Mite-sensitive Asthmatic Patients Research Articles

IFN-γ stimulation of dental follicle mesenchymal stem cells modulates immune response of CD4+ T lymphocytes in Der p1+ asthmatic patients in vitro

BackgroundHouse dust mite (Dermataphagoides pteronyssinus) is a widespread risk factor in the development of asthma. CD4+ T lymphocytes have an important role in the pathogenesis of allergic asthma by polarizing to Th2 cells. ObjectiveWe aimed to evaluate the immunoregulatory effects of dental follicle mesenchymal stem cells with and without IFN-γ stimulation on peripheral blood mononuclear cells of house dust mite sensitive asthmatic patients, and compared those with Dexamethasone as a systemic steroid. Material and methodsPBMC of asthmatic patients and healthy individuals separately cultured with or without DF-MSCs in the presence and absence of IFN-γ or Der p1 or Dexamethasone for 72h. CD4+ T proliferation, cell viability, CD4+CD25+FoxP3+ Treg cell frequency and cytokine profiles of PBMC were evaluated via flow cytometry. ResultsDF-MSCs suppressed proliferation of CD4+ T lymphocytes (pCDmix<0.01, pDerp1<0.01, pIFN<0.005) by increasing the number of FoxP3 expressing CD4+CD25+ T regulatory cells (pCDmix<0.005, pDerp1<0.01, pIFN<0.001) and suppressed lymphocyte apoptosis (pCDmix<0.05, pDerp1<0.05, pIFN<0.05), while Dexamethasone increased the apoptosis and decreased Treg cell frequency in asthmatic patients. IFN-γ stimulation increased the suppressive effect of DF-MSCs and also enhanced the frequency of FoxP3 expressing CD4+CD25+ T regulatory cells. The cytokine levels were regulated by DF-MSCs by reducing IL-4 cytokine levels (pCDmix<0.01, pDerp1<0.05, pIFN<0.05) and upregulating IFN-γ levels (pCDmix<0.01, pDerp1<0.05, pIFN<0.005) in asthmatic patients. ConclusionIFN-γ stimulated DF-MSCs were found to have a high modulatory effect on CD4+ T cell responses, while Dexamethasone had an apoptotic effect on CD4+ T cells in asthmatic patients. DF-MSCs may be a new cell-based therapy option for allergic diseases including asthma.

TLR2 Agonists Enhance CD8+Foxp3+ Regulatory T Cells and Suppress Th2 Immune Responses during Allergen Immunotherapy

Pam3CSK4, a synthetic TLR2 ligand, has been shown to expand CD4+ regulatory T cells (Treg cells). Less is known about the function of CD8+ Treg cells than about the function of CD4+ Treg cells generated during allergen-specific immunotherapy (IT). This study investigated whether Dermatophagoides pteronyssinus-specific IT could expand the CD8+CD25+Foxp3+ Treg population and whether Pam3CSK4 could enhance the Treg population. PBMCs were isolated from healthy control subjects and from mite-sensitive asthmatic patients during IT at three specific times: before IT and 6 mo and 1 y after the maximum-tolerated dose. This study was performed without a placebo-controlled group. D. pteronyssinus-specific IT induced a significant increase in CD8+Foxp3+ Treg cells expressing intracellular IL-10 and granzyme B. Costimulation of PBMCs with Pam3CSK4 and D. pteronyssinus 2 expanded the CD8+CD25+Foxp3+ Treg population and inhibited D. pteronyssinus 2-induced IL-4 production. Pam3CSK4-treated CD8+CD25+ Treg cells directly suppressed CD4+ T cell proliferation by cell-contact inhibition. TUNEL revealed that CD8+CD25+ Treg cells, but not CD4+CD25+ Treg cells, directly induced CD4+CD45ROhi+ apoptosis. Our results provide direct evidence that Pam3CSK4 induces an immunomodulatory effect by inducing CD8+ Treg cells; therefore, it may be a good adjuvant for the treatment of mite allergies.

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.

