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Preventive Effects of Lactobacillus rhamnosus (Lcr35) through the suppression of inflammatory cytokines in Mouse Model with Atopic Dermatitis
Preventive Effects of Lactobacillus rhamnosus (Lcr35) through the suppression of inflammatory cytokines in Mouse Model with Atopic Dermatitis H. Kim1, Y. Kim1, M. Kang1, J. Seo2, H. Kim2, J. Yu3, S. Hong2; 1Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, REPUBLIC OF KOREA, 2Research Center for Standardization of Allergic Diseases, Childhood Asthma, Atopy Center, Department of Pediatrics, Asan Medical Center, University ofUlsan College of Medicine, Seoul, REPUBLIC OF KOREA, 3Childhood Asthma, Atopy Center, Department of Pediatrics, Asan Medical Center, University ofUlsan College of Medicine, Seoul, REPUBLIC OF KOREA. RATIONALE: Probiotics have been regarded to induce immune regulation or tolerance in allergic diseases. This study was to investigate the protective effects on atopic dermatitis (AD) by oral administration of Lactobacillus rhamnosus (Lcr35) in AD mouse model. METHODS: To develop the mouse model of AD, ovalbumin (OVA) was sensitized through epicutaneous for 1 week at three times followed by twice intervals for 2 weeks after each sensitization in hairless mice. To evaluate the preventive effects of Lcr35, 1 x 109 CFU of it was administrated orally everyday from 1-week before first sensitization to the end of the study. Evaluation of clinical signs (itchy frequency and erythema), transepidermal water loss (TEWL), histopathology (H&E stain) and immunohistochemistry of inflammatory cytokines on skin were performed. In addition, serum total IgE and OVA-specific IgE were measured to confirm systemic immunization. RESULTS: The administration of Lcr35 attenuated the phenotype of AD in mouse model: clinical signs, TEWL on skin, inflammation of skin on histopathology compared to positive control. And Lcr35 treated group showed decrease of the production of serum total and OVA-specific IgE. In addition, the expression of IL-4 and thymic stromal lymphopoietin (TSLP) on immunohistochemistry of skin were suppressed in Lcr35 treated group. CONCLUSION: Oral application of Lcr35 attenuates the development of major characteristics of AD and suppresses the expression of inflammatory cytokines of IL-4 and TSLP on skin in AD mouse model. These findings suggest that probiotics inhibit AD through suppression of inflammatory cytokine in the skin.
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Vitamin D As An Immunomodulator In Ova Induced Allergic Mice S. Park1,2, B. Kang1, S. Ahn2, J. Kim1,2, B. Son1,2, D. Lim1,2; 1Department of Pediatrics, Inha University Hospital, Incheon, REPUBLIC OF KOREA, 2Environmental Health Center for Allergic Rhinitis, Inha University Hospital, Ministry of Environment, Seoul, REPUBLIC OF KOREA. RATIONALE: We investigated how vitamin D affected allergic inflammation in this study. METHODS: BALB/c female mice aging 6-8 wks were inoculated with ovualbumin (OVA) subcutaneously on day 0 and 14, and intranasally on day 21, 22 and 23 (OVA group). Thereafter 10 mice were subcutaneously inoculated with vitamin D (100 ng/mL) after 10 minutes of OVA inoculation on day 0, 14, 21, 22 and 23 (OVA/Vitamin D group 1), and 10 mice were inoculated the same way only on day 21, 22 and 23 (OVA/ Vitamin D group 2). After 24 hours of vitamin D inoculation, Penh was checked and bronchoalveolar lavage fluid (BALF) was analyzed. RESULTS: The eosinophil count in BALF were 12.34 6 7.44 3 104 cells/ mL in the OVA group, 1.87 6 0.69 3 104 cells/mL in the OVA/Vit D group 1 and 2.13 6 0.54 3 104 cells/mL in the OVA/Vit D group 2, showing significantly reduced counts in the OVA/Vit D groups (P<0.05). IL-4 concentration were 30.28 6 6.28 vs 10.94 6 3.16, 13.78 6 0.51 pg/mL. IL-5 concentration were 26.02 6 0.01 vs 3.39 6 0.02, 8.49 6 0.01 pg/mL. IL-4 and IL-5 concentration were both significantly reduced in the OVA/Vit D groups (P<0.05). IL-10 concentration were 21.71 6 2.15 vs 16.94 6 2.83, (P<0.005) 66.25 6 0.69 pg/mL. Penh at methacholine concentration 50 mg/ mL were 9.52 6 5.31 vs 3.34 6 1.35(P<0.05), 6.87 6 4.01, respectively. CONCLUSIONS: We concluded that vitamin D modulated allergic inflammation by regulatory T cells.
