Serological analysis of allergic components of house dust mite provides more insight in epidemiological characteristics and clinical symptom development in North China.

Background: House dust mite (HDM) is the most common airborne source causing complex allergy symptoms. There are geographic differences in the allergen molecule sensitization profiles. Serological testing with allergen components may provide more clues for diagnosis and clinical management. Objective: This study aims to investigate the sensitization profile of eight HDM allergen components in a large number of patients enrolled in the clinic and to analyze the relation of gender, age, and clinical symptoms in North China. Methods: The 548 serum samples of HDM-allergic patients (ImmunoCAP® d1 or d2 IgE ≥0.35) were collected in Beijing City and divided in four different age groups and three allergic symptoms. The specific IgE of HDM allergenic components, Der p 1/Der f 1, Der p 2/Der f 2, Der p 7, Der p 10, Der p 21, and Der p 23, was measured using the micro-arrayed allergen test kit developed by Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. The new system was validated by comparing to single-component Der p 1, Der p 2, and Der p 23 tests by ImmunoCAP in 39 sera. The epidemiological study of these IgE profiles and the relation to age and clinical phenotypes were analyzed. Results: A greater proportion of male patients was in the younger age groups, while more female patients were in the adult groups. Both the sIgE levels and the positive rates (approximately 60%) against Der p 1/Der f 1 and Der p 2/Der f 2 were higher than for the Der p 7, Der p 10, and Der p 21 components (below 25%). The Der f 1 and Der p 2 positive rates were higher in 2-12-year-old children. The Der p 2 and Der f 2 IgE levels and positive rates were higher in the allergic rhinitis group. The positive rates of Der p 10 increased significantly with age. Der p 21 is relevant in allergic dermatitis symptom, while Der p 23 contributes to asthma development. Conclusion: HDM groups 1 and 2 were the major sensitizing allergens, with group 2 being the most important component relevant to respiratory symptoms in North China. The Der p 10 sensitization tends to increase with age. Der p 21 and Der p 23 might be associated with the development of allergic skin disease and asthma, respectively. Multiple allergen sensitizations increased the risk of allergic asthma.

Heat shock cognate 70 protein like-2 protein in camphor pollen is one of the major culprits of asthma.

AIMS: In recent decades, Cinnamomum camphora have gradually become the main street trees in Shanghai. This study aims to investigate the allergenicity of camphor pollen. MAIN METHODS: A total of 194 serum samples from patients with respiratory allergy were collected and analyzed. Through protein profile identification and bioinformatics analysis, we hypothesized that heat shock cognate protein 2-like protein (HSC70L2) is the major potential allergenic protein in camphor pollen. Recombinant HSC70L2 (rHSC70L2) was expressed and purified, and a mouse model of camphor pollen allergy was established by subcutaneous injection of total camphor pollen protein extract (CPPE) and rHSC70L2. KEY FINDINGS: Specific IgE was found in the serum of 5 patients in response to camphor pollen and three positive bands were identified by Western blotting. Enzyme-linked immunosorbent assay (ELISA), Immune dot blot and Western blot experiments confirmed that CPPE and rHSC70L2 can cause allergies in mice. Moreover, rHSC70L2 induces polarization of peripheral blood CD4+ T cells to Th2 cells in patients with respiratory allergies and mice with camphor pollen allergy. Finally, we predicted the T cell epitope of the HSC70L2 protein, and through the mouse spleen T cell stimulation experiment, we found that the 295EGIDFYSTITRARFE309 peptide induced T cells differentiation to Th2 and macrophages differentiation to the alternatively activated (M2) state. Moreover, 295EGIDFYSTITRARFE309 peptide increased the serum IgE levels in mice. SIGNIFICANCE: The identification of HSC70L2 protein can provide novel diagnostic and therapeutic targets for allergies caused by camphor pollen.

Long-term Prophylaxis with Androgens in the management of Hereditary Angioedema (HAE) in emerging countries.

Hereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterized by repetitive subcutaneous or submucosal angioedema, activation of the kinin system, and increased vascular permeability. C1-inhibitor (C1-INH) deficiency, the main mechanism of HAE pathogenesis, occurs when abnormal activation of plasma kallikrein, bradykinin, and factor XII, or mutation of genes such as SERPING1 cause quantitative or functional C1-INH defects. Although androgens are not approved for HAE treatment in many countries, they are widely used in China and Brazil to reduce the frequency and severity of HAE attacks. The long-term adverse effects of androgen treatment are concerning for both physicians and patients. Virilization, weight gain, acne, hirsutism, liver damage, headache, myalgia, hematuria, menstrual disorders, diminished libido, arterial hypertension, dyslipidemia, and anxiety/depression are commonly observed during long-term treatment with androgens. These adverse effects can affect the quality of life of HAE patients and often lead to treatment interruption, especially in women and children. In-depth studies of the pathogenesis of HAE have led to the approval of alternative treatment strategies, including plasma-derived C1 inhibitor, recombinant human C1 inhibitor, plasma Kallikrein inhibitor (ecallantide; lanadelumab), and bradykinin B2 receptor antagonist (icatibant), some of which have achieved satisfactory results with mostly non-serious side effects. Therefore, a new standard of medical care may expand possibilities for the management of HAE in emerging countries.

Urine protein in patients with type I hypersensitivity is indicative of reversible renal tube injury.

AIMS: In our clinical work, some patients with type I hypersensitivity could be detected protein in their urine. This study focused on the early renal injury in patients with type I hypersensitivity. MAIN METHODS: From 43 type I hypersensitivity patients with proteinuria, 10 patients were randomly selected for mass spectrometry analysis of 24-h urine together with 5 healthy volunteers. Mice were vaccinated with Dermatophagoides farina (Der f) and ovalbumin (OVA) were used as antigen to establish the type I hypersensitivity animal models. KEY FINDINGS: The urine protein of hypersensitivity patients was significantly increased in the alpha-1-microglobulin/ bikunin precursor (Protein AMBP) (t = 3.140, P = 0.008), retinol binding protein 4 (RBP4) (t = 2.426, P = 0.031), kininogen-1 (t = 2.501, P = 0.027), and transferrin appeared only in patients' urine. After immunizing mice with antigens, significant increases of the total serum immunoglobulin E (IgE) were observed in both Der f (86.92 ± 36.01 U/mL, t = 5.231, P = 0.0004) and OVA group (34.65 ± 24.72 U/mL, t = 2.891, P = 0.0161) compared with the negative control group (2.68 ± 0.47 U/mL). Meanwhile, definite eosinophil infiltration around the impaired renal tubules as well as the bronchus in Der f mice were observed, and urine protein appeared. After stopping the allergen stimulation, proteinuria disappeared. Instead, when the mice were treated with the antigen again, proteinuria reappeared. SIGNIFICANCE: Our findings suggest that renal tubular damage in patients with type I hypersensitivity is reversible, and proteinuria disappears with allergy symptoms remission.

Expert consensus on the use of omalizumab in chronic urticaria in China.

Chronic urticaria (CU) is a debilitating skin disease that lasts for more than 6 weeks with wheals and/or angioedema, including chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIndU). In China, the prevalence of this disease is high, more than 1%, and on the rise. CU has a major impact on the quality of life (QoL) of patients who frequently experience sleep disturbance, depression, and anxiety. Nearly one-third of patients with CSU, in China, are resistant to second-generation H1-antihistamines (sgAHs), even at a fourfold dose (second line; off-label). Omalizumab is approved for the treatment of CSU treatment in Europe and shows remarkable efficacy and safety. In China, regulatory approval for the use of omalizumab is pending, and its use in clinical practice varies widely. Consensus on omalizumab CU treatment in China is urgently needed. The aim of this article is to propose a practical omalizumab treatment algorithm for the management of antihistamine-resistant CSU and CIndU in adults and special population including children and adolescents, and pregnant or breast feeding women, to guide daily clinical practice in China. In the development of this consensus, an expert group including mainly dermatologists, allergists, but also pulmonologists, ENTs, immunologists, and pediatricians in Allergic Disease Prevention and Control Committee, Chinese Preventive Medicine Association, reviewed the existing evidence and developed consensus on the use of omalizumab in CU patients from China. The goal of this consensus is to assist clinicians in making rational decisions in the management of refractory CU with omalizumab. The key clinical questions covered by the treatment algorithm are: 1) Omalizumab treatment routine strategy in both CSU and CIndU patients; 2) Recommended dose and treatment duration for different age stratification; 3) Treatment duration for CU patients with other allergic comorbidities; 4) Recommendation on omalizumab stopping strategy.

