An-Soo Jang

ADAM33 polymorphism : association with bronchial hyper-responsiveness in Korean asthmatics

Background A disintegrin and metalloprotease 33 (ADAM33) is expressed in the lung by fibroblasts and bronchial smooth muscle cells. Given its structure and cellular provenance, ADAM33 may be associated with airway remodelling and bronchial hyper‐responsiveness. Single nucleotide polymorphisms (SNPs) and haplotypes of the ADAM33 gene have previously been associated with asthma susceptibility in the Caucasian population.

Identification of asthma clusters in two independent Korean adult asthma cohorts

Asthma is a heterogeneous airway disease with various clinical phenotypes. It is crucial to clearly identify clinical phenotypes to achieve better asthma management. We used cluster analysis to classify the clinical groups of 724 asthmatic patients from the Cohort for Reality and Evolution of Adult Asthma in Korea (COREA), and in 1843 subjects from another independent Korean asthma cohort of Soonchunhyang University Asthma Genome Research Centre (SCH) (Bucheon, Republic of Korea). Hierarchical cluster analysis was performed by Wards method, followed by &kgr;-means cluster analysis. Cluster analysis of the COREA cohort indicated four asthma subtypes: 1) smoking asthma; 2) severe obstructive asthma; 3) early-onset atopic asthma; and 4) late-onset mild asthma. An independent cluster analysis of the SCH cohort also indicated four clusters that were similar to the COREA clusters. Our results indicate that adult Korean asthma patients can be classified into four distinct clusters.

The interactive effect of Ras, HER2, P53 and Bcl-2 expression in predicting the survival of non-small cell lung cancer patients

In patients with non-small cell lung cancer (NSCLC), tumor expression of P21-Ras, HER2, P53, and Bcl-2 has been reported as independent predictors of prognosis. However, the prognostic information carried by these proteins has usually been determined separately, and their potential interaction has not been taken into account. We conducted immunostaining for P21-Ras, HER2, P53 and Bcl-2 on 238 cases of NSCLC in a Korean population with 203 squamous cell carcinomas, and 35 adenocarcinomas. P21-Ras, HER2, P53 or Bcl-2 was expressed at high levels in 54.6, 42.0, 18.1 and 71.8% of the NSCLC studied, respectively. A total of 59 tumors (24.8%) expressed only one protein, while 70 (29.4%) expressed two, 59 (24.8) expressed three, and 17 tumors (7.1%) expressed all four proteins. Univariate analysis testing the association of marker expression with survival found Bcl-2 expression to be significantly associated with a poor prognosis, as well as the co-expression of Bcl-2 + HER2, Bcl-2 + HER2 + P53, and Bcl-2 + HER2 + P53 + P21-ras with an increasing hazard ratio. By multivariate analysis controlling for age, tumor stage and tumor type, only the combination of Bcl-2 + HER2 expression was an independent marker of poor prognosis (hazard ratio = 1.91, P = 0.003). Thus, a prospective analysis of the co-expression of Bcl-2 + HER2 in NSCLC patients may identify patients with a poor prognosis who may benefit from more aggressive therapy.

Modulation of cytokine and nitric oxide by mesenchymal stem cell transfer in lung injury/fibrosis

BackgroundNo effective treatment for acute lung injury and fibrosis currently exists. Aim of this study was to investigate the time-dependent effect of bone marrow-derived mesenchymal stem cells (BMDMSCs) on bleomycin (BLM)-induced acute lung injury and fibrosis and nitric oxide metabolites and inflammatory cytokine production.MethodsBMDMSCs were transferred 4 days after BLM inhalation. Wet/dry ratio, bronchoalveolar lavage cell profiles, histologic changes and deposition of collagen were analyzed.ResultsNitrite, nitrate and cytokines were measured weekly through day 28. At day 7, the wet/dry ratio, neutrophilic inflammation, and amount of collagen were elevated in BLM-treated rats compared to sham rats (p = 0.05-0.002). Levels nitrite, nitrate, IL-1β, IL-6, TNF-α, TGF-β and VEGF were also higher at day 7 (p < 0.05). Degree of lymphocyte and macrophage infiltration increased steadily over time. BMDMSC transfer significantly reduced the BLM-induced increase in wet/dry ratio, degree of neutrophilic infiltration, collagen deposition, and levels of the cytokines, nitrite, and nitrate to those in sham-treated rats (p < 0.05). Fluorescence in situ hybridization localized the engrafted cells to areas of lung injury.ConclusionSystemic transfer of BMDMSCs effectively reduced the BLM-induced lung injury and fibrosis through the down-regulation of nitric oxide metabolites, and proinflammatory and angiogenic cytokines.

