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Cord Blood CD34 + Hemopoietic Progenitor Eosinophilic Lineage Commitment Assessed by Q-PCR
Abstracts S225
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Cord Blood CD34 1 Hemopoietic Progenitor Eosinophilic Lineage Commitment Assessed by Q-PCR A. K. Ellis1,2, L. Crawford2, J. A. Denburg2; 1Queen’s University, Kingston, ON, Canada, 2McMaster University, Hamilton, ON, Canada. RATIONALE: We have recently demonstrated multiplex Q-PCR analyses of mRNA expression of key eosinophil-lineage commitment events in cord blood (CB) non-adherent mononuclear cells (NAMNCs) after stimulation with IL-5. We hypothesized that the majority of these events were occurring in the differentiating pluripotent CD341 cell population. The objective was to ascertain the kinetic patterns of expression of CB eosinophil-lineage specific genes, namely GATA-1, MBP, and IL-5Ra, in a purified CB CD341 fraction. METHODS: CB mononuclear cells were isolated from random fresh and frozen samples, separated by magnetic cell-sorting into a highly- purified CD341 population, and then incubated in the presence of 1 ng/mL rhIL-5. At various time-points post-stimulation, RNA was isolated, reverse transcribed, and expression of GATA-1, IL-5Ra, and MBP mRNA determined, utilizing multiplex Q-PCR. Relative expression ratios of stimulated and un-stimulated cells were calculated using the DDCt method. RESULTS: Stimulation of CD341 cells with IL-5 resulted in up-regulation of GATA-1 mRNA, peaking between 24 and 48 h, an identical pattern to that seen with NAMNCs (r 5 0.963); MBP mRNAwas up-regulated progressively, maximally at 96 h correlation with NAMNC, r 5 0.978). Total IL-5R mRNA was stable at all time-points, with differential expression of mRNA for IL-5R soluble (Sol-) and transmembrane (Tm-) isoforms: At 24 h, the ratio of Tm-IL-5Ra to Sol-IL5Ra was 1.5:1, but this substantially reversed by 72 h, with a Sol-IL-5Ra to Tm-IL-5Ra ratio of 9.4:1. CONCLUSIONS: Multiplex Q-PCR analyses of mRNA from CB CD341 cells stimulated with IL-5 demonstrate sequential expression of critical eosinophil lineage-specific events, providing potential surrogate molecular markers of CB eosinophil differentiation.
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Productive Influenza Infection Of Human Mast Cells Is Highly Diminished Compared With Epithelial Cells C. Marcet, C. St. Laurent, N. Singh, A. D. Befus; University of Alberta, Edmonton, AB, Canada. RATIONALE: Aside from being critical effector cells in allergy, mast cells (MC) are important in innate immune responses against bacteria and viruses. MC exist in abundance close to lung epithelial cells (EC), which are the primary target of influenza infection. Our aim is to study the infectivity of influenza A virus (FluA) in MC and potential host factors that might regulate virus replication. METHODS: Human MC lines (HMC-1 and LAD-2) and primary MC cultured from human peripheral blood (PBMC) were exposed to FluA (PR/34/ 8). Epithelial cell lines (MDCK and Calu-3) were used as comparative cell types. FluA RNA expression was determined by RT-PCR. Presence of intracellular FluA particles was visualized by electron microscopy. Total FluA particles in culture supernatants were determined by ability to agglutinate human red blood cells (RBC) using hemagglutination (HA) assay. Infectious FluA particles were determined by ability of supernatants to infect Calu-3 cells. RESULTS: Production of viral transcripts stopped at 4 days post-infection in MC, whereas in EC transcripts were produced for at least 6 days. FluA virus recovery at 4 days post-infection was significantly lower (p 5 0.015) in MC culture supernatants (27 6 13 HAU/ml) than in EC (480 6 160 HAU/ml). Infectious FluA was released from EC but not MC. We also have preliminary data that NO production, which may have antiviral effects, is upregulated in MC exposed to FluA. CONCLUSIONS: Our evidence supports that FluA undergoes a non-productive infection in MC, suggesting that MC may have antiviral mechanisms, such as NO production, to restrict FluA infection.
