responded to allergen challenge with an isolated early asthmatic response (single responders, SR). In 22 patients dual asthmatic response was demonstrated (dual responders, DR). At T(0) neither the platelet count nor the mean plasma level of beta-TG in DR or SR were different from HC, the mean plasma level of PF-4 in SR was significantly greater than in HC (P=0.01) or DR (P=0.001), the mean plasma level of sP-selectin was significantly greater in DR than in HC (0.0002) but not statistically different from SR (P=0.055). A significant decrease in the platelet count and increase in the plasma level of all the studied markers was seen at T(EAR), which was followed by a gradual return to the baseline values in the SR. Elevated plasma levels of platelet activation markers and decreased platelet count were seen in the DR even at T(24). Strong correlation was found between the increase in plasma concentration of beta-TG at T(EAR) and the maximum fall in forced expiratory volume in 1 s at T(LAR) (r=-0.57; P=0.0006). In allergic asthma patients development of prolonged airway inflammation after allergen challenge is associated with intravascular platelet activation.

A double‐blind, placebo‐controlled study of house dust mite immunotherapy in Chinese asthmatic patients

The purpose of this study was to determine if house dust mite immunotherapy with Alutard SQ is effective in improving symptom control and reducing rescue medication use in Chinese patients with mild to moderate allergic asthma. This is a double-blind, placebo-controlled study involving 132 asthmatic subjects aged 6-45 years recruited from three different regions of Mainland China. Subjects were given a 52-week course of immunotherapy with Dermatophagoides pteronyssinus extract (Alutard Der p, ALK-Abelló, Hørsholm, Denmark) or placebo while their dose of inhaled corticosteroids (ICS) was maintained. 129 subjects (64 in active group) completed the study. The symptom scores began to diverge at week 29 with the immunotherapy group showing a significantly lower score until week 48 (P = 0.018). Immunotherapy resulted in a significant decline in symptom (P = 0.002) and medication (P = 0.007) scores during the second half of the treatment period. Both groups showed significant improvement in peak flow rate and bronchial hyperresponsiveness. Serum eosinophil cationic protein (ECP) also decreased in both groups of subjects, but peripheral blood eosinophil count remained unchanged. Skin test response decreased in actively treated subjects only, but Der p-specific immunoglobulin E (IgE) remained unchanged. Immunotherapy resulted in a significantly greater improvement in self-evaluation scores (P < 0.01). One year treatment with Alutard SQ house dust mite immunotherapy significantly reduced symptoms and medication use in asthmatic subjects. This was associated with a greater subjective improvement in asthma control.

Bacterial 16S ribosomal DNA in house dust mite cultures

Allergen extracts prepared from Dermatophagoides farinae contain significantly more endotoxin than Dermatophagoides pteronyssinus extracts, and extracts from both mite extracts contain more endotoxin than pollen extracts. Attempts to culture bacteria from mite cultures have failed to establish the sources of the endotoxin. To determine the bacterial sources of endotoxin in mite extracts. Live mites of both species were obtained from 2 sources, DNA was extracted from the mites, and DNA encoding bacterial 16S ribosomal RNA was amplified by using specific primers. The amount of bacterial DNA in each mite DNA sample was determined by quantitative PCR using an internal standard, and sequence homologies were determined from amplifications performed by using a high-fidelity DNA polymerase. DNA from D farinae appeared to contain between 11-fold and 24-fold more 16S ribosomal gene copies than the genomic DNA from D pteronyssinus (P < or = .003). Sequence analysis indicated the dominant presence of at least 3 phylogenetic clusters of Bartonella species (henselae, quintana, vinsonii, and grahamii), as well as uncharacterized alpha-proteobacteria, from both D farinae and D pteronyssinus. In a few clones, sequences from Escherichia coli, Pseudomonas species, and Acinetobacter species were also identified. House dust mite DNA contains evidence of Bartonella and other Gram-negative species. These Gram-negative species are likely to be the sources of the endotoxin found in mite allergenic extracts.

