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Response of Peripheral Blood Invariant Natural Killer T Cells to Diesel Exhaust Particles in Mild Atopic Asthmatics
Abstracts S261
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Parvovirus B19: A Useful Tool in the Study of the Hygiene Hypothesis? T. A. O’Bryan, W. F. Wright; Penn State College of Medicine, Hershey, PA. RATIONALE: Exposure to certain environmental factors during childhood may influence the developing immune system, causing predisposing or protective effects toward development of inflammatory disorders. Parvovirus B19 (B19) is a common infection with a peak incidence during childhood. This study examines the hypothesis that past B19 infection is associated with altered levels of subclinical inflammatory activity in presumably healthy adults. METHODS: Anti-parvovirus B19 IgG antibody (B19 IgG) and high sensitivity C-reactive protein (CRP) were determined in serum samples from adult blood bank donors. CRP values of B19 IgG positive and B19 IgG negative groups were compared using the Mann-Whitney U test. A logistic regression model examined the association of B19 IgG and CRP levels controlling for age and gender. RESULTS: 282 blood bank donors (44% male, mean age: 45.5 6 15.2 years) were analyzed. Among donors aged 17-49 years (n 5 152), B19 IgG positive samples (57.9%) were associated with significantly lower CRP levels compared with B19 IgG negative samples (median CRP: 1.30 mg/L vs. 2.65 mg/L; p 5 .012 unadjusted; p 5 .022 adjusted for gender and age). Among donors aged > 49 years, median CRP levels were identical (2.75 mg/L) by B19 IgG status. CONCLUSIONS: The association of B19 IgG antibody with lower CRP levels in serum of younger adults supports the hypothesis that infection in childhood may have long term beneficial adaptive immune responses. This relationship may help understanding certain components of the ‘‘hygiene hypothesis’’ which suggest that exposure in childhood to certain infectious agents decreases susceptibility to allergic diseases. Funding: NIH Grant M01-RR10732 and C06RR16499
1010
Response of Peripheral Blood Invariant Natural Killer T Cells to Diesel Exhaust Particles in Mild Atopic Asthmatics R. M. Zemble1, R. L. Miller2; 1University of Pennsylvania Health System, Philadelphia, PA, 2Columbia University College of Physicians and Surgeons, New York, NY. RATIONALE: CD41 natural killer T (NKT) cells may play a pathologic role in asthma. Because exposure to diesel exhaust particles (DEP) has been associated with an increased rate of asthma, the response of peripheral NKT cells to DEP exposure in vitro was examined. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from a group of mild atopic asthmatic and control subjects and incubated in the absence or presence of varying doses of DEP for 18-20 hours. PBMCs were stained with anti-CD4 antibodies, CD1D tetramers loaded with an analogue of a-galactosylceramide (PBS57), and 49 ,6-diamidino-2-phenylindole. Cells were collected by flow cytometry and changes in NKT cell frequency were analyzed using FloJo software. The frequency and the relative percentage change in frequency of CD41 CD1D tetramer1 cells were compared. RESULTS: A trend towards higher frequency of NKT cells in peripheral blood of asthmatics (0.023% of CD41 cells) compared to controls (0.010%) under basal conditions was found (p 5 0.12). The frequency of NKT cells among asthmatics was unchanged after DEP incubation (1420 ug/ml) (0.015% of CD41 cells vs. 0.013%, p 5 0.77). Decreases in the frequency of NKT cells after incubation with DEP occurred in 7/8 control samples and 4/8 asthmatic samples. The relative percentage change in the frequency of NKT cells after exposure to DEP was 0.44% in asthmatics vs. -37.74% in controls (p 5 0.029). CONCLUSIONS: Despite a small trend towards a higher frequency of NKT cells in peripheral blood of mild atopic asthmatics compared to controls, few alterations were found following in vitro exposure to the common asthma trigger DEP. Funding: P30 ES009089
1011