Protective effects of a recombinant fragment of human surfactant protein D in a murine model of pulmonary hypersensitivity induced by dust mite allergens

Lung surfactant protein D (SP-D) is a carbohydrate pattern recognition immune molecule. It can interact with a range of pathogens, stimulate immune cells and manipulate cytokine profiles during host's immune response. SP-D has also been shown to interact, via its carbohydrate recognition domains, with glycoprotein allergens of house dust mite ( Dermatophagoides pteronyssinus, Derp), inhibiting specific IgE isolated from mite-sensitive asthmatic patients from binding these allergens, and blocking subsequent histamine release from sensitized basophils. In the present study, we have examined the protection offered by various doses of intranasal administration of a recombinant fragment of human SP-D (rhSP-D) in a murine model of pulmonary hypersensitivity to Derp allergens which showed characteristic high levels of specific IgE antibodies, peripheral blood eosinophilia, pulmonary infiltrates and a Th2 cytokine response. Treatment of Derp mice with rhSP-D led to significant reduction in Derp-specific IgE levels, blood eosinophilia and pulmonary cellular infiltration. The levels of IL-4 and IL-5 were decreased, while those of IL-12 and IFN-γ were raised in the supernatant of the cultured splenocytes, indicating a Th2 to Th1 polarization. These results suggest that SP-D has a protective role in the modulation of allergic sensitization and in the development of allergic reactions to Derp allergens and highlight potential of the rhSP-D as a therapeutic for pulmonary hypersensitivity.

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae.

The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. The study indicated that 98-kDa mite paramyosin is an important allergen.

Seasonal variation of IgG subclass antibodies to house dust mite in sera from mite-sensitive asthmatic patients.

House dust mite is an important cause of bronchial asthma. Seasonal variation of environmental house dust mite allergen levels and the specific IgE antibody to house dust mite have been reported. We studied the changes in IgG subclass antibodies to house dust mite associated with seasonal variation of house dust mite allergen levels in houses of mite-sensitive asthmatic patients. In 14 mite-sensitive asthmatic patients, house dust mite allergen (Der f 1) contents in bedding were measured monthly, and IgG subclass antibodies to house dust mite, Dermatophagoides farinae (D. farinae), were determined by enzyme-linked immunosorbent assay (ELISA) every 3 months from July to December. The concentration of Der f 1 in dust from bedding reached maximum levels in August and September, and significantly decreased in November and December compared with August and September (P < .05). Levels of D. farinae-specific IgG4 antibodies significantly decreased in December compared with September (P < .05) with no statistically significant change between September and June (P > .05). Levels of D. farinae-specific IgG2 antibodies decreased significantly in December compared with June (P < .05). The levels of IgG1 and IgG3 antibodies to D. farinae showed no significant differences during the study period. These findings suggest that seasonal changes in natural exposure to house dust mite allergen might lead to concurrent changes in specific IgG4 antibodies to house dust mite in mite-sensitive asthmatic patients and each IgG subclass antibodies to house dust mite might have a different kinetics.

Domestic allergens in public places III: house dust mite, cat, dog and cockroach allergens in British hospitals.

Exposure and sensitization to indoor allergens is a major cause of asthma. This study investigated the levels of house dust mite, cat, dog and cockroach allergens in the dust and air in hospitals and the effects of regular vacuum cleaning on allergen levels in hospital chairs. Der p 1, Fel d 1, Can f 1 and Bla g 2 were measured in the dust collected by vacuuming upholstered chairs and a 1 m2 area of carpet and mattress in 14 hospitals. Air samples were collected using an air sampler (flow rate 60 L/min) on 10 separate days for 4 h in the outpatient department in one of the hospitals during busy clinics when patients were waiting for their appointments. In addition, dust samples were collected on four occasions, at 4-weekly intervals, from 36 fabric covered chairs in the outpatient area of a busy chest clinic by vacuuming each chair for 2 min. During the intervening weeks, 18 of the chairs (active group) were each cleaned by vacuuming for 1 min, three times per week. Der p 1, Fel d 1, Can f 1 and Bla g 2 were assayed using monoclonal antibody-based ELISA. In total, 83 carpets, 69 mattresses and 42 upholstered chairs were sampled. The levels of dust mite allergen Der p 1 and cockroach allergen Bla g 2 found in the hospital setting were low. High levels of Fel d 1 (GM 22.9 microg/g, range 4.5-58) and Can f 1 (GM 21.6 microg/g, range 4-63) were found in upholstered chairs. Airborne Can f 1 was detected on every occasion (range 0.12-0.56 ng/m3), whilst detectable airborne Fel d 1 was found on 7 out of the 10 sampling days (range 0.09-0.22 ng/m3). Der p 1 and Bla g 2 were below the detection limit in all airborne samples. Following repeated vacuuming the mean cat and dog allergen levels decreased significantly (P<0.001) and were almost fivefold lower in the vacuumed chairs compared with the control group. Low levels of mite allergen are unlikely to be of any clinical significance to mite-sensitive asthmatic patients. However, upholstered chairs in hospitals constitute a significant reservoir of cat and dog allergen. Inhalation of airborne allergen in patients attending their hospital appointment may exacerbate asthma in those highly allergic to cats or dogs. These results question the wisdom of introducing soft furnishings and carpets into hospitals. Three-times weekly vacuuming significantly reduces allergen levels in upholstered chairs.