Intranasal mite allergen induces allergic asthma-like responses in NC/Nga mice

Airway responses induced by intranasal administration of mite allergen without adjuvant were studied in NC/Nga mice. A crude extract of Dermatophagoides farinae (Df) was administered for 5 consecutive days and a single intranasal challenge booster dose was given 1 week after the last sensitization. 24 h after the single challenge, the airway hyperresponsiveness (AHR) was measured and the bronchoalveolar lavage fluid (BALF) was analyzed for numbers of eosinophils and neutrophils, and both cytokine and chemokine levels. There were marked increases in number of eosinophils in the BALF, AHR, Th2 cytokines (IL-5 and IL-13), and chemokine (eotaxin-1 and eotaxin-2) levels in the BALF following Df exposure. C57BL/6N, A/J, BALB/c, and CBA/JN mouse strains were also exposed to Df crude extract, but all of the measured responses were strongest in NC/Nga mice. Furthermore, Df-exposed NC/Nga mice showed the goblet cell hyperplasia, pulmonary eosinophilic inflammation, and increases in both total serum IgE and Df-specific IgG1. After intranasal exposure of NC/Nga mice to crude extract of Dermatophagoides pteronyssinus, the BALF eosinophilia and AHR were similar to responses induced by Df. None of the study parameters were increased in response to intranasal exposure to ovalbumin. These data demonstrated that NC/Nga mice developed allergic asthma-like responses after intranasal exposure to mite allergens.

Expression of CD163 on peripheral blood monocytes in allergic asthma patients during specific immunotherapy (SIT)

RATIONALE: CD163 is expressed on alternatively activated mononuclear phagocytes. The aim of this study was to evaluate the expression of CD163 and CD36 on peripheral blood monocytes of allergic asthma patients during SIT with house dust mite allergen extracts.METHODS: 25 mild allergic asthma (AA) patients with positive skin prick tests and a positive bronchial provocation test with Dermatophagoides pteronyssinus extract were selected for the study. 10 nonatopic healthy persons (HP) were used as a control group. SIT was performed on 20 (AA-IT) patients using Novo Helisen Depot. 5 patients (AA) who declined SIT were considered a control group. Blood was collected before, 3 months and 12 months after initiation of SIT. FACS analysis was performed on whole blood samples using labeled monoclonal antibodies against CD14, CD36 and CD163 (Pharmingen, USA).RESULTS: Before SIT more CD14 cells co-expressing CD163 (CD14+/CD163+) were found in AA-IT (47.4 +/- 19.6%) and AA (43.2 +/- 16%) than in HP (25.6 +/- 8.5; p< 0.01). In AA-IT patterns during SIT the number of CD14+/CD163+ cells increased reaching 54.8 +/- 11.6 % at 3 months and 49.1 +/- 8.9% at 12 months (p<0.01). The maximum increase in CD14+/CD163+ cells was associated with maximum improvement in FEV1. No change was found in the number of CD14+/CD163+ cells in AA during 12 month period. The number of CD14+/CD36+ monocytes decreased in AA-IT but did not change in AA.CONCLUSIONS: In house dust mite sensitive asthmatics, SIT is associated with alternative activation of peripheral blood monocytes. RATIONALE: CD163 is expressed on alternatively activated mononuclear phagocytes. The aim of this study was to evaluate the expression of CD163 and CD36 on peripheral blood monocytes of allergic asthma patients during SIT with house dust mite allergen extracts. METHODS: 25 mild allergic asthma (AA) patients with positive skin prick tests and a positive bronchial provocation test with Dermatophagoides pteronyssinus extract were selected for the study. 10 nonatopic healthy persons (HP) were used as a control group. SIT was performed on 20 (AA-IT) patients using Novo Helisen Depot. 5 patients (AA) who declined SIT were considered a control group. Blood was collected before, 3 months and 12 months after initiation of SIT. FACS analysis was performed on whole blood samples using labeled monoclonal antibodies against CD14, CD36 and CD163 (Pharmingen, USA). RESULTS: Before SIT more CD14 cells co-expressing CD163 (CD14+/CD163+) were found in AA-IT (47.4 +/- 19.6%) and AA (43.2 +/- 16%) than in HP (25.6 +/- 8.5; p< 0.01). In AA-IT patterns during SIT the number of CD14+/CD163+ cells increased reaching 54.8 +/- 11.6 % at 3 months and 49.1 +/- 8.9% at 12 months (p<0.01). The maximum increase in CD14+/CD163+ cells was associated with maximum improvement in FEV1. No change was found in the number of CD14+/CD163+ cells in AA during 12 month period. The number of CD14+/CD36+ monocytes decreased in AA-IT but did not change in AA. CONCLUSIONS: In house dust mite sensitive asthmatics, SIT is associated with alternative activation of peripheral blood monocytes.

