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Antibody Responses to Rhinovirus VP1 in Asthmatic Children
J ALLERGY CLIN IMMUNOL MARCH 2009
726 Late-Breaking Abstracts
rious to the host. Therefore, gaining insight on how to limit IL-5R signaling is important for controlling the intensity and duration of eosinophilderived inflammatory signals. In this study, regulation of IL-5R trafficking and signal termination by IL-5-induced tyrosine phosphorylation was examined. METHODS: Two different approaches were used to inhibit IL-5R tyrosine phosphorylation: 1) ‚c tyrosine phosphorylation-deficient (Y to F) mutants; and 2) JAK inhibitor 1. Deconvolution microscopy and biochemical methods were used to examine the trafficking, degradation and protein/protein interactions of activated IL-5Rs in the absence of tyrosine phosphorylation. RESULTS: Our data reveal that mutation of c Y-612 to phenylalanine (which is a STAT5 and SHP2 binding site) significantly altered the intracellular trafficking of activated IL-5 receptors. Whereas WT IL-5Rs trafficked to lysosomes for their degradation, c Y612F mutants were misrouted to a perinuclear region in what appeared to be an aggresome-like structure. Moreover, compared to WT IL-5Rs, c Y612F mutants showed delayed degradation kinetics and impaired protein/protein interactions with various signaling molecules. CONCLUSION: These data reveal that tyrosine phosphorylation of c Y612 is a new regulatory site for IL-5R trafficking and function. The data further establish that c Y612 phosphorylation is an important sorting signal for proper receptor routing to the lysosomes, and alterations in this sorting signal results in misrouting, delayed receptor degradation, and altered protein interactions.
Natural Anti-Amyloid-beta Antibodies in Intravenous Immunoglobulin (IVIG) Preparation Attenuate Amyloid betaInduced Neurotoxicity in vitro C. Hermann, H. P. Schwarz, H. Ehrlich, B. M. Reipert, K. Olas; Baxter Bioscience, Vienna, AUSTRIA. RATIONALE: Natural antibodies against neurotoxic amyloid beta 1-42 (Aß42) were detected in IVIG. Accumulation of Aß42 in the brain is a key pathogenic event in Alzheimer’s disease (AD). The purpose of this study was to investigate if IVIG preparations can prevent in vitro neurotoxicity induced by Aß42. METHODS: Gammagard Liquid, a highly purified IVIG, contains natural antibodies binding to different conformers of Aß. To examine the potential of IVIG to prevent Aß42- induced neurotoxicity we developed and validated an in vitro assay using pheochromocytoma PC-12 cells, and synthetic Aß42. We assessed the fibrillization state of Aß42 preparation by fluorimetric Thioflavine T binding assay, SDS-PAGE and Western Blot. The analysis of cytotoxicity was done by photometric measurement of Lactate Dehydrogenase released into cell supernatants. RESULTS: We found that Aß42 was predominantly oligomeric and induced concentration-dependently cytotoxicity in PC-12 cells. The cytotoxic effect of Aß42 was cell-specific with neuronal cells being most susceptible. Preincubation of Aß42 with monoclonal anti-Aß42 antibodies decreased the Aß42 induced cytotoxicity in PC-12 cells. 6E10, a monoclonal antibody that shows therapeutic effects in murine models of AD, was most effective. A control murine IgG was not effective. Preincubation of Aß42 with IVIG decreased the cytotoxicity by up to 60%. The potency of different lots was comparable. A control human IgG1 antibody had no effect. CONCLUSIONS: Our data indicate that natural anti-Aß42 antibodies contained in IVIG are neuroprotective in vitro. Furthermore, different lots of IVIG should contain comparable levels of neuroprotective antibodies which is an important finding for the clinical application of IVIG.