Association of serum chemokine ligand 21 levels with asthma control in adults.

OBJECTIVES: The chemokine ligand (CCL) 21 regulates the maturation, migration, and function of dendritic cells, and has been implicated in the pathogenesis of asthma. This study aimed to investigate the association between serum CCL21 levels and asthma control. METHODS: The serum levels of CCL21 and other inflammatory cytokines were analyzed in patients with asthma (n=44) and healthy controls (n=35) by enzyme-linked immunosorbent assay. IgE levels and eosinophil counts were determined by turbidimetric inhibition immunoassay and fully automatic blood analysis, respectively. The Asthma Control Test (ACT) questionnaire was used, and spirometry and fractional exhaled nitric oxide (FENO) measurements were performed. A multiple unpaired Student's t-test was performed to analyze the differences in CCL21 and interleukin levels between patients with asthma and healthy controls. The correlation of CCL21 levels with disease severity was evaluated using the Pearson's rank correlation test. RESULTS: Serum CCL21 levels were lower in patients with asthma (254.78±95.66 pg/mL) than in healthy controls (382.95±87.77 pg/mL) (p<0.001). Patients with asthma had significantly higher levels of IL-1ß (19.74±16.77 vs. 2.63±5.22 pg/mL), IL-6 (7.55±8.65 vs. 2.37±2.47 pg/mL), and tumor necrosis factor-α (12.70±12.03 vs. 4.82±3.97 pg/mL) compared with the controls. CCL21 levels were positively correlated with the ACT score (rs=0.1653, p=0.0062), forced expiratory volume in 1s (FEV1)/forced vital capacity (rs=0.3607, p<0.0001), and FEV1 (rs=0.2753, p=0.0003), and negatively correlated with FENO (rs=0.1060, p=0.0310). CCL21 levels were negatively correlated with serum IgE levels (rs=0.1114, p=0.0268) and eosinophil counts (rs=0.3476, p<0.0001). CONCLUSIONS: Serum CCL21 levels may be a new biomarker for assessing asthma control.

China Consensus Document on Allergy Diagnostics.

The prevalence of allergic diseases has increased dramatically in recent years in China, affecting the quality of life in 40% of the population. The identification of allergens is the key to the diagnosis of allergic diseases. Presently, several methods of allergy diagnostics are available in China, but they have not been standardized. Additionally, cross-sensitization and co-sensitization make allergy diagnostics even more complicated. Based on 4 aspects of allergic disease (mechanism, diagnosis procedures, allergen detection in vivo and in vitro as well as the distribution map of the most important airborne allergens in China) and by referring to the consensus of the European Society of Allergy and Clinical Immunology, the World Allergy Organization, and the important literature on allergy diagnostics in China in recent years, we drafted this consensus of allergy diagnostics with Chinese characteristics. It aims to standardize the diagnostic methods of allergens and provides a reference for health care givers. The current document was prepared by a panel of experts from the main stream of professional allergy associations in China.