Efficacy and safety of specific immunotherapy with a modified mite extract

Background In specific immunotherapy (SIT), modified extracts have been used to allow safe administration of higher allergen doses. Schedules reaching maintenance doses in approximately 1 month, which may have greater efficacy, have even been proposed. Aims To assess the safety and efficacy of SIT with modified (depigmented and polymerized) Dermatophagoides pteronyssinus extract in the treatment of allergic rhinitis. Material and methods Fifty patients with moderate-to-severe persistent allergic rhinitis and who were monosensitized to Dermatophagoides were included in this controlled, pragmatic, 1-year open study. The patients were randomly allocated to receive treatment with a Dermatophagoides pteronyssinus 100% modified allergen vaccine (active group, n = 25) or pharmacological treatment only (control group, n = 25). All SIT-related adverse reactions were recorded. Efficacy was assessed primarily through the results of nasal allergen challenges, through visual analog scale (VAS) and symptom scores. Results In SIT-treated patients, significant improvements were found in symptom scores (mean reduction > 40 %), VAS scores (mean improvement > 20%) and nasal challenges (mean increase in allergen concentration threshold > 500%). For symptom and VAS scores, statistically significant differences between control and SIT-treated patients were recorded at 12 months. In nasal challenges statistically significant differences were observed as early as at 6 months. Control patients showed no significant differences during the study period. Local reactions were observed in 28 % of SIT-treated patients (total 24 reactions). There was only one immediate grade I systemic reaction, which was successfully treated with an antihistamine. Conclusions SIT with this modified extract appears to be a relatively safe treatment, which can rapidly improve nasal allergenic tolerance, reduce symptom scores and improve subjective self-evaluation measured through VAS, reflecting a general improvement in patients’ well-being.

The prevalence of positive reactions in the atopy patch test with aeroallergens and food allergens in subjects with atopic eczema: a European multicenter study

The atopy patch test (APT) was proposed to evaluate IgE-mediated sensitizations in patients with atopic eczema (AE). The prevalence and agreement with clinical history and specific IgE (sIgE) of positive APT reactions was investigated in six European countries using a standardized method. A total of 314 patients with AE in remission were tested in 12 study centers on clinically uninvolved, non-abraded back skin with 200 index of reactivity (IR)/g of house dust mite Dermatophagoides pteronyssinus, cat dander, grass, and birch pollen allergen extracts with defined major allergen contents in petrolatum. Extracts of egg white, celery and wheat flour with defined protein content were also patch tested. APT values were evaluated at 24, 48, and 72 h according to the European Task Force on Atopic Dermatitis (ETFAD) guidelines. In addition, skin-prick test (SPT) and sIgE and a detailed history on allergen-induced eczema flares were obtained. Previous eczema flares, after contact with specific allergens, were reported in 1% (celery) to 34% (D. pteronyssinus) of patients. The frequency of clear-cut positive APT reactions ranged from 39% with D. pteronyssinus to 9% with celery. All ETFAD intensities occured after 48 and 72 h. Positive SPT (16-57%) and elevated sIgE (19-59%) results were more frequent. Clear-cut positive APT with all SPT and sIgE testing negative was seen in 7% of the patients, whereas a positive APT without SPT or sIgE for the respective allergen was seen in 17% of the patients. APT, SPT and sIgE results showed significant agreement with history for grass pollen and egg white (two-sided Pr > /Z/ < or = 0.01). In addition, SPT and sIgE showed significant agreement with history for the other aeroallergens. With regard to clinical history, the APT had a higher specificity (64-91% depending on the allergen) than SPT (50-85%) or sIgE (52-85%). Positive APT were associated with longer duration of eczema flares and showed regional differences. In 10 non-atopic controls, no positive APT reaction was seen. Aeroallergens and food allergens are able to elicit eczematous skin reactions after epicutaneous application. As no gold standard for aeroallergen provocation in AE exists, the relevance of aeroallergens for AE flares may be evaluated by APT in addition to SPT and sIgE. The data may contribute to the international standardization of the APT.