869
Vascular Endothelial Cell Growth Factor (VEGF) Production by Staphylococcal Enterotoxin B (SEB) and IL-1b-Stimulated Human Keratinocytes is Inhibited by Pimecrolimus M. Frieri, A. Capetandes; Nassau University Medical Center, East Meadow, NY. RATIONALE: Atopic dermatitis (AD), an inflammatory skin disease involves keratinocyte release of cytokines. Topical pimecrolimus inhibits T cell-dependent proinflammatory cytokines We demonstrated keratinocyte release of TNFa and inhibition of GMCSF by pimecrolimus. (Frieri M et al. JACI 119: S263, 2007; J Inv Med 55: S340, 2007). VEGF in AD lesions causes vessel hyperpermeability, endothelial cell proliferation and VEGFmRNA is upregulated in epidermal keratinocytes and microvessels in skin culture. METHODS: Confluent monolayers of human keratinocytes (HK) were stimulated with cytomix (100 mg/ml of SEB and 10U/ml rhIL-1b). HK were stimulated with cytomix 1/2 102821029 M pimecrolimus for 24 hours in serum free media (SFM). Conditioned media was assayed by ELISA .Cells were analyzed for cell viability using the MTT assay. VEGF data (pg/ml) was normalized to the MTT data (A550 nm). Parametric data is shown as the mean 1/22SD and analyzed by ANOVA and the SNK post hoc test. Power for all studies was 0.5-0.8 at a 5 0.05. RESULTS: HK showed >95% viability by the MTT test in SFM. HK viability decreased to 50-70% with cytomix. Cytomix stimulated normalized VEGF secretion over SFM by approximately 2-fold. In five experiments (n 5 28) 1028 and 1029 M pimecrolimus with cytomix decreased normalized VEGF secretion by 1.5-2.0- fold relative to cytomix alone (P 5 0.0003, ANOVA). CONCLUSIONS: 1028 and 1029 M pimecrolimus inhibited increased secretion of cytomix stimulated normalized VEGF by HK cells in SFM. Calcineurin pathways mediate multiple VEGF functions in proliferation, migration, vascular permeability and pimecrolimus may improve AD skin by inhibiting these events.
870
Cross-reactivity Between Blo t 13 Allergen and Homologues From the Mite D. farinae (Der f 13) and Human FABPs L. Puerta1, D. Mercado1, S. L. Chan2, S. Jimenez1, A. Labrada3, J. F. Glatz4, F. T. Chew2, L. Caraballo1; 1University of Cartagena, Cartagena, Colombia, 2National University of Singapore, Singapore, Singapore, 3BIOCEN, La Havana, Cuba, 4Maastrich University, Maastrich, Netherlands. RATIONALE: Fatty Acid Binding Protein family (FABP) has conserved molecular structure and biological function, which suggests cross-reactivity among their members such as group 13 mite allergens and human FABP. METHODS: ELISA was performed with the recombinant allergens Blo t 13, Der f 13 (isoforms DF414 and DF 1096) and the human FABP from heart (H-FABP), brain (B-FABP) and adipocyte (A-FABP) using sera from asthmatic allergy patients and the monoclonal antibodies anti-Blo t 13 (5G3) and anti H-FABP (67D3 and 66E2). ELISA cross-inhibitions were done using sera from asthmatic patients. RESULTS: IgE reactivity to H-FABP, B-FABP and A-FABP was detected in two of twelve sera from asthmatic allergic patients sensitized to Blo t 13. The IgE reactivity to Blo t 13 in solid phase was inhibited 32.6% by preadsorption with H-FABP, and 33.84% with B-FABP. The inhibition of IgE binding to B-FABP by Blo t 13 was 85.61%. The maximum inhibition of IgE binding to Blo t 13 in solid phase by DF414 was 44.6%; when DF414 was in solid phase the maximum inhibition produced by Blo t 13 was 79.4%. The monoclonal 5G3 reacted with DF414 and DF1096 but not with human FABPs. The monoclonal 67D3 reacted strongly against DF1096. CONCLUSIONS: IgE antibody reactivity against the human FABPs was detected in sera from asthmatic allergic patients, which seems to be induced by exposure to Blo t 13. DF414 and Blo t 13 showed a moderate cross-reactivity. The clinical significance of these findings is unknown.
