Production of Egg Yolk Antibodies Specific to House Dust Mite Proteins

PURPOSE: House dust mites (HDMs) are an important source of indoor allergens associated with asthma, rhinitis and atopic dermatitis. Chicken immunoglobulin (Ig) Y is known to be a good alternative to mice and rabbit antibody production. In this study, we produced IgYs specific to HDMs and investigated their IgE immunoreactivities. MATERIALS AND METHODS: Total IgYs were isolated from the yolks of White Leghorn hens immunized with either Dermatophagoides pteronyssinus or D. farinae protein extract. Control antibodies were separated from the yolks of immunized hens with phosphate buffered saline. IgYs specific to HDMs were analyzed using enzyme-linked immunosorbent assay and Western blotting analysis. RESULTS: The concentration of egg IgY specific to D. farinae in an immunized hen increased and the highest achieved was 661.3 ug/mg (per an egg) on day 47, compared with 760 ug/mg IgY specific to D. pteronyssinus on day 16. The D. pteronyssinus or D. farinae-specific IgY was detected by binding of each mite proteins, and their immunoreactivities were elevated dependent of the specific IgY concentration. CONCLUSION: IgY specific to HDMs may be a promising antibody for immunological diagnosis as well as identification of possible resistance relating to HDM allergy.ope

Involvement of human histamine N-methyltransferase gene polymorphisms in susceptibility to atopic dermatitis in Korean children.

PURPOSE: Histamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children. METHODS: We genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed. RESULTS: Of the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype. CONCLUSIONS: Polymorphisms in HNMT appear to confer susceptibility to AD in Korean children.ope

The effect of heat treatment or hydrolysis on cow’s milk protein distributions and antigenicities

Purpose: Cow`s milk protein is one of the most common and strongest food allergen. We investigated the effects of heat treatment on the distribution and antigenicities of major allergens from cow`s milk. We also compared the protein distribution and antigenicities among cow`s milk formula and its substitutes. Methods: We heated α-casen, β-lactoglobulin (BLG), α-lactalbumin (ALA), and crude extract of cow`s milk in 100°C boiling water for 1 hour. We prepared crude extracts from cow`s milk formula, partially hydrolyzed milk formula (pHF) and extensively hydrolyzed milk formula (eHF). The protein compositions of all the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicities were determined by IgE immunoblotting with pooled serum collected from 11 patients with milk allergy. Results: After heating, no significant alteration was found in casein, and the aggregates of ALA and BLG were detected with molecular weights of about 30 and 45 kDa, respectively. The antigenicities of newly detected aggregates were increased. The new aggregates of BLG with increased antigenicities were also found in heated milk total protein. Major milk allergens were not found in pHF, and residual components with a molecular weight below 10 KDa did not show IgE-binding activity. We failed to observe the residual components and antigenicities of eHF. Conclusion: Changes in protein distribution and antigenicity of milk total protein induced by heat treatment may not be significantly different from those of each major allergen. The residual components of pHF could have little IgE-binding capacity, and there may be few or no antigenic components in eHF.ope

Increased inflammatory mediator in exhaled breath condensate from asthmatic children

Purpose: There has recently been increasing interest in the use of exhaled breath condensate (EBC) as a simple noninvasive means for understanding the physiology of asthma. The aim of this study was to evaluate the levels of leukotriene B4 (LTB4) and eosinophil cationic protein (ECP) in the EBC of asthmatic children. Methods: We measured LTB4 and ECP levels in EBC from children aged 6.14 years, including healthy children (n=25) and asthmatic children (n=25). We also measured serum LTB4 and serum ECP. Pulmonary function tests and methacholine challenge tests were performed on all subjects. Results: Exhaled LTB4 levels were increased significantly in patients with asthma compared to normal subjects (7.1±3.7 pg/mL vs. 2.2±1.7 pg/mL, P<0.05). Serum LTB4 levels were not significantly different in patients with asthma compared to normal subjects (674.7±484.1 pg/mL vs. 487.1±272.0 pg/mL, P=0.156,) and no significant correlations were found between exhaled and serum LTB4 concentrations in children with asthma (r=0.052, P=0.758). Exhaled ECP levels were not significantly different in patients with asthma compared to normal subjects (P=0.419). Serum ECP levels were significantly increased in patients with asthma compared to normal subjects (44.37±32.14 μg/L vs. 16.40±13.23 μg/L, P=0.001). Conclusion: We found significantly elevated LTB4 levels in the EBC of asthmatic children. Our results suggest that EBC may be one of the supportive tools to measure airway inflammation in children with asthma.ope