Toll-like Receptor 8 (TLR8)-Mediated Inhibition of Th2 Cytokine Responses to Ragweed Antigen in Peripheral Blood Mononuclear Cells (PBMC) of Ragweed Allergic Individuals J. K. Lawson1, B. Yu1, L. L. Thomas2, A. Landay2, J. N. Moy1; 1Rush University Medical Center and Stroger Hospital of Cook County, Chicago, IL, 2Rush University Medical Center, Chicago, IL. RATIONALE: Because TLR8 agonists are potent stimuli for IFN-g production, we examined the ability of TLR8 agonist 3M-002, an imidazoquinoline class compound, to inhibit production of Th2 cytokines in vitro by PBMC of ragweed allergic subjects. METHODS: PBMC isolated from ragweed allergic subjects and nonallergic controls were incubated with medium alone, ragweed extract (1 mg/ml), 3M-002 (1 mM and 3 mM), or ragweed plus 3M-002. After 6 days, the levels of IL-5, IL-13, and IFN-g in cell culture supernatants were measured by cytometric bead assay. RESULTS: Incubation of PBMC from 6 allergic and 2 control subjects with 3M-002 stimulated a concentration-dependent increase in IFN-g production (spontaneous: 228 6 153 pg/ml*; 1 mM 3M-002: 548 6 288 pg/ml; 3 mM 3M-002: 1263 6 540 pg/ml*; *p 5 0.05). Ragweed extract stimulated increased levels of IL-5 production (193 6 88 pg/ml ) by PBMC of ragweed allergic subjects, which were reduced to 86 6 48 pg/ml and 17 6 9 pg/ml ( p 5 0.03) in the presence of 1 mM and 3 mM 3M-002, respectively. Ragweed extract also stimulated IL-13 production (225 6 88 pg/mlà) by PBMC of the ragweed allergic subjects, which was reduced to 177 6 72 pg/ml and 69 6 15 pg/mlà (àp 5 0.03) in the presence of 1 mM and 3 mM 3M-002, respectively. In contrast to its inhibitory effect on ragweedstimulated IL-5 and IL-13 production, 3M-002 enhanced IFN-g production in the presence of ragweed (ragweed alone: 178 6 83, ragweed 1 1 uM 3M-002: 806 6 292, ragweed 1 3 uM 3M-002: 2023 6 799). CONCLUSIONS: The TLR8 agonist, 3M-002 is an effective stimulus for IFN-g production by PBMC of both atopic and non-atopic individuals. Moreover, 3M-002 effectively suppresses the ragweed-induced production of Th2 cytokines by PBMC of ragweed allergic individuals. Funding: Rush University Medical Center
1012
Allergen-Specific Regulatory T Cells (Tregs) in Milk Allergic Children M. Moloney, N. Wanich, S. Noone, A. Nowak-Wegrzyn, H. Sampson, W. Shreffler; Mount Sinai School of Medicine, New York, NY. RATIONALE: Antigen-specific Tregs may regulate tolerance to dietary antigens. An important subset of Tregs can be defined as CD251 CD127-FoxP31. We sought to assess the relationship between milk allergy and allergen-specific Tregs in children and whether resolution of milkallergy is associated with an increase of antigen specific Tregs. METHODS: Venous blood samples from pediatric subjects undergoing milk challenge as part of a prospective natural history study were obtained. PBMCs were labeled with CFSE and cultured for seven days with medium, purified caseins, or anti-CD3/-CD28 beads with and without exogenous IL2. Live (Aqua LIVE/DEADnegative) proliferating (CFSElow) CD4 T cells (CD31CD41) were assessed for surface expression of CD25 and CD127 and intracellular expression of FoxP3 by seven-color flow cytometry. RESULTS: Allergen-induced proliferating T cells were identified by CFSE dilution in milk allergic patients and controls. Most casein-specific T cells were CD25 and FoxP3 positive, consistent with previous reports. However there was a distinct CD25bright CD127negative FoxP3bright population that was greatly enhanced by exogenous IL-2, consistent with previous reports of in vitro anergy, and therefore has phenotypic and functional qualities of Tregs. As a percentage of CD4 T cells, the frequency of casein-specific Tregs typically exceeds the frequency of polyclonally induced Tregs. In vitro suppressor function and correlation with disease status are currently being investigated. CONCLUSIONS: Allergen-specific CD4 T cells with a Treg phenotype are present in the peripheral blood from human subjects and may play a role in regulating immunity to dietary antigens. Funding: 2007 AAAAAI/Elliot & Roslyn Jaffe 3rd Year Fellowship Food Allergy Research Award and NIAID P01-AI44236