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Expression of Vitamin D Receptor and CYP24A1 Enzyme in Airway Epithelium in Allergic Asthma T. O. Makinde, R. Gaurav, R. Steininger, D. K. Agrawal; Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE. RATIONALE: Vitamin D deficiency is associated with increased AHR and decreased pulmonary function. However, its role in the initiation of inflammation and development of airway remodeling is poorly understood. In this study, we examined the expression pattern of Vitamin D receptor (VDR) and CYP24A1 and their correlation with the degree of inflammation and airway remodeling in the lungs of ovalbumin (OVA)-sensitized and challenged (OVA-treated) mice. METHODS: Lung tissues were isolated from four groups of Balb/c mice: PBS and OVA-treated mice on days 33, 45 and 80. H&E, PAS, and trichrome stainings were used to show the severity of lung histopathology. The expression patterns of VDR and CYP24A1 in the airways were examined using immunofluorescence. BEAB-2b human airway epithelial cells were stimulated with TGF-b1, TGF-b2, IL-4 and IFN-g alone and in combination. Stimulated cells were analyzed for mRNA transcripts of VDR and CYP24A1. RESULTS: VDR was constitutively expressed, while CYP24A1 was barely detectable in airway epithelium in PBS-treated mice. However, expression of both VDR and CYP24A1 was significantly increased in the airway epithelium of OVA-treated mice. There was differential VDR expression within the inflammatory cell population in OVA-treated mice. Expression of both VDR and CYP24A1 was increased in BEAB-2b cells following TGF-b1 stimulation. CONCLUSIONS: Pro-inflammatory cytokines, including TGF-b1, significantly affect the expression of both VDR and CYP24A1, the catabolizing enzyme for active vitamin D, in airway epithelial cells. It is, however, unclear whether such an increase in VDR and CYP24A1 is a defensive/compensatory mechanism to protect airway epithelium against the detrimental effects of inflammatory cytokines.
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Challenge With Ragweed Pollen Extract (RWPE) In Allergic Rhinitis Induces Rapid Increase In 8-hydroxydeoxyguanosine (8-OHdG), IL-10 and G-CSF H. Qi, D. Redding, A. Kurosky, B. Singh, J. Lane, M. Bain, I. Boldogh, T. Hazra, S. Sur; University of Texas Medical Branch, Galveston, TX. RATIONALE: RWPE contains NADPH oxidases that rapidly induce oxidative stress in the airways that is critical for allergic inflammation in sensitized mice. However response to oxidative stress is also dependent on host-specific factors. To identify these host factors, we performed saline or RWPE challenge in subjects with allergic rhinitis, and quantified 8-OHdG levels and 48 cytokines as markers of innate oxidative stress and host response respectively. METHODS: Six RWPE allergic subjects were challenged intranasally with saline and RWPE, and symptom scores were recorded. Small strips of filter paper were applied to the nose 30 min post-challenge and stored. Cytokines were recovered from these filter strips, and 48 human cytokines were measured by Bio-Plex cytokine array. Nasal lavage fluid was also obtained 30 min post-challenge, and 8-OHdG in these fluids was measured by ELISA. RESULTS: RWE nasal challenge increased the patient symptom score at 30 min post-challenge (2.3 6 0.8 saline, 6.2 6 0.7 RWPE, p<0.05). In nasal lavage fluid, RWPE challenge increased 8-OHdG levels (0.2 6 0.1 saline, 0.3 6 0.1 RWPE, ng/ml, p<0.05). In nasal filter paper eluate, RWPE challenge increased levels of IL-10 (40 6 40 saline, 72 6 39 RWPE, pg/ml, p<0.05) and G-CSF (61 6 39 saline, 154 6 105 RWPE pg/ ml, p<0.05). CONCLUSIONS: RWPE nasal challenge in allergic rhinitis patients increased symptoms, and levels of 8-OHdG, IL-10 and G-CSF. These observations suggest that RWPE rapidly induces ROS and DNA damage, but simultaneously increases anti-inflammatory cytokines IL-10 and GCSF that may suppress host response to allergens.
SATURDAY
Abstracts AB51
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
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Vitamin D As An Immunomodulator In Ova Induced Allergic Mice S. Park1,2, B. Kang1, S. Ahn2, J. Kim1,2, B. Son1,2, D. Lim1,2; 1Department of Pediatrics, Inha University Hospital, Incheon, REPUBLIC OF KOREA, 2Environmental Health Center for Allergic Rhinitis, Inha University Hospital, Ministry of Environment, Seoul, REPUBLIC OF KOREA. RATIONALE: We investigated how vitamin D affected allergic inflammation in this study. METHODS: BALB/c female mice aging 6-8 wks were inoculated with ovualbumin (OVA) subcutaneously on day 0 and 14, and intranasally on day 21, 22 and 23 (OVA group). Thereafter 10 mice were subcutaneously inoculated with vitamin D (100 ng/mL) after 10 minutes of OVA inoculation on day 0, 14, 21, 22 and 23 (OVA/Vitamin D group 1), and 10 mice were inoculated the same way only on day 21, 22 and 23 (OVA/ Vitamin D group 2). After 24 hours of vitamin D inoculation, Penh was checked and bronchoalveolar lavage fluid (BALF) was analyzed. RESULTS: The eosinophil count in BALF were 12.34 6 7.44 3 104 cells/ mL in the OVA group, 1.87 6 0.69 3 104 cells/mL in the OVA/Vit D group 1 and 2.13 6 0.54 3 104 cells/mL in the OVA/Vit D group 2, showing significantly reduced counts in the OVA/Vit D groups (P<0.05). IL-4 concentration were 30.28 6 6.28 vs 10.94 6 3.16, 13.78 6 0.51 pg/mL. IL-5 concentration were 26.02 6 0.01 vs 3.39 6 0.02, 8.49 6 0.01 pg/mL. IL-4 and IL-5 concentration were both significantly reduced in the OVA/Vit D groups (P<0.05). IL-10 concentration were 21.71 6 2.15 vs 16.94 6 2.83, (P<0.005) 66.25 6 0.69 pg/mL. Penh at methacholine concentration 50 mg/ mL were 9.52 6 5.31 vs 3.34 6 1.35(P<0.05), 6.87 6 4.01, respectively. CONCLUSIONS: We concluded that vitamin D modulated allergic inflammation by regulatory T cells.