옥사졸론에 의해서 유도된 피부염에서 면역 조절이상과 피부장벽의 이상의 인터루킨17에 의한 조절

Dept. of Medical Science/석사BACKGROUND: Atopic dermatitis (AD) and contact dermatitis (CD) are known as T cell-mediated eczematous disorders. Interleukin (IL)-17 is expressed by Th17 cells, and involved in the recruitment of inflammatory cells into the AD and CD skin. In this study, we investigated whether IL-17 regulates immune dysregulation and skin barrier dysfunction in oxazolone-induced AD and CD-like murine model.MATERIALS AND METHODS: IL-17-/- and wild type mice were used to make oxazolone-induced AD and CD-like murine model. The ear swelling was measured by micrometer, and the skin biopsies were obtained for RNA isolation and histology. Quantitative real-time PCR analysis was performed to quantify mRNA expression of Th2 cytokines and antimicrobial peptides (AMPs). A vapometer, electron and confocal microscopy were used to evaluate structural changes in skin barrier.RESULT: Our data showed that AD and CD responses were alleviated in IL-17-/-mice. It was evidenced by reduced ear swelling, inflammatory cell infiltration and Th2 cytokines as inflammatory dysregulation in both IL-17-/-mice AD and CD. Skin barrier dysfunction was also characterized by reduced transepidermal water loss (TEWL) and lamellar bodies clustering, while increased lipid distribution in both IL-17-/-mice AD and CD. However, the inductions of AMP and barrier genes were significantly diminished in IL-17-/-mice AD only.CONCLUSION: These studies indic4ate that IL-17 gene may play a role to modulate immune dysregulation and skin barrier dysfunction in oxazolone-induced AD and CDope

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BACKGROUND: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. PURPOSE: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. METHOD: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. RESULTS: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown. CONCLUSION: This study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cellsope

Involvement of IL-10 gene promoter polymorphisms in the susceptibility for childhood asthma

Asthma and atopy have a complex background that may result from the interaction of genes and the environment. Interleukin (IL)-10 is known to play various roles in immune-regulating and anti-inflammatory responses. The aim of this study was to evaluate the possible effect of the IL-10 promoter polymorphisms on susceptibility to childhood asthma. We recruited 333 patients with atopic asthma, 55 with nonatopic asthma, and 248 normal controls. We performed a genetic association study of three genetic polymorphisms (IL-10 -1082A>G, IL-10 -819T>C, and IL-10 -592A>C) of the IL-10 promoter. There was no difference between atopic asthma, nonatopic asthma, and normal controls with respect to allele, genotype, or haplotype frequencies of these IL-10 polymorphisms. However, the -1082A>G polymorphism and ATA haplotype in the IL-10 promoter gene were associated with airway hyper responsiveness (AHR) and the -819T>C, -592A>C, and ATA and ACC haplotypes were also shown to be related to serum eosinophil cationic protein (ECP). Our results suggest that the polymorphisms within the IL-10 promoter may have a disease-modifying effect in the asthmatic airway.ope

Association of genetic variation in chitotriosidase with atopy in Korean children

BACKGROUND: The atopic diseases, which are the most common chronic diseases of childhood, are complex genetic diseases that involve the contribution of multiple genetic factors to disease pathophysiology. Chitotriosidase is involved in innate immunity, but the association of chitotriosidase with allergic diseases remains unclear. OBJECTIVE: To examine the contribution of genetic variation of the chitotriosidase-encoding gene CHIT1 to atopic phenotypes in a Korean cohort of children. METHODS: We identified CHIT1 variations in a Korean population and conducted association analyses using 295 atopic and 242 nonatopic children. An independent replication study was performed using DNA samples from 148 atopic and 243 nonatopic children. All children were unrelated. We performed Western blot analysis in each genotype in vitro to see whether the CHIT1 A442G variation affects the final protein expression levels. RESULTS: In the case-control association analysis, atopy was significantly associated with a single A442G (rs1065761) polymorphism in CHIT1 (odds ratio = 1.32, P = .01). Children with the c.442G risk allele had significantly higher blood eosinophils (P = .001), total serum IgE (P = .007), and eosinophil cationic protein (P = .02) levels. The results of the replication stage analysis confirmed a significant association between the A442G polymorphism and childhood atopy. The joint analysis of the exploratory and replication studies displayed a stronger significant association. The relative protein expression levels of chitotriosidase were significantly higher in both cell lysate and media with the G transfection compared with the wild type. CONCLUSION: These results indicate that the nonsynonymous A442G polymorphism in CHIT1 is associated with risk of atopy.ope