Atopy patch test with house dust mite allergen--an IgE-mediated reaction?

The eczema reaction in the atopy patch test (APT) is proposed to be immunoglobulin (Ig)E mediated, but can take place also in individuals lacking allergen-specific IgE in serum. The purpose of this study was to evaluate the importance of allergen-specific serum IgE for the APT reaction. Ten patients with reproducible positive APT to extract of Dermatophagoides pteronyssinus, five patients with (group A) and five patients without (group B) detectable serum-IgE to D. pteronyssinus, were tested with extract of D. pteronyssinus on normal skin for 6, 24, 48 and 72 h. Skin biopsies were taken and analysed for cell infiltrates, eosinophils (EG2), IgE, FcepsilonRI, CD1a, CD4, CD8 and metalloproteinase 9 (MMP9). The number of IgE+, CD4+, EG2+ and MMP9+ cells increased with time in group A. FcepsilonRI+ cells and CD8+ cells increased with time in both groups. A correlation was found between the levels of D. pteronyssinus-specific serum-IgE and the score of dermal cell infiltrates at 72 h. The three patients with the highest values of allergen-specific IgE also had the highest expression of EG2+ cells and the highest APT scores. Our study strengthens the hypothesis that the IgE molecule has a key role, at least as an amplifier, in the APT reaction.

IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract.

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-beta secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 +/- 3,519 vs 29,280 +/- 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

Characterization of house dust for peptidase activity from fungal and mite allergens using monoclonal antibodies and specific peptidase assays

Rationale Exposure to active peptidase enzymes is an occupational hazard. Active peptidases are known to be a major concern in the pathology of allergies in airways of the lungs. The study set out to detect and characterize domestic dust samples for house dust mite ( Dermatophagoides pteronyssinus) and fungal ( Aspergillus fumigatus) allergens and the contribution of peptidase activity from these allergens. Methods Domestic dust samples were collected and extracted in a Tris buffer at pH 7.5. Extracts were subsequently assessed by commercially available ELISA and a peptidase activity assay using peptide pNA substrates. These were compared to the peptidase activities and allergen contents of standard extracts of Dermatophagoides pteronyssinus and Aspergillus fumigatus. Samples were further characterized by screening the extracts with a variety of peptide substrates and peptidase inhibitors. Results ELISA results demonstrated the presence of allergens from Dermatophagoides pteronyssinus and Aspergillus fumigatus in a variety of the dust samples that were tested. Further characterization of these samples with a panel of peptide pNA substrates and peptidase inhibitors demonstrated peptidase activities can contributed to fungal and dust mite allergens. The significance of peptidase activities from both Dermatophagoides pteronyssinus and Aspergillus fumigatus allergens will be discussed. Conclusions Presence of both house dust mite and fungal allergens and peptidases were demonstrated in domestic dust samples using both mABs and specific peptidase action. Exposure to these peptidases can be a health hazard and contribute to allergic conditions.

Expression, in basophils, of activation molecule CD63 in patients with mites rhinitis submitted to specific immunotherapy