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Antibody Responses to Rhinovirus VP1 in Asthmatic Children B. J. Hales1, S. Lee1,2, L. J. Pearce1, W. Smith1, J. Goldblatt2, P. N. Le Souëf2, I. A. Laing1,2; 1Telethon Institute for Child Health Research,
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Centre for Child Health Research, UWA, Perth, AUSTRALIA, 2School of Paediatrics and Child Health, UWA, Perth, AUSTRALIA. RATIONALE: Rhinovirus (RV) is strongly associated with asthma exacerbations. It is hypothesised that the conserved epitopes of the capsid viral protein 1 (VP1) can be used to measure RV-specific IgG1 (Th1), IgE and IgG4 (Th2) antibody responses that may either contribute to, or be markers of immune responses that mediate the immunopathogenesis of asthma. METHODS: Microtitre assays were used to quantitate the antibody isotype response to recombinant rhinovirus VP1 proteins in plasma from house dust mite (HDM)-allergic asthmatic children either admitted to the emergency department for asthma exacerbations (n=38) or recruited from a tertiary hospital respiratory outpatient clinic (n=65). RESULTS: Anti-VP1 IgG1antibody was readily detected although children with acute asthma (AA) had significantly lower IgG1 titres (p<0.001) to VP1 compared to stable asthmatic (SA) children. The AA children also had a lower prevalence of IgE (p<0.001) and IgG4 (p<0.01) antibodies to VP1 and lower anti-VP1 IgE titres (p<0.001). VP1-specific IgE antibody were found for both groups and a third of the children had titres above 5 ng/ml. CONCLUSIONS: High titres of IgG1 antibody are induced to epitopes of VP1 in almost all children showing it is highly suitable for measuring RV-specific antibody responses. Although a high proportion of children were infected with RV on admission to hospital, the AA children had lower anti-VP1 responses implying their antiviral antibody responses may be impaired or underdeveloped. The VP1-specific IgE could play a direct role in the asthmatic response but requires further investigation.
– [Withdrawn]
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Elevation Of Anti-inflammatory Mediator Sphingosine-1phosphate Abrogate Inflammation In Allergic Rhinitis A. KleinJan, M. van Nimwegen, K. Leman, H. C. Hoogsteden; Erasmus MC, Rotterdam, NETHERLANDS. RATIONALE: Sphingosine-1-phosphate (S1P) is an anti-inflammatory mediator and an important molecule for the egress of lymphocytes from lymphoid tissue into the circulation. S1P breakdown occurs rapidly due to sphingosine-lyase(SL) in tissue’s, which can be inhibited by 2-acetyl-4tetrahydroxybutyl(THI). The anti-inflammatory role of a S1P-analogFTY720 and THI was studied in an ovalbumin(OVA) driven model of allergic rhinitis(AR). METHODS: OVA-alum sensitized animals were intranasal challenged with OVA during a 3 week period of 3 consecutive days per week. Animals were treated with (optima)l doses of 6ug FTY720 or 7.5ug THI or combined FTY720-THI or diluent. Nasal mucosal inflammation and cytokine production in OVA restimulated cervical lymph node cell suspensions (CLNs) were evaluated. RESULTS: FTY720 treatment showed significant less eosinophils and mast cells compared to diluent in the nasa[l mucosa of AR animals. IL-4, IL-5, IL-10, IL-13 and OVA specific IgE reduced significantly in FTY720 treated animals. Topical treatment with THI showed a strong reduction in mast cell numbers. Combined treatment of FTY720 and THI reduced eosinophil numbers and mast cell numbers in the nasal mucosa and reduced Th2 cytokines in CLNs in AR. Moreover, FTY720 showed to be potent enough to suppress the inflammation in a model of persistent AR. CONCLUSIONS: Elevation of the anti-inflammatory mediator S1P by adding a synthetic analog or by inhibiting the breakdown of natural occurred S1P has significant advantage to abrogate inflammation in AR. Our results may be translatable into new locally acting drugs as a powerful strategy for the treatment of AR in humans as an alternative for topical steroids treatment.
LB12
726 Late-Breaking Abstracts
rious to the host. Therefore, gaining insight on how to limit IL-5R signaling is important for controlling the intensity and duration of eosinophilderived inflammatory signals. In this study, regulation of IL-5R trafficking and signal termination by IL-5-induced tyrosine phosphorylation was examined. METHODS: Two different approaches were used to inhibit IL-5R tyrosine phosphorylation: 1) ‚c tyrosine phosphorylation-deficient (Y to F) mutants; and 2) JAK inhibitor 1. Deconvolution microscopy and biochemical methods were used to examine the trafficking, degradation and protein/protein interactions of activated IL-5Rs in the absence of tyrosine phosphorylation. RESULTS: Our data reveal that mutation of c Y-612 to phenylalanine (which is a STAT5 and SHP2 binding site) significantly altered the intracellular trafficking of activated IL-5 receptors. Whereas WT IL-5Rs trafficked to lysosomes for their degradation, c Y612F mutants were misrouted to a perinuclear region in what appeared to be an aggresome-like structure. Moreover, compared to WT IL-5Rs, c Y612F mutants showed delayed degradation kinetics and impaired protein/protein interactions with various signaling molecules. CONCLUSION: These data reveal that tyrosine phosphorylation of c Y612 is a new regulatory site for IL-5R trafficking and function. The data further establish that c Y612 phosphorylation is an important sorting signal for proper receptor routing to the lysosomes, and alterations in this sorting signal results in misrouting, delayed receptor degradation, and altered protein interactions.