Mesenchymal Stem Cell Transfer Suppresses Airway Remodeling in a Toluene Diisocyanate-Induced Murine Asthma Model

Purpose Severe asthma is characterized by high medication requirements to maintain good disease control or by persistent symptoms despite high medication use. The transfer of bone marrow-derived mesenchymal stem cells (BMDMSCs) to the injured lungs is a possible treatment for severe asthma. This study investigated the therapeutic effects of BMDMSCs in airway remodeling and inflammation in an experimental toluene diisocyanate (TDI)-induced asthma animal model of severe asthma. Methods BMDMSCs were transferred into rats after TDI inhalation. Bronchoalveolar lavage (BAL) cell profiles, histological changes including an inflammatory index and goblet cell hyperplasia, and the airway response to methacholine using plethysmography were analyzed. Smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA) protein expression were observed in lung tissue using immunohistochemical staining. The collagen content was measured in lung tissue sections and lung extracts using Massons trichrome staining and an immunoassay kit. Results The numbers of inflammatory cells in BAL fluid, histological inflammatory index, airway response to methacholine, number of goblet cells, and amount of collagen were increased in TDI-treated rats compared with sham rats (P=0.05-0.002). BMDMSC transfer significantly reduced the TDI-induced increase in the inflammatory index and numbers of eosinophils and neutrophils in BAL fluid to levels seen in sham-treated rats (P<0.05). BMDMSC transfer significantly reduced the number of goblet cells, collagen deposition, and immune staining for SMA and PCNA with concomitant normalization of the airway response to methacholine. Conclusions The systemic transfer of BMDMSCs effectively reduced experimental TDI-induced airway inflammation and remodeling and airway hyperreactivity.

Association of IL-17RB Gene Polymorphism With Asthma

BACKGROUND Interleukin (IL)-17E is a member of the IL-17 family, which induces IL-4, IL-5, IL-13, and eotaxin in experimental animals via IL-17 receptor B (IL-17RB). The activation of IL-17RB amplifies allergic-type inflammatory responses by inducing Jun kinase (or JNK), p38 mitogen-activated protein kinase (or MAPK), and nuclear factor-kappaB. OBJECTIVES We examined the association of polymorphisms in the IL-17RB gene with asthma susceptibility and investigated the effects of those polymorphisms on the transcription of various IL-17RB isoforms. METHODS In total, 954 asthmatic patients or 265 healthy control subjects were screened for polymorphisms in IL-17RB by single-base extension. The messenger RNA expression IL-17RB in B-cell lines derived from patients was also measured by reverse transcription-polymerase chain reaction. RESULTS Direct sequencing of 24 unrelated Korean DNA samples revealed 18 genetic variants, including four insertion/deletions and 14 single-nucleotide polymorphisms (SNPs). Six of the SNPs (-1465G>A, +5661G>A, +6297T>C [Y123Y], +13797C>T, +18661C>T, and +18965G>A) were used to screen a larger group of subjects. Intronic polymorphism +5661G>A was significantly associated with the development of asthma (p = 0.001); moreover, a minor allele of IL-17RB +5661G>A appeared at a lower frequency in the asthmatic patients than in the healthy control subjects (0.13 vs 0.19, respectively). The IL-17RB messenger RNA expression in B cells homozygous for IL-17RB+ 5661GG was significantly higher than that in B cells homozygous for IL-17RB+5661AA (p = 0.002). CONCLUSIONS A rare allele of IL-17RB +5661G>A may have a protective role against the development of asthma via regulation at the level of transcription. The SNPs identified in this study may be used to develop markers to assess the risk of asthma.

A promoter SNP rs4073T>A in the common allele of the interleukin 8 gene is associated with the development of idiopathic pulmonary fibrosis via the IL-8 protein enhancing mode

BackgroundInterleukin-8 (IL-8) is a potent chemo-attractant cytokine responsible for neutrophil infiltration in lungs with idiopathic pulmonary fibrosis (IPF). The IL-8 protein and mRNA expression are increased in the lung with IPF. We evaluated the effect of single nucleotide polymorphisms (SNPs) of the IL-8 gene on the risk of IPF.MethodsOne promoter (rs4073T>A) and two intronic SNPs (rs2227307T>G and rs2227306C>T) of the IL-8 genes were genotyped in 237 subjects with IPF and 456 normal controls. Logistic regression analysis was applied to evaluate the association of these SNPs with IPF. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay, and promoter activity was assessed using the luciferase reporter assay.ResultsThe minor allele frequencies of rs4073T>A and rs2227307T>G were significantly lower in the 162 subjects with surgical biopsy-proven IPF and 75 subjects with clinical IPF compared with normal controls in the recessive model (OR = 0.46 and 0.48, p = 0.006 and 0.007, respectively). The IL-8 protein concentration in BAL fluids significantly increased in 24 subjects with IPF compared with 14 controls (p = 0.009). Nine IPF subjects homozygous for the rs4073 T>A common allele exhibited higher levels of the IL-8 protein compared with six subjects homozygous for the minor allele (p = 0.024). The luciferase activity of the rs4073T>A common allele was significantly higher than that of the rs4073T>A minor allele (p = 0.002).ConclusionThe common allele of a promoter SNP, rs4073T>A, may increase susceptibility to the development of IPF via up-regulation of IL-8.