Association of serum chemokine ligand 21 levels with asthma control in adults

OBJECTIVES: The chemokine ligand (CCL) 21 regulates the maturation, migration, and function of dendritic cells, and has been implicated in the pathogenesis of asthma. This study aimed to investigate the association between serum CCL21 levels and asthma control. METHODS: The serum levels of CCL21 and other inflammatory cytokines were analyzed in patients with asthma (n=44) and healthy controls (n=35) by enzyme-linked immunosorbent assay. IgE levels and eosinophil counts were determined by turbidimetric inhibition immunoassay and fully automatic blood analysis, respectively. The Asthma Control Test (ACT) questionnaire was used, and spirometry and fractional exhaled nitric oxide (FENO) measurements were performed. A multiple unpaired Student's t-test was performed to analyze the differences in CCL21 and interleukin levels between patients with asthma and healthy controls. The correlation of CCL21 levels with disease severity was evaluated using the Pearson's rank correlation test. RESULTS: Serum CCL21 levels were lower in patients with asthma (254.78±95.66 pg/mL) than in healthy controls (382.95±87.77 pg/mL) (p<0.001). Patients with asthma had significantly higher levels of IL-1β (19.74±16.77 vs. 2.63±5.22 pg/mL), IL-6 (7.55±8.65 vs. 2.37±2.47 pg/mL), and tumor necrosis factor-α (12.70±12.03 vs. 4.82±3.97 pg/mL) compared with the controls. CCL21 levels were positively correlated with the ACT score (rs=0.1653, p=0.0062), forced expiratory volume in 1s (FEV1)/forced vital capacity (rs=0.3607, p<0.0001), and FEV1 (rs=0.2753, p=0.0003), and negatively correlated with FENO (rs=0.1060, p=0.0310). CCL21 levels were negatively correlated with serum IgE levels (rs=0.1114, p=0.0268) and eosinophil counts (rs=0.3476, p<0.0001). CONCLUSIONS: Serum CCL21 levels may be a new biomarker for assessing asthma control.

Artemisia Pollen Extracts Exposed to Diesel Exhaust Enhance Airway Inflammation and Immunological Imbalance in Asthmatic Mice Model.

BACKGROUND: Vehicle-induced air pollution may increase the prevalence and severity of asthma. Pollens are important sources of outdoor allergens associated with asthma. Outdoor pollution may influence the structure of pollen grains, resulting in enhanced immune reactions. OBJECTIVE: This study aims to investigate the impact that artemisia pollen extracts exposed to diesel emissions (APEDE) may induce - allergic airway inflammation, pulmonary pathology and immune imbalance - in mice. METHODS: Sixty male Balb/c mice were equally randomized into 5 groups, sensitized with 30 µL artemisia pollen extracts (APE) or APEDE adsorbed on 2 mg aluminum hydroxide gel by intraperitoneal injection on day 0, 7, 14, and 22, and challenged intranasally once per day with 30 µL APE or APEDE from day 29 to 36. The controlling group used phosphate-buffered saline as control. RESULTS: In mice immunized and challenged by APEDE, the clinical phenotype of eosinophils, neutrophils in bronchoalveolar lavage fluid (BALF), tracheal wall thickness, airway smooth muscle thickness and airway resistance increased significantly. Pathophysiological parameters such as interleukin (IL)-17A and tumour necrosis factor-α production in BALF and serum, and the ratio of Th17/Treg cells in CD4+ cells increased significantly, while IL-10 in BALF and serum and the ratio of Treg cells decreased significantly. It was further found that the expression of oxidative stress marker 3-nitrotyrosine (3-NT) and the activation of nuclear factor kappa B (NF-κB) were significantly increased. The correlation analysis showed that the expression of 3-NT was positively correlated with the activation of NF-κB. CONCLUSION: Our findings suggested that pollens exposed to diesel exhaust enhance allergic responses, which may contribute to an increased prevalence of allergic diseases in urban environments with serious exhaust emissions.

Invasive mucinous adenocarcinoma with lepidic-predominant pattern coexisted with tuberculosis: a case report.