Seasonal variation of skin reactivity and specific IgE antibody to house dust mite.

House dust mite is an important cause of bronchial asthma worldwide. While the allergen is present all year-round, a seasonal variation of house dust mite allergen levels has been found. There have been few reports, however, on seasonal variation of specific immune response to house dust mite. We studied the changes in skin reactivity and specific IgE antibody to house dust mite associated with seasonal variation of house dust mite allergen levels in houses of mite-sensitive asthmatic patients. In 14 mite-sensitive asthmatic patients, house dust mite allergen (Der f 1) contents in bedding were measured monthly. Skin reactivity on prick test and specific IgE antibody to house dust mite, Dermatophagoides farinae (D. farinae), were determined every 3 months from july to December. The concentration of Der f 1 in dust from bedding reached maximum levels in August and September, and significantly decreased in November and December compared with August and September (P < .05). Skin reactivity (a ratio of mean wheal diameter of allergen to histamine) to D. farinae decreased significantly in December compared with September (P < .05). Serum levels of total IgE and specific IgE antibody to D. farinae decreased significantly in December compared with September (P < .05). These findings suggest that seasonal changes in natural exposure to house dust mite allergen might lead to concurrent changes in skin reactivity and specific IgE antibody to house dust mite in mite-sensitive asthmatic patients.

Seasonal variation in bronchial hyperreactivity (BHR) in allergic patients.

As summarized in Table 1, the literature consistently supports the hypothesis that allergic asthmatic patients have seasonal BHR changes that parallel allergen exposure. These seasonal changes appear to be preventable by treatment with corticosteroids (systemic, inhaled, or nasal), disodium cromoglycate, and immunotherapy. Studies have almost exclusively focused on pollens, though similar limited data exist for dust mites. Though the dust mite is a perennial allergen, mite levels are well known to fluctuate with seasonal temperature and humidity trends (44-46), and therefore, seasonal BHR variation in mite-sensitive asthmatic patients is not surprising. Allergenic mold species have not been studied in this regard. In allergic rhinitis patients, the data are less consistent (see Table 2). However, the studies that failed to identify a seasonal BHR difference were either small or had other design limitations. The seasonal changes identified by the larger analyses were similar to those identified for asthmatic patients. Thus, although confirmatory studies would be helpful, it seems likely that in the absence of clinical asthma, allergic rhinitis patients with baseline BHR have allergen-related seasonal changes in BHR. The BHR effects of seasonal changes in air pollution and viral URIs are not known, since they have not yet been directly studied. However, interesting recent reports have identified possible synergistic effects of air pollution exposure on BHR and allergic responses. Similarly, the availability of new viral identification techniques has resulted in the discovery that viral infection may be more prevalent during clinical asthma exacerbation than previously realized. Therefore, air pollution and viral infections may well influence BHR seasonally, and (along with allergens) may contribute to seasonal asthma morbidity and mortality peaks. The mechanism(s) underlying seasonal BHR changes is (are) not known. One plausible possibility with regard to allergen-driven BHR changes involves a type I hypersensitivity late-phase reaction. Characterized by recruitment of eosinophils, lymphocytes, and other cells that are central components of allergic inflammation and are not normally found in the lower airways, this reversible inflammatory process could in turn act, presumably via chemical mediators, on the airway smooth muscle. This may cause bronchoconstriction, but may also increase responsiveness to bronchoconstrictive stimuli independent of bronchoconstriction. This explanation for seasonal BHR changes is supported by findings of blood eosinophil (31,47) and BAL eosinophilic cationic protein (31) level changes that parallel BHR. Prevention of seasonal BHR changes using anti-inflammatory medications (32,33,35) also supports this hypothesis (30) however, and the complex potential interactions between infectious agents and air pollutants on seasonal BHR changes have yet to be studied directly. Therefore, although BHR indeed may predictably vary season to season in allergic individuals, additional investigation is needed to better characterize the reasons for this phenomenon. Further insight in this area may help address the reasons why there are often seasonal epidemics in asthma morbidity and mortality.