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Expression of Vitamin D Receptor and CYP24A1 Enzyme in Airway Epithelium in Allergic Asthma T. O. Makinde, R. Gaurav, R. Steininger, D. K. Agrawal; Center for Clinical and Translational Science and Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE. RATIONALE: Vitamin D deficiency is associated with increased AHR and decreased pulmonary function. However, its role in the initiation of inflammation and development of airway remodeling is poorly understood. In this study, we examined the expression pattern of Vitamin D receptor (VDR) and CYP24A1 and their correlation with the degree of inflammation and airway remodeling in the lungs of ovalbumin (OVA)-sensitized and challenged (OVA-treated) mice. METHODS: Lung tissues were isolated from four groups of Balb/c mice: PBS and OVA-treated mice on days 33, 45 and 80. H&E, PAS, and trichrome stainings were used to show the severity of lung histopathology. The expression patterns of VDR and CYP24A1 in the airways were examined using immunofluorescence. BEAB-2b human airway epithelial cells were stimulated with TGF-b1, TGF-b2, IL-4 and IFN-g alone and in combination. Stimulated cells were analyzed for mRNA transcripts of VDR and CYP24A1. RESULTS: VDR was constitutively expressed, while CYP24A1 was barely detectable in airway epithelium in PBS-treated mice. However, expression of both VDR and CYP24A1 was significantly increased in the airway epithelium of OVA-treated mice. There was differential VDR expression within the inflammatory cell population in OVA-treated mice. Expression of both VDR and CYP24A1 was increased in BEAB-2b cells following TGF-b1 stimulation. CONCLUSIONS: Pro-inflammatory cytokines, including TGF-b1, significantly affect the expression of both VDR and CYP24A1, the catabolizing enzyme for active vitamin D, in airway epithelial cells. It is, however, unclear whether such an increase in VDR and CYP24A1 is a defensive/compensatory mechanism to protect airway epithelium against the detrimental effects of inflammatory cytokines.
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Challenge With Ragweed Pollen Extract (RWPE) In Allergic Rhinitis Induces Rapid Increase In 8-hydroxydeoxyguanosine (8-OHdG), IL-10 and G-CSF H. Qi, D. Redding, A. Kurosky, B. Singh, J. Lane, M. Bain, I. Boldogh, T. Hazra, S. Sur; University of Texas Medical Branch, Galveston, TX. RATIONALE: RWPE contains NADPH oxidases that rapidly induce oxidative stress in the airways that is critical for allergic inflammation in sensitized mice. However response to oxidative stress is also dependent on host-specific factors. To identify these host factors, we performed saline or RWPE challenge in subjects with allergic rhinitis, and quantified 8-OHdG levels and 48 cytokines as markers of innate oxidative stress and host response respectively. METHODS: Six RWPE allergic subjects were challenged intranasally with saline and RWPE, and symptom scores were recorded. Small strips of filter paper were applied to the nose 30 min post-challenge and stored. Cytokines were recovered from these filter strips, and 48 human cytokines were measured by Bio-Plex cytokine array. Nasal lavage fluid was also obtained 30 min post-challenge, and 8-OHdG in these fluids was measured by ELISA. RESULTS: RWE nasal challenge increased the patient symptom score at 30 min post-challenge (2.3 6 0.8 saline, 6.2 6 0.7 RWPE, p<0.05). In nasal lavage fluid, RWPE challenge increased 8-OHdG levels (0.2 6 0.1 saline, 0.3 6 0.1 RWPE, ng/ml, p<0.05). In nasal filter paper eluate, RWPE challenge increased levels of IL-10 (40 6 40 saline, 72 6 39 RWPE, pg/ml, p<0.05) and G-CSF (61 6 39 saline, 154 6 105 RWPE pg/ ml, p<0.05). CONCLUSIONS: RWPE nasal challenge in allergic rhinitis patients increased symptoms, and levels of 8-OHdG, IL-10 and G-CSF. These observations suggest that RWPE rapidly induces ROS and DNA damage, but simultaneously increases anti-inflammatory cytokines IL-10 and GCSF that may suppress host response to allergens.
SATURDAY
Abstracts AB51
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2