We observed a rare case of invasive mucinous adenocarcinoma (IMA) with a lepidic-predominant pattern accompanied by pulmonary tuberculosis. An 85-year-old man with repeated cough and sputum was admitted to Xinhua Hospital. T-SPOT test result was 212 pg/ml (reference value of negative is < 14 pg/ml), Mycobacterium tuberculosis culture was positive, and tuberculin skin test (PPD) was negative (skin induration < 5 mm). The patient was treated with several courses of antibiotics and anti-tuberculosis treatments. Repeated chest CT scans showed disease progression. Bronchoscopy yielded negative results. PET-CT scans showed negative results. A percutaneous lung biopsy revealed mucin-secreting cells lining the alveolar walls. IMA with a lepidic-predominant pattern was diagnosed after invasiveness was found after experimental treatments. Simultaneous occurrence of pulmonary tuberculosis and lung cancer are common; however, the present case of IMA having a lepidic-predominant pattern and coexisting with active tuberculosis has not been reported yet.

Chinese Guideline on allergen immunotherapy for allergic rhinitis.

The present document is based on a consensus reached by a panel of experts from Chinese Society of Allergy (CSA) and Chinese Allergic Rhinitis Collaborative Research Group (C2AR2G). Allergen immunotherapy (AIT), has increasingly been used as a treatment for allergic rhinitis (AR) globally, as it has been shown to provide a long-term effect in improving nasal and ocular symptoms, reducing medication need, and improving quality of life. AIT is currently the only curative intervention that can potentially modify the immune system in individuals suffering from AR and prevent the development of new sensitization and the progression of disease from AR to asthma. Although the use of AIT is becoming more acceptable in China, to date no AR immunotherapy guideline from China is available for use by the international community. This document has thus been produced and covers the main aspects of AIT undertaken in China; including selection of patients for AIT, the allergen extracts available on the Chinese market, schedules and doses of allergen employed in different routes of AIT, assessment of effect and safety, patients' administration and follow-up, and management of adverse reactions. The Chinese guideline for AR immunotherapy will thus serve as a reference point by doctors, healthcare professionals and organizations involved in the AIT of AR in China. Moreover, this guideline will serve as a source of information for the international community on AIT treatment strategies employed in China.

Species and quantity of airborne pollens in Shanghai as monitored by gravitational and volumetric methods.

BACKGROUND: The prevalence of allergic diseases has markedly increased in the last decades. It is therefore important to assess the distribution of airborne pollen, the most important aeroallergen, for allergic disease prevention and control. OBJECTIVE: To identify the species and quantity of airborne pollens, and observe their distribution characteristics in Shanghai, using gravitational (Durham Sampler) and volumetric (Rotorod Sampler 40) methods simultaneously. In addition, the correlation between both methods was analyzed to provide effective preventive measures for pollen-sensitized individuals. METHOD: Pollen counts were monitored in the same area from November 1, 2009 to October 31, 2010 by samplers set at the same height and site. Pollen concentrations as well as any association between the two methods were determined. RESULTS: Two pollen concentration peaks in Shanghai were observed from March to May (spring) and September to October (autumn). In spring, tree pollen was the main species, with a predominance of Broussonetia. In autumn, grass pollen predominated, with mostly Humulus. Thirty-two species were identified by both gravitational and volumetric methods. Five and seven additional species were identified exclusively by the gravitational and volumetric methods, respectively. Pollen counts obtained from both devices were significantly correlated (P<0.05). CONCLUSIONS: Two methods were used simultaneously for the first time to monitor pollen counts in central urban Shanghai, showing two annual peaks. Broussonetia and Humulus were the predominant spring and autumn pollens, respectively. Pollen counts obtained by both methods were clearly correlated. Regional airborne pollen monitoring offers preventive measures for sensitized individuals and provides useful clinical information.

Short-term association between particular matter air pollution and pediatric clinical visits for wheezing in a subarea of Shanghai.