Proteomic Identification of Macrophage Migration-inhibitory Factor upon Exposure to TiO2 Particles

Inhalation of particulate matter aggravates respiratory symptoms in patients with chronic airway diseases, but the mechanisms underlying this response remain poorly understood. We used a proteomics approach to examine this phenomenon. Treatment of epithelial cells with BSA-coated titanium dioxide (TiO2) particles altered 20 protein spots on the two-dimensional gel, and these were then analyzed by nano-LC-MS/MS. These proteins included defense-related, cell-activating, and cytoskeletal proteins implicated in the response to oxidative stress. The proteins were classified into four groups according to the time course of their expression patterns. For validation, RT-PCR was performed on extracts of in vitro TiO2-treated cells, and lung issues from TiO2-treated rats were analyzed by immunohistochemical staining and enzyme immunoassay. TiO2 treatment was found to increase the amount of mRNA for macrophage migration-inhibitory factor (MIF). MIF was expressed primarily in epithelium and was elevated in lung tissues and bronchoalveolar lavage fluids of TiO2-treated rats as compared with sham-treated rats. Carbon black and diesel exhaust particles also induced expression of MIF protein in the epithelial cells.

Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity.

Objective Aspirin affects interleukin-4 (IL-4) synthesis; however, the genetic role of IL-4 has not been evaluated in asthmatics with aspirin hypersensitivity. The objective of the study was to examine the influence of single nucleotide polymorphisms (SNPs) in IL-4 gene on aspirin hypersensitivity in asthmatics at the genetic and molecular levels. Methods Aspirin-intolerant (AIA, n=103) and aspirin-tolerant asthmatics (n=270) were genotyped and functional promoter assays were performed. Results Of 15 SNPs tested, seven (−589T>C (rs2243250) in promoter, −33T>C (rs2070874) in the 5′-untranslated region, +4047A>G (rs2243266), +4144C>G (rs2243267), +4221C>A (rs2243268), +4367G>A (rs2243270), and +5090A>G (rs2243274) in introns) were significantly associated with AIA risk. The frequency of the rare allele (C) of −589T>C was higher in the AIA group than in the aspirin-tolerant asthmatic group (Pcorr=0.016), and a gene dose-dependent decline in forced expiratory volume in 1 s was noted after an aspirin challenge (P=0.0009). Aspirin unregulated IL-4 mRNA production in Jurkat T and K562 leukemia cells. A reporter plasmid assay revealed that aspirin augmented IL-4 promoter transactivation with the −589T>C C and −33T>C C alleles, compared with that bearing the −589T>C T and −33T>C T alleles. Further, electrophoretic mobility shift assay showed the formation of nuclear complexes with −33T>C and −589T>C allele-containing probes; this was augmented by aspirin. The complexes formed with the −33T>C and −589T>C probes were shifted by treatment with anti-CCAAT-enhancer-binding proteins &bgr; and anti-nuclear factor of activated T-cells antibodies, respectively, indicating the inclusion of these transcription factors. Conclusion Aspirin may regulate IL4 expression in an allele-specific manner by altering the availability of transcription factors to the key regulatory elements in the IL4 promoter, leading to aspirin hypersensitivity.

Elevation of S100 calcium binding protein A9 in sputum of neutrophilic inflammation in severe uncontrolled asthma

BACKGROUND Neutrophilic airway inflammation is frequently observed in severe uncontrolled asthma (UA) and controlled asthma (CA). However, there is no sputum biomarker to differentiate the 2 conditions. OBJECTIVE To identify biomarkers of severe uncontrolled asthma with neutrophilic airway inflammation. METHODS Sputum with a neutrophil content larger than 70% was pooled from 5 patients with severe UA and from 10 patients with CA. Two-dimensional electrophoresis was adopted for differential display proteomics, and candidate proteins were identified using matrix-assisted laser adsorption/ionization-time of flight mass spectrometric analysis. S100 calcium binding protein A9 (S100A9) was identified by western blot and its level was measured in sputum from asthmatics with varying disease severity, patients with chronic obstructive lung disease, and normal controls using enzyme-linked immunosorbent assay. RESULTS Fourteen protein spots exhibited differences in relative intensity between patients with severe UA and those with CA. Matrix-assisted laser adsorption/ionization-time of flight/time of flight of these spots showed an increase in human neutrophil peptide-2, S100A9, β-amylase, neutrophil gelatinase-associated lipocalin, 4-aminobutyrate transaminase, and cystatin SA in patients with UA compared with patients with CA. There was a decrease in the plunc precursor, complement C3 component, immunoglobulin heavy-chain variable region, glial fibrillary acidic protein isoform-1, IgM κIIIb SON, MLL-AF4 der(11) fusion protein, cytokeratin-8, and recombinant IgG4 heavy chain. S100A9 was detected at a higher level in western blots of neutrophilic sputum from patients with severe UA vs CA. S100A9 levels were significantly increased, as measured by enzyme-linked immunosorbent assay, in neutrophilic UA compared with CA, eosinophilic UA and CA, and chronic obstructive lung disease. CONCLUSION S100A9 in sputum may be a biomarker of neutrophilic inflammation in severe UA.