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867
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Cord Blood CD34 1 Hemopoietic Progenitor Eosinophilic Lineage Commitment Assessed by Q-PCR A. K. Ellis1,2, L. Crawford2, J. A. Denburg2; 1Queen’s University, Kingston, ON, Canada, 2McMaster University, Hamilton, ON, Canada. RATIONALE: We have recently demonstrated multiplex Q-PCR analyses of mRNA expression of key eosinophil-lineage commitment events in cord blood (CB) non-adherent mononuclear cells (NAMNCs) after stimulation with IL-5. We hypothesized that the majority of these events were occurring in the differentiating pluripotent CD341 cell population. The objective was to ascertain the kinetic patterns of expression of CB eosinophil-lineage specific genes, namely GATA-1, MBP, and IL-5Ra, in a purified CB CD341 fraction. METHODS: CB mononuclear cells were isolated from random fresh and frozen samples, separated by magnetic cell-sorting into a highly- purified CD341 population, and then incubated in the presence of 1 ng/mL rhIL-5. At various time-points post-stimulation, RNA was isolated, reverse transcribed, and expression of GATA-1, IL-5Ra, and MBP mRNA determined, utilizing multiplex Q-PCR. Relative expression ratios of stimulated and un-stimulated cells were calculated using the DDCt method. RESULTS: Stimulation of CD341 cells with IL-5 resulted in up-regulation of GATA-1 mRNA, peaking between 24 and 48 h, an identical pattern to that seen with NAMNCs (r 5 0.963); MBP mRNAwas up-regulated progressively, maximally at 96 h correlation with NAMNC, r 5 0.978). Total IL-5R mRNA was stable at all time-points, with differential expression of mRNA for IL-5R soluble (Sol-) and transmembrane (Tm-) isoforms: At 24 h, the ratio of Tm-IL-5Ra to Sol-IL5Ra was 1.5:1, but this substantially reversed by 72 h, with a Sol-IL-5Ra to Tm-IL-5Ra ratio of 9.4:1. CONCLUSIONS: Multiplex Q-PCR analyses of mRNA from CB CD341 cells stimulated with IL-5 demonstrate sequential expression of critical eosinophil lineage-specific events, providing potential surrogate molecular markers of CB eosinophil differentiation.
868
Productive Influenza Infection Of Human Mast Cells Is Highly Diminished Compared With Epithelial Cells C. Marcet, C. St. Laurent, N. Singh, A. D. Befus; University of Alberta, Edmonton, AB, Canada. RATIONALE: Aside from being critical effector cells in allergy, mast cells (MC) are important in innate immune responses against bacteria and viruses. MC exist in abundance close to lung epithelial cells (EC), which are the primary target of influenza infection. Our aim is to study the infectivity of influenza A virus (FluA) in MC and potential host factors that might regulate virus replication. METHODS: Human MC lines (HMC-1 and LAD-2) and primary MC cultured from human peripheral blood (PBMC) were exposed to FluA (PR/34/ 8). Epithelial cell lines (MDCK and Calu-3) were used as comparative cell types. FluA RNA expression was determined by RT-PCR. Presence of intracellular FluA particles was visualized by electron microscopy. Total FluA particles in culture supernatants were determined by ability to agglutinate human red blood cells (RBC) using hemagglutination (HA) assay. Infectious FluA particles were determined by ability of supernatants to infect Calu-3 cells. RESULTS: Production of viral transcripts stopped at 4 days post-infection in MC, whereas in EC transcripts were produced for at least 6 days. FluA virus recovery at 4 days post-infection was significantly lower (p 5 0.015) in MC culture supernatants (27 6 13 HAU/ml) than in EC (480 6 160 HAU/ml). Infectious FluA was released from EC but not MC. We also have preliminary data that NO production, which may have antiviral effects, is upregulated in MC exposed to FluA. CONCLUSIONS: Our evidence supports that FluA undergoes a non-productive infection in MC, suggesting that MC may have antiviral mechanisms, such as NO production, to restrict FluA infection.