Rationale Basophil activation by flow cytometry (BAFC) allows the study of type I hypersensitivity. In this study we evaluated the effect of specific immunotherapy (SIT) in patients with allergic rhinitis on BAFC. Methods 3 groups of patients with allergic rhinitis confirmed by clinically history, skin tests and specific IgE (sIgE). Group I: 12 patients (15-55 years old), 5 females and 7 males with SIT, performed with a modified extract of Dermatophagoides pteronyssinus, Depigoid ®. Group II: 10 patients (15-55 years old) 4 females and 6 males, with SIT modified to 1/10 of the concentration. Control group: 10 patients (15-55 years old), 4 females and 6 males, with respiratory allergy without SIT. Blood was collected in all patients before SIT (T0) and after 6 months of SIT (T1). “In vitro” evaluation was performed using Basotest to evaluate BAFC-double labeled with CD63 FITC/IgE PE and -ELISA-UniCAP, to evaluate sIgE. Results Group I: the mean of % of BA was: T0-74%; T1-6, 3%. The mean of sIgE values was: T0- 38,4 Ku/l; T1-34,64 KU/l. Group II: % of BA: T0-72%; T1-5.8%. sIgE T0-43,08 KU/l; T1-39,24 KU/l. Control group: % of BA: T0- 81%; T1- 78,3%. sIgE: T0-38,90 KU/l; T1-38,64KU/l. Conclusions Results obtained in BAFC in group I and group II showed a significant decrease between T0 and T1 which not occurred in the control group. These changes precede specific IgE variation which is not significant. BAFC could be a useful tool, in a short time, to evaluate the effect of SIT.

Nasal provocation tests with Dermatophagoides pteronyssinus, D. farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae

Rationale The objective of this study was to perform nasal provocation tests with standardized extracts of Dermatophagoides pteronyssinus, D. farinae, Lepidoglyphus destructor and Tyrophagus putrescentiae in a group of patients sensitized to Dermatophagoides spp. Methods A total of 25 patients with allergic rhinitis with or without asthma were entered in the study. All patients had positive skin tests to Dermatophagoides spp. Three groups of patients were studied. Group A consisted of 10 patients, who underwent nasal provocation tests with standardized extracts of D. pteronyssinus and D. farinae (100 HEP/ml). Group B consisted of 10 patients, 9 of whom were skin prick test positive to L. destructor. All 10 patients underwent a nasal provocation test with L. destructor and 5 with D. pteronyssinus. Group C, consisted of 5 patients, 2 of whom were skin test positive to T. putrescentiae. All individuals underwent nasal provocation tests with T. putrescentiae and Dermatophagoides spp. Specific IgE was measured by the CAP system. Results Group A: 7 patients had a positive provocation test with D. pteronyssinus and 8 with D. farinae. Group B: 6 patients had a positive provocation test with L. destructor. Group C: 3 patients had a positive provocation test with T. putrescentiae. Conclusions We confirm a high prevalence of positive nasal challenges to L. destructor and T. putrescentiae in patients sensitized to these mites and to Dermatophagoides spp. The clinical relevance of these mites species is suggested. In general, there was a good correlation between specific IgE levels and positive provocation tests.

Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression.

Neonates are known to have poor cellular immunity, especially poor Th1 response. We investigated how neonatal mononuclear cells raised different Th1/Th2 reactions in response to different antigens. Employing Dermatophagoides pteronyssinus (Der p) extract and varicella zoster virus (VZV) as antigens, we assessed Th1/Th2 reactions as demonstrated by IL-4/IFNgamma production and mRNA expression, and transcriptional factors T-bet/GATA-3 mRNA expression in mononuclear cells from human umbilical cord blood (CBMC). Results showed that VZV induced a dramatic increase of IFNgamma production by adult peripheral blood mononuclear cells (PBMC), whereas VZV did not drive CBMC to release significant IFNgamma production (1614.7+/-362.0 vs. 49.0+/-29.3,p<0.005). However, Der p induced higher IFNgamma production by CBMC than VZV (298.1+/-171.8 vs. 49.0+/-29.3, P=0.047). In contrast, VZV did not induce significant IL-4 production either by CBMC or by PBMC. Der p induced a comparative IL-4 production by CBMC and PBMC (2.58+/-0.84 vs. 2.04+/-0.37, p>0.05). A real-time RT-PCR analysis of IL-4 and IFNgamma mRNA expression showed that VZV induced a significantly higher IFNgamma, but not IL-4, mRNA expression in PBMC than CBMC. Der p did not induce significant difference of IFNgamma or IL-4 mRNA expression in PBMC and CBMC. VZV enhanced Th1-related transcription factor T-bet mRNA expression, in association with later down-regulation of Th2-related GATA-3 mRNA expression in PBMC. However, VZV did not up-regulate T-bet or down-regulate GATA-3 expression significantly in CBMC. In contrast, Der p induced an early GATA-3 expression and later T-bet expression in CBMC. These results suggest that different antigens trigger various Th1/Th2 reactions in PBMC and CBMC resulting from kinetic changes of T-bet/GATA-3 expression.