To assess the association between the concentration of ambient particulate matter (PM) and the pediatric clinical visits for wheezing among children under 3 years old, data of daily air pollutants (PM2.5, PM10, NO2, SO2, CO), meteorological reports, along with the number of daily clinical visits of the children with wheezing at the Pediatric Department of Shanghai Renji Hospital (South Campus) were collected from January through December 2014. Correlation between the levels of air pollutants and the number of clinical patients for wheezing were analyzed by a time series analysis with a generalized addictive model (GAM). During the study period, the daily average concentrations of PM2.5, PM10, NO2, SO2, and CO were 51.84 ± 32.51, 72.69 ± 41.15, 43.25 ± 18.07, 17.45 ± 10.42, and 0.82 ± 0.26 µg/m(3), respectively, which were abnormally higher compared to the standard defined by the Chinese Ministry of Environment Protection. The average number of daily clinical patients with wheezing was 23 ± 14 persons/day. The number of clinical visit by children with wheezing was significantly correlated with concentration of PM2.5 or PM10 when the effect of SO2 and NO2 was adjusted (P < 0.05). It was also found that exposure-response relationship was a linear non-threshold mode when it was analyzed by the GAM, and the percent of the clinical visits of children with wheezing increased from 0 to nearly 20 % with every interquartile increase of PM2.5. The visiting number of children at a pediatric outpatient clinic increased due to the increase of PM2.5 in Pujiang, Shanghai, China.

Semi-depot house-dust mite allergen extract for Chinese with allergic rhinitis and asthma.

BACKGROUND: Real-world data of the subcutaneous immunotherapy (SCIT) with semi-depot house-dust mite (HDM) allergen extract (a HDM allergen extract that contains a 50%-50% mixture of Dermatophagoides pteronyssinus and Dermatophagoides farina) for allergic rhinitis and asthma was unavailable in China until recently. AIM: To investigate the effectiveness and safety of a HDM-SCIT for allergic rhinitis and asthma in Chinese patients. METHODS: A multicenter, single-arm, open-label, self-controlled study. Chinese patients with allergic rhinitis or allergic asthma and with a history of symptoms from HDM exposure were included and received allergen-specific immunotherapy for 1 year by subcutaneous injection of HDM-SCIT. The primary outcome measure was the percentage of patients with an improvement in symptom severity assessed at 12 months after initiation of the treatment. The occurrence of adverse events and compliance of treatment were also evaluated. RESULTS: A total of 272 outpatients were included for effectiveness analysis. The subject-evaluated improvement rate in the visual analog scale (VAS) was 76.1% and 71.3% at 6 and 12 months, respectively; corresponding values for investigator-evaluated VAS were 77.9% and 71.7%, respectively (p < 0.0001). Symptom score changes were -2.43 and -3.79 at 6 and 12 months, respectively (both p < 0.0001); the VAS improvement rate and symptom score change did not differ significantly between children and adolescents and/or adults. Good injection schedule adherence was found in 98.8% of the patients. No study drug-related serious adverse events or serious systemic allergic reactions occurred. CONCLUSION: HDM-SCIT was safe and effective in the treatment of allergic rhinitis and asthma in a Chinese population, with good compliance.

Changes in levels of IL-9, IL-17, IFN-γ, dendritic cell numbers and TLR expression in peripheral blood in asthmatic children with Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (MP) infection in children with asthma resulted in a more severe allergic state compared with a non-MP infected group. The infection rate of children with asthma was higher than that of the other groups, suggesting that being asthmatic may be a predisposing factor for MP infection and that the infection itself is an important co-factor in the disease progression of asthma. The number of dendritic cells (DCs) and the expression of TLR2 and TLR4 were compared in 22 asthmatic patients with MP infection, 22 asthmatic patients without MP infection, and 17 normal children as controls. The percentages of DCs in the peripheral blood of the three groups showed significant differences between asthmatic children with MP infection and controls, and asthmatic children without MP and controls (P<0.05), whereas no difference was found between asthmatic children with and without MP infection. The asthmatic children with MP infection group showed increased expression of TLR-2 and TLR-4 on DCs (P<0.01). Asthmatic patients infected with MP showed that DCs and TLRs (TLR-2, TLR-4) might play an important role in asthma pathogenesis with MP infection. The cytokines produced by the T-cell subsets in asthmatic children with MP infection showed a significant increase in IL-9 (P<0.01) and a decrease in IFN-γ (P<0.05) levels post-MP infection, while the IL-17 level remained stable (P>0.05), indicating a shift towards Th1/Th9 in the presence of MP infection.