TUESDAY
1009
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Parvovirus B19: A Useful Tool in the Study of the Hygiene Hypothesis? T. A. O’Bryan, W. F. Wright; Penn State College of Medicine, Hershey, PA. RATIONALE: Exposure to certain environmental factors during childhood may influence the developing immune system, causing predisposing or protective effects toward development of inflammatory disorders. Parvovirus B19 (B19) is a common infection with a peak incidence during childhood. This study examines the hypothesis that past B19 infection is associated with altered levels of subclinical inflammatory activity in presumably healthy adults. METHODS: Anti-parvovirus B19 IgG antibody (B19 IgG) and high sensitivity C-reactive protein (CRP) were determined in serum samples from adult blood bank donors. CRP values of B19 IgG positive and B19 IgG negative groups were compared using the Mann-Whitney U test. A logistic regression model examined the association of B19 IgG and CRP levels controlling for age and gender. RESULTS: 282 blood bank donors (44% male, mean age: 45.5 6 15.2 years) were analyzed. Among donors aged 17-49 years (n 5 152), B19 IgG positive samples (57.9%) were associated with significantly lower CRP levels compared with B19 IgG negative samples (median CRP: 1.30 mg/L vs. 2.65 mg/L; p 5 .012 unadjusted; p 5 .022 adjusted for gender and age). Among donors aged > 49 years, median CRP levels were identical (2.75 mg/L) by B19 IgG status. CONCLUSIONS: The association of B19 IgG antibody with lower CRP levels in serum of younger adults supports the hypothesis that infection in childhood may have long term beneficial adaptive immune responses. This relationship may help understanding certain components of the ‘‘hygiene hypothesis’’ which suggest that exposure in childhood to certain infectious agents decreases susceptibility to allergic diseases. Funding: NIH Grant M01-RR10732 and C06RR16499
1010
Response of Peripheral Blood Invariant Natural Killer T Cells to Diesel Exhaust Particles in Mild Atopic Asthmatics R. M. Zemble1, R. L. Miller2; 1University of Pennsylvania Health System, Philadelphia, PA, 2Columbia University College of Physicians and Surgeons, New York, NY. RATIONALE: CD41 natural killer T (NKT) cells may play a pathologic role in asthma. Because exposure to diesel exhaust particles (DEP) has been associated with an increased rate of asthma, the response of peripheral NKT cells to DEP exposure in vitro was examined. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from a group of mild atopic asthmatic and control subjects and incubated in the absence or presence of varying doses of DEP for 18-20 hours. PBMCs were stained with anti-CD4 antibodies, CD1D tetramers loaded with an analogue of a-galactosylceramide (PBS57), and 49 ,6-diamidino-2-phenylindole. Cells were collected by flow cytometry and changes in NKT cell frequency were analyzed using FloJo software. The frequency and the relative percentage change in frequency of CD41 CD1D tetramer1 cells were compared. RESULTS: A trend towards higher frequency of NKT cells in peripheral blood of asthmatics (0.023% of CD41 cells) compared to controls (0.010%) under basal conditions was found (p 5 0.12). The frequency of NKT cells among asthmatics was unchanged after DEP incubation (1420 ug/ml) (0.015% of CD41 cells vs. 0.013%, p 5 0.77). Decreases in the frequency of NKT cells after incubation with DEP occurred in 7/8 control samples and 4/8 asthmatic samples. The relative percentage change in the frequency of NKT cells after exposure to DEP was 0.44% in asthmatics vs. -37.74% in controls (p 5 0.029). CONCLUSIONS: Despite a small trend towards a higher frequency of NKT cells in peripheral blood of mild atopic asthmatics compared to controls, few alterations were found following in vitro exposure to the common asthma trigger DEP. Funding: P30 ES009089
1011