Skin Tests and Conjunctival and Bronchial Challenges with Extracts of Blomia tropicalis and Dermatophagoides pteronyssinus in Patients with Allergic Asthma and/or Rhinoconjunctivitis

Background:Blomia tropicalis and Dermatophagoides pteronyssinus are important mite species in Tenerife, Canary Islands, Spain. Several studies have demonstrated a variable degree of allergenic cross-reactivity in vitro. However, only a few have addressed their allergenic cross-reactivity using challenge tests. Objective: The objective of this study was to conduct conjunctival and bronchial challenge tests with B. tropicalis and D. pteronyssinus extracts in a group of 42 patients with allergic asthma and/or rhinoconjunctivitis sensitised to house dust mites (31 females, 11 males; mean age 21.7 ± 7.02 years). Methods: Prick tests using standardised extracts and specific IgE determinations using the CAP system were performed. Bronchial and/or conjunctival challenges were conducted using freeze-dried extracts of both mite species. A patient was considered sensitive to a mite species if she/he had a positive prick and/or CAP test result. A total of 32 conjunctival and 15 bronchial challenges were performed with both mite species. Results: Prick tests were positive to B. tropicalis in 23 patients (54.7%) and to D. pteronyssinus in 41 (97.6%). One patient (2.4%) was exclusively sensitive to B. tropicalis. The CAP test was positive for B. tropicalis in 28 patients and for D. pteronyssinus in 41. Conjunctival challenges to B. tropicalis were positive in 20 patients (18 sensitised and 2 non-sensitised) and negative in 12 (5 sensitised and 7 non-sensitised patients). Conjunctival challenges with D. pteronyssinus were positive in all 31 D. pteronyssinus-sensitised patients who underwent conjunctival challenges. Bronchial challenges with B. tropicalis were positive in 9 sensitised patients and negative in 6 patients (2 sensitised and 4 non-sensitised). Bronchial challenges with D. pteronyssinus were positive in all patients except 1, who only reacted to B. tropicalis. Conclusions: Allergens of the mite species B. tropicalis induce positive conjunctival and bronchial challenges in B. tropicalis-sensitised individuals. Our results suggest that although there is a low to moderate degree of in vivo cross-reactivity between B. tropicalis and D. pteronyssinus, B. tropicalis seems to be a relevant source of allergens in areas where patients are exposed.

Comparison of Allergen-Induced Late Inflammatory Reactions in the Nose and in the Skin in House Dust Mite-Allergic Patients with or without Asthma

Background: It remains to be established which factors contribute to the occurrence of asthma in allergic individuals. We hypothesized that differences in the late allergic inflammatory reaction to allergen between asthmatic and non-asthmatic house dust mite-allergic individuals might contribute to the difference in the clinical presentation of allergy. Aim: To compare allergen-induced changes in parameters for cellular inflammation during the phase of the late allergic reaction in the skin and nose, in house dust mite-allergic individuals with or without asthma. Material and Methods: Nasal and dermal allergen challenges with house dust mite (Dermatophagoides pteronyssinus) extract were performed in 52 house dust mite-allergic individuals, of whom 26 had mild to moderate persistent asthma and 26 had perennial rhinitis without current or past asthmatic symptoms. Serial nasal lavage samples were analyzed for the presence of inflammatory cells (eosinophils and neutrophils) and soluble markers associated with cellular inflammation [interleukin-5 (IL-5), interleukin-8 (IL-8), eosinophil cationic protein (ECP) and myeloperoxidase (MPO)]. Macroscopic late phase skin reactions were studied after intracutaneous skin tests with house dust mite extract. Results: Fixed dose nasal allergen provocation elicited a similar degree of immediate allergic reaction as judged by plasma protein exudation and histamine concentrations in asthma and non-asthmatic rhinitis. Subsequently, no differences between groups were found during the phase of the late allergic reaction (4–24 h) in inflammatory cell influx, plasma protein leakage, ECP or MPO. Likewise, there were no differences in levels of chemotactic cytokines IL-5 and IL-8. In agreement with the results of nasal challenge, the late skin reaction after dermal challenge with a fixed allergen dose and after an allergen dose 10,000 times above the skin threshold for an early skin reaction did not differ between the groups. Conclusion: House dust mite-allergic patients with or without asthma have very similar late allergic inflammatory reactions in the skin and in the nose after allergen challenge. Hence, it is unlikely that the occurrence of pulmonary symptoms in asthma is explained by a general tendency of asthmatics to have an enhanced late allergic cellular inflammatory response. Nasal and dermal allergen provocations are adequate models to study allergen-induced inflammation but probably lack the pivotal link which is essential for the development of asthma.