Purification and IgE binding capacity of Der s 3, a major allergen from Dermatophagoides siboney

Sensitization to the house dust mite Dermatophagoides siboney has been demonstrated in asthmatic patients. Previously, Dermatophagoides siboney group 1 and group 2 allergens, named Der s 1 and Der s 2, respectively, have been purified. The aim of this study was to purify and to study the IgE reactivity of 30 kDa component, suspected to correspond to group 3 allergens. The protein was purified by affinity chromatography using anti-Der f 3 monoclonal antibodies and semi-preparative SDS-PAGE. The IgE binding capacity of the purified fractions was tested with sera from 106 mite-sensitive asthmatic patients using a modified chemiluminiscent method. Affinity chromatography resulted in fractions containing the 30 kDa component which was further purified to homogeneity by SDS-PAGE. Seventy-three per cent of the sera showed IgE reactivity to this protein, indicating that it is a major allergen. The protein also reacted with anti Der f 3 polyclonal antibodies and had tryptic activity. There were differences in the reactivity to Der s 3 according to the age of the patients. Based on the frequency of IgE reactions and the reactivity with antibodies directed to Der f 3, it is proposed to name this 30 kDa allergen from D. siboney, Der s 3.

MHC class II restriction for T cell proliferative response to mite antigen.

We investigated the immunoreguratory role of the major histocompatibility complex in peripheral blood lymphocytes' proliferative response to mite antigen. Peripheral blood lymphocytes of Japanese asthmatic patients were incubated with antigen obtained from Dermatophagoides pteronyssinus with a molecular weight of about 15,000, with and without 0.05 microgram/mL of monoclonal antibody against HLA class I or class II for seven days at 37 degrees C humidified in 5% CO2 and 95% air. High and low responders to the mite body antigen were found among the patients while there were no high responders among the healthy individuals tested. In the mite-sensitive asthmatic patients, CD4+ T cells were the population that responded to the antigen. Depletion of CD8+ T cells from the peripheral blood lymphocytes of mite-insensitive individuals caused high responsiveness to the antigen, indicative that the mite-specific CD4+ T cells controlled the high responsiveness and the antigen-specific CD8+ T cells, the low responsiveness. Anti-HLA-DR monoclonal antibody inhibited responsiveness. In contrast, anti-HLA-DQ monoclonal antibody produced high responsiveness in low responders to mite antigen. High T cell proliferative responsiveness to mite antigen was restricted by HLA-DR antigen through CD4+ T cells in high responders, whereas HLA-DQ antigen is a restriction antigen of low responsiveness through CD8+ T cells in low responders and non-atopic individuals.

Clinical efficacy of reduction in house‐dust mite exposure in specially designed, mechanically ventilated “healthy” homes

In temperate climates, energy-conserving measures may increase indoor humidity, enhancing house-dust mite (HDM) growth. Movement of families to "healthy" homes with mechanical ventilation systems reduced HDM exposure. The effect on asthma control of moving to the "healthy" homes was studied in 14 asthmatic patients allergic to HDM. Base-line evaluations of lung function, asthma symptoms, and medication requirements were made before moving and again after 5 and 15 months' residence. A control group of 11 mite-sensitive asthmatic patients who did not move were examined contemporaneously with the study group at base line and at the first follow-up investigation. After 5 months, the residents of the "healthy" homes improved in forced expiratory volume in 1 s (FEV1), medicine score, and serum IgE. These changes were significantly different from control group measurements. After 15 months, statistically significant improvements from base line were found in FEV1, average daily peak expiratory flow values, medicine score, symptom score, and serum IgE. Insignificant trends toward improvement were seen in provocation concentration of histamine and blood eosinophils. A significant relation was found between reduction in medicine score and fall in HDM exposure. The present study shows that a specific HDM-avoidance procedure can result in an overall, clinical improvement in HDM-sensitive asthmatic patients.