Toll-like Receptor 8 (TLR8)-Mediated Inhibition of Th2 Cytokine Responses to Ragweed Antigen in Peripheral Blood Mononuclear Cells (PBMC) of Ragweed Allergic Individuals J. K. Lawson1, B. Yu1, L. L. Thomas2, A. Landay2, J. N. Moy1; 1Rush University Medical Center and Stroger Hospital of Cook County, Chicago, IL, 2Rush University Medical Center, Chicago, IL. RATIONALE: Because TLR8 agonists are potent stimuli for IFN-g production, we examined the ability of TLR8 agonist 3M-002, an imidazoquinoline class compound, to inhibit production of Th2 cytokines in vitro by PBMC of ragweed allergic subjects. METHODS: PBMC isolated from ragweed allergic subjects and nonallergic controls were incubated with medium alone, ragweed extract (1 mg/ml), 3M-002 (1 mM and 3 mM), or ragweed plus 3M-002. After 6 days, the levels of IL-5, IL-13, and IFN-g in cell culture supernatants were measured by cytometric bead assay. RESULTS: Incubation of PBMC from 6 allergic and 2 control subjects with 3M-002 stimulated a concentration-dependent increase in IFN-g production (spontaneous: 228 6 153 pg/ml*; 1 mM 3M-002: 548 6 288 pg/ml; 3 mM 3M-002: 1263 6 540 pg/ml*; *p 5 0.05). Ragweed extract stimulated increased levels of IL-5 production (193 6 88 pg/ml ) by PBMC of ragweed allergic subjects, which were reduced to 86 6 48 pg/ml and 17 6 9 pg/ml ( p 5 0.03) in the presence of 1 mM and 3 mM 3M-002, respectively. Ragweed extract also stimulated IL-13 production (225 6 88 pg/mlà) by PBMC of the ragweed allergic subjects, which was reduced to 177 6 72 pg/ml and 69 6 15 pg/mlà (àp 5 0.03) in the presence of 1 mM and 3 mM 3M-002, respectively. In contrast to its inhibitory effect on ragweedstimulated IL-5 and IL-13 production, 3M-002 enhanced IFN-g production in the presence of ragweed (ragweed alone: 178 6 83, ragweed 1 1 uM 3M-002: 806 6 292, ragweed 1 3 uM 3M-002: 2023 6 799). CONCLUSIONS: The TLR8 agonist, 3M-002 is an effective stimulus for IFN-g production by PBMC of both atopic and non-atopic individuals. Moreover, 3M-002 effectively suppresses the ragweed-induced production of Th2 cytokines by PBMC of ragweed allergic individuals. Funding: Rush University Medical Center
1012
Allergen-Specific Regulatory T Cells (Tregs) in Milk Allergic Children M. Moloney, N. Wanich, S. Noone, A. Nowak-Wegrzyn, H. Sampson, W. Shreffler; Mount Sinai School of Medicine, New York, NY. RATIONALE: Antigen-specific Tregs may regulate tolerance to dietary antigens. An important subset of Tregs can be defined as CD251 CD127-FoxP31. We sought to assess the relationship between milk allergy and allergen-specific Tregs in children and whether resolution of milkallergy is associated with an increase of antigen specific Tregs. METHODS: Venous blood samples from pediatric subjects undergoing milk challenge as part of a prospective natural history study were obtained. PBMCs were labeled with CFSE and cultured for seven days with medium, purified caseins, or anti-CD3/-CD28 beads with and without exogenous IL2. Live (Aqua LIVE/DEADnegative) proliferating (CFSElow) CD4 T cells (CD31CD41) were assessed for surface expression of CD25 and CD127 and intracellular expression of FoxP3 by seven-color flow cytometry. RESULTS: Allergen-induced proliferating T cells were identified by CFSE dilution in milk allergic patients and controls. Most casein-specific T cells were CD25 and FoxP3 positive, consistent with previous reports. However there was a distinct CD25bright CD127negative FoxP3bright population that was greatly enhanced by exogenous IL-2, consistent with previous reports of in vitro anergy, and therefore has phenotypic and functional qualities of Tregs. As a percentage of CD4 T cells, the frequency of casein-specific Tregs typically exceeds the frequency of polyclonally induced Tregs. In vitro suppressor function and correlation with disease status are currently being investigated. CONCLUSIONS: Allergen-specific CD4 T cells with a Treg phenotype are present in the peripheral blood from human subjects and may play a role in regulating immunity to dietary antigens. Funding: 2007 AAAAAI/Elliot & Roslyn Jaffe 3rd Year Fellowship Food Allergy Research Award and NIAID P01-AI44236
TUESDAY
1009