Lack of correlation between bronchial late allergic reaction to Dermatophagoides pteronyssinus and in vitro immunoglobulin E reactivity to histamine-releasing factor derived from mononuclear cells

Activity of immunoglobulin (Ig)E-dependent histamine-releasing factor (HRF) is dependent on the IgE molecules bound to the surface of basophils. Sera capable of passively sensitizing basophils to release histamine to HRF were designated IgE+ sera. IgE+ and HRF have been suggested to play a role in late allergic reaction (LAR). The working hypothesis was tested that IgE+ induces a LAR. Further, activity of HRF produced by mononuclear cells (HRF(mn)) was compared with that of recombinant HRF p23. Atopic patients (n = 82) were bronchially provoked with Dermatophagoides pteronyssinus extract and the change in forced expiratory volume in 1 second was monitored. A LAR was defined as forced expiratory volume in 1 second as percentage of baseline < 80% 4 to 10 hours after allergen challenge. The presence of HRF-responsive IgE in serum was determined using basophils sensitized in vitro by serum. The presence of HRF(mn)-responsive IgE (IgE(mn+)) in serum was shown not be essential for a LAR: 63% of the patients with a LAR had no IgE(mn+) in their serum. Further, 71% of patients with IgE(mn+) did not have a LAR. HRF(mn) and recombinant HRF p23 were not equivalent in the bioassay: serum of 38 of 82 atopic patients sensitized basophils to release histamine to HRF(mn), whereas this was found with serum of 1 of 82 patients to HRF p23. The results do not support the hypothesis that IgE(mn+) induces a LAR, but do not exclude the alternative hypothesis that HRFs are released during a LAR and contribute to asthma severity.

Matrix Metalloproteinase-9, but not Tissue Inhibitor of Matrix Metalloproteinase-1, Increases in the Sputum from Allergic Asthmatic Patients After Allergen Challenge

The aim of the study was to determine whether allergen inhalation modulates the levels of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the induced sputum recovered from patients during a late-phase reaction. Eight allergic asthma patients and five healthy control subjects inhaled a dose of Dermatophagoides pteronyssinus extract corresponding to the provocative concentration of the allergen causing a 20% fall in FEV(1) and saline solution. Lung function was carefully monitored for 6 h, and an induced sputum test was performed at 6 h after sham challenge or allergen challenge. The total and differential cell counts were analyzed, and the levels of MMP-9 (by enzyme-linked immunosorbent assay [ELISA] and zymography), TIMP-1 (by ELISA), and albumin (by rocket immunoelectrophoresis) were measured. The sputum eosinophil counts (p < 0.01) and MMP-9 levels (p < 0.05) increased significantly in atopic asthma patients after undergoing the allergen challenge but did not in the control subjects. By contrast, TIMP-1 and albumin levels were not significantly increased in any group. MMP-9 levels, measured after the allergen challenge in asthmatic patients, were significantly correlated with FEV(1) variations after allergen inhalation (r = 0.51; p < 0.05) and with the sputum neutrophil percentage (r = 0.71; p < 0.01). The levels of MMP-9, but not TIMP-1, increase after inhaled allergen challenge in the sputum of allergic asthmatic patients. This protease increase may lead to a transient imbalance between MMP-9 and TIMP-1 favoring proteolytic extracellular matrix degradation.

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