Foxp3 regulates ratio of Treg and NKT cells in a mouse model of asthma.

Chronic inflammatory disorder of the airways causes asthma. Regulatory T cells (Treg cells) and Natural killer T cells (NKT cells) both play critical roles in the pathogenesis of asthma. Activation of Treg cells requires Foxp3, whereas whether Foxp3 may regulate the ratio of Treg and NKT cells to affect asthma is uncertain. In an ovalbumin (OVA)-induced mouse model of asthma, we either increased Treg cells by lentivirus-mediated forced expression of exogenous Foxp3, or increased NKT cells by stimulation with its activator α-GalCer. We found that the CD4+CD25+ Treg cells increased by forced Foxp3 expression, and decreased by α-GalCer, while the CD3+CD161+ NKT cells decreased by forced Foxp3 expression, and increased by α-GalCer. Moreover, forced Foxp3 expression, but not α-GalCer, significantly alleviated the hallmarks of asthma. Furthermore, forced Foxp3 increased levels of IL_10 and TGFß1, and α-GalCer increased levels of IL_4 and INFγ in the OVA-treated lung. Taken together, our study suggests that Foxp3 may activate Treg cells and suppress NKT cells in asthma. Treg and NKT cells may antagonize the effects of each other in asthma.

The effect of stimuli on basophil-mediated atopic responses during asthmatic lying-in women and in newborns.

Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.

The effect of peptidoglycan stimulation on basophil-mediated atopic responses during pregnancy and in newborns.

BACKGROUND: Toll-like receptor (TLR) molecules play critical roles in directing the course of atopic diseases by recognizing specific microbial products that activate immune effector cell function. OBJECTIVE: We determined if basophils harvested from neonates genetically predisposed to atopic disease had different levels of TLR2 expression and determined whether putative TLR2 ligands mediated cytokine secretion. METHODS: Blood samples were collected from 10 asthmatic and 12 healthy women and their newborns. Basophil histamine was measured using the human basophil degranulation test and TLR2 expression was determined using nucleic acid hybridization in situ and flow cytometry. IL-4 levels were quantified by ELISA following allergen stimulation. RESULTS: The basophil degranulation index (DI) in granulocytes harvested from peripheral blood of asthmatic women was assessed following stimulation with either peptidoglycan (PGN) or Dermatophagoides farinae (Df) extract. The DI was significantly higher in atopic women than in healthy controls. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared with basophils purified from children born to nonatopic controls. Finally, TLR2 expression at the protein and mRNA levels was upregulated in cord blood basophils from neonates born to mothers with asthma following stimulation with PGN but not Df. CONCLUSION: These data suggested that TLR2-mediated innate immune responses play a role in augmenting allergic reactions through the modulation of basophil cytokine secretion and histamine release. Microbial components may activate basophils through TLR2 (especially for genetically predisposed infants) to release cytokines associated with an increased incidence of allergic diseases.

Method for umbilical cord blood-derived basophils by FCM.

We have devised a method for the purification of human basophils from umbilical cord blood by FCM. Umbilical cord blood was collected from six healthy, full-term deliveries. After separation of red blood cells, mononuclear cells were isolated by Ficoll-Hypaque. Six samples followed by positive selection using flowcytometry (FCM) for CD203c(+)CD45(int+) cells. Purity and recovery of cells were measured. Purity and recovery of basophils by FCM with CD203c(+)CD45(int+) markers were 95.02 +/- 2.94% and 61.42 +/- 5.95%. Cell sorting for CD203c(+)CD45(int+) cells by FCM is an improved method for obtaining pure umbilical cord blood-derived basophils. We established cord blood-derived basophil purification technique as a source from which active basophils can be isolated for immunochemical characterization.