Augmentation of Leukotriene C4 Production by Gamma Interferon in Leukocytes Challenged with an Allergen

Leukocytes from mite-sensitive asthmatic patients were challenged with an allergen and the supernatant assayed for histamine, immunoreactive leukotriene C4 (i-LTC4), and gamma-interferon (gamma-IFN). In addition, the concentration of gamma-IFN in the plasma of patients with bronchial asthma was assayed. Significantly higher concentrations (p less than 0.01) of gamma-IFN were detected in patients' plasma (0.27 +/- 0.32 U/ml, n = 61) than in that of healthy controls (0.04 +/- 0.06 U/ml, n = 19). The leukocytes produced 7.3 +/- 6.7 ng of i-LTC4/10(6) basophils (n = 32), and histamine release was 41.1 +/- 37.1% of total histamine. There was a significant correlation (p less than 0.01) between the capacity of leukocytes to release i-LTC4 and gamma-IFN. The capacity of leukocytes to release histamine and to produce gamma-IFN was not significantly correlated (r = 0.263). About twice as much i-LTC4 was generated from leukocytes pretreated for 24 h with 0.1-1.0 U/ml of recombinant human gamma-IFN, but histamine release was not changed. Another type of IFN, alpha-IFN, did not alter the capacity of leukocytes to release histamine as well as gamma-IFN upon preincubation for 24 h at 1-1,000 U/ml. Pretreatment with 1-10 U/ml of beta-IFN slightly enhanced the capacity upon challenge with 10 ng/ml of mite allergen. The enhancing effect of gamma-IFN on i-LTC4 generation was decreased by treatment with H-7, a nonspecific inhibitor of protein kinase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergen induced histamine release and immunoreactive-leukotriene C 4 generation from leukocytes in mite sensitive asthmatic patients

Leukocytes from mite sensitive asthmatic patient were challenged with the allergen and the supernatant was assayed for histamine and immunoreactive-leukotriene C 4 (i-LTC 4). The release of histamine was quantitated by an automated fluorometric technique and i-LTC 4 was determined using a commercial radioimmunoassay kit. The results of analysis of the supernatant by high speed liquid chromatography, together with observations of modulation of the formation by agents, indicated that i-LTC 4 consisted of LTC 4 with a little amount of LTD 4. i-LTC 4 was generated as a result of basophil activation but not derived from the other cells such as monocytes and eosinophils. Allergen induced a concentration-dependent release of histamine and i-LTC 4 and the maximal release of histamine and i-LTC 4 occurred at the same dose of the allergen. At optimal concentration of the allergen, basophils produced 20.4 ± 17.9 ng of i-LTC 4/10 6-cells (mean ± S.D., n=39) and histamine release was 55.6 ± 20.1% of total histamine. There was a significant correlation in the capacity of leukocytes to release histamine and i-LTC 4 (r=0.47, p<0.01). We found a correlation between maximal histamine release or cell sensitivity, allergen concentration for 50% histamine release, and a ratio of specific IgE to mite to total IgE in the serum, but the amount of i-LTC 4 failed to correlate significantly with the ratio. The releasability and the cell sensitivity of asthmatic patients' cells to the allergen for histamine release paralleled the severity to symptoms, but this correlation was not significant in i-LTC 4 generation.

Lymphocyte responsiveness to Dermatophagoides farinae extract in mite-sensitive patients: effect of immunotherapy on cellular proliferative response and specific immunoglobulin E antibody (RAST score)

Lymphocyte responsiveness in vitro and serum levels of specific IgE antibody to mite extracts ( Dermatophagoides farinae) were evaluated in mite-sensitive asthmatic patients and in nonatopic individuals. Lymphocytes from untreated mite-sensitive patients (n = 16) responded to mite extracts with greater 3H-thymidine uptake than those from nonatopic individuals (n = 20; p < 0.01). Specific IgE antibody and total IgE levels were significantly higher in mite-sensitive patients than those in nonatopic individuals. These results suggest that mite-sensitive patients respond to mite allergen with exaggerated lymphocyte proliferation as well as with enhanced specific IgE antibody production. Lymphocyte response to mite extracts was significantly lower in the patients with long-term dust immunotherapy than that in the untreated patients (p < 0.05), but there was no significant difference in the total IgE and the specific IgE antibody levels between them. Lymphocyte response to Candida albicans was not significantly affected in these patients. These results suggest that as far as mite allergy is concerned, there is some difference in the effect of immunotherapy between lymphocyte proliferative response and specific IgE antibody to mite allergen.