869
Vascular Endothelial Cell Growth Factor (VEGF) Production by Staphylococcal Enterotoxin B (SEB) and IL-1b-Stimulated Human Keratinocytes is Inhibited by Pimecrolimus M. Frieri, A. Capetandes; Nassau University Medical Center, East Meadow, NY. RATIONALE: Atopic dermatitis (AD), an inflammatory skin disease involves keratinocyte release of cytokines. Topical pimecrolimus inhibits T cell-dependent proinflammatory cytokines We demonstrated keratinocyte release of TNFa and inhibition of GMCSF by pimecrolimus. (Frieri M et al. JACI 119: S263, 2007; J Inv Med 55: S340, 2007). VEGF in AD lesions causes vessel hyperpermeability, endothelial cell proliferation and VEGFmRNA is upregulated in epidermal keratinocytes and microvessels in skin culture. METHODS: Confluent monolayers of human keratinocytes (HK) were stimulated with cytomix (100 mg/ml of SEB and 10U/ml rhIL-1b). HK were stimulated with cytomix 1/2 102821029 M pimecrolimus for 24 hours in serum free media (SFM). Conditioned media was assayed by ELISA .Cells were analyzed for cell viability using the MTT assay. VEGF data (pg/ml) was normalized to the MTT data (A550 nm). Parametric data is shown as the mean 1/22SD and analyzed by ANOVA and the SNK post hoc test. Power for all studies was 0.5-0.8 at a 5 0.05. RESULTS: HK showed >95% viability by the MTT test in SFM. HK viability decreased to 50-70% with cytomix. Cytomix stimulated normalized VEGF secretion over SFM by approximately 2-fold. In five experiments (n 5 28) 1028 and 1029 M pimecrolimus with cytomix decreased normalized VEGF secretion by 1.5-2.0- fold relative to cytomix alone (P 5 0.0003, ANOVA). CONCLUSIONS: 1028 and 1029 M pimecrolimus inhibited increased secretion of cytomix stimulated normalized VEGF by HK cells in SFM. Calcineurin pathways mediate multiple VEGF functions in proliferation, migration, vascular permeability and pimecrolimus may improve AD skin by inhibiting these events.
870
Cross-reactivity Between Blo t 13 Allergen and Homologues From the Mite D. farinae (Der f 13) and Human FABPs L. Puerta1, D. Mercado1, S. L. Chan2, S. Jimenez1, A. Labrada3, J. F. Glatz4, F. T. Chew2, L. Caraballo1; 1University of Cartagena, Cartagena, Colombia, 2National University of Singapore, Singapore, Singapore, 3BIOCEN, La Havana, Cuba, 4Maastrich University, Maastrich, Netherlands. RATIONALE: Fatty Acid Binding Protein family (FABP) has conserved molecular structure and biological function, which suggests cross-reactivity among their members such as group 13 mite allergens and human FABP. METHODS: ELISA was performed with the recombinant allergens Blo t 13, Der f 13 (isoforms DF414 and DF 1096) and the human FABP from heart (H-FABP), brain (B-FABP) and adipocyte (A-FABP) using sera from asthmatic allergy patients and the monoclonal antibodies anti-Blo t 13 (5G3) and anti H-FABP (67D3 and 66E2). ELISA cross-inhibitions were done using sera from asthmatic patients. RESULTS: IgE reactivity to H-FABP, B-FABP and A-FABP was detected in two of twelve sera from asthmatic allergic patients sensitized to Blo t 13. The IgE reactivity to Blo t 13 in solid phase was inhibited 32.6% by preadsorption with H-FABP, and 33.84% with B-FABP. The inhibition of IgE binding to B-FABP by Blo t 13 was 85.61%. The maximum inhibition of IgE binding to Blo t 13 in solid phase by DF414 was 44.6%; when DF414 was in solid phase the maximum inhibition produced by Blo t 13 was 79.4%. The monoclonal 5G3 reacted with DF414 and DF1096 but not with human FABPs. The monoclonal 67D3 reacted strongly against DF1096. CONCLUSIONS: IgE antibody reactivity against the human FABPs was detected in sera from asthmatic allergic patients, which seems to be induced by exposure to Blo t 13. DF414 and Blo t 13 showed a moderate cross-reactivity. The clinical significance of these findings is unknown.
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