Natural Anti-Amyloid-beta Antibodies in Intravenous Immunoglobulin (IVIG) Preparation Attenuate Amyloid betaInduced Neurotoxicity in vitro C. Hermann, H. P. Schwarz, H. Ehrlich, B. M. Reipert, K. Olas; Baxter Bioscience, Vienna, AUSTRIA. RATIONALE: Natural antibodies against neurotoxic amyloid beta 1-42 (Aß42) were detected in IVIG. Accumulation of Aß42 in the brain is a key pathogenic event in Alzheimer’s disease (AD). The purpose of this study was to investigate if IVIG preparations can prevent in vitro neurotoxicity induced by Aß42. METHODS: Gammagard Liquid, a highly purified IVIG, contains natural antibodies binding to different conformers of Aß. To examine the potential of IVIG to prevent Aß42- induced neurotoxicity we developed and validated an in vitro assay using pheochromocytoma PC-12 cells, and synthetic Aß42. We assessed the fibrillization state of Aß42 preparation by fluorimetric Thioflavine T binding assay, SDS-PAGE and Western Blot. The analysis of cytotoxicity was done by photometric measurement of Lactate Dehydrogenase released into cell supernatants. RESULTS: We found that Aß42 was predominantly oligomeric and induced concentration-dependently cytotoxicity in PC-12 cells. The cytotoxic effect of Aß42 was cell-specific with neuronal cells being most susceptible. Preincubation of Aß42 with monoclonal anti-Aß42 antibodies decreased the Aß42 induced cytotoxicity in PC-12 cells. 6E10, a monoclonal antibody that shows therapeutic effects in murine models of AD, was most effective. A control murine IgG was not effective. Preincubation of Aß42 with IVIG decreased the cytotoxicity by up to 60%. The potency of different lots was comparable. A control human IgG1 antibody had no effect. CONCLUSIONS: Our data indicate that natural anti-Aß42 antibodies contained in IVIG are neuroprotective in vitro. Furthermore, different lots of IVIG should contain comparable levels of neuroprotective antibodies which is an important finding for the clinical application of IVIG.
LB9
Antibody Responses to Rhinovirus VP1 in Asthmatic Children B. J. Hales1, S. Lee1,2, L. J. Pearce1, W. Smith1, J. Goldblatt2, P. N. Le Souëf2, I. A. Laing1,2; 1Telethon Institute for Child Health Research,
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Centre for Child Health Research, UWA, Perth, AUSTRALIA, 2School of Paediatrics and Child Health, UWA, Perth, AUSTRALIA. RATIONALE: Rhinovirus (RV) is strongly associated with asthma exacerbations. It is hypothesised that the conserved epitopes of the capsid viral protein 1 (VP1) can be used to measure RV-specific IgG1 (Th1), IgE and IgG4 (Th2) antibody responses that may either contribute to, or be markers of immune responses that mediate the immunopathogenesis of asthma. METHODS: Microtitre assays were used to quantitate the antibody isotype response to recombinant rhinovirus VP1 proteins in plasma from house dust mite (HDM)-allergic asthmatic children either admitted to the emergency department for asthma exacerbations (n=38) or recruited from a tertiary hospital respiratory outpatient clinic (n=65). RESULTS: Anti-VP1 IgG1antibody was readily detected although children with acute asthma (AA) had significantly lower IgG1 titres (p<0.001) to VP1 compared to stable asthmatic (SA) children. The AA children also had a lower prevalence of IgE (p<0.001) and IgG4 (p<0.01) antibodies to VP1 and lower anti-VP1 IgE titres (p<0.001). VP1-specific IgE antibody were found for both groups and a third of the children had titres above 5 ng/ml. CONCLUSIONS: High titres of IgG1 antibody are induced to epitopes of VP1 in almost all children showing it is highly suitable for measuring RV-specific antibody responses. Although a high proportion of children were infected with RV on admission to hospital, the AA children had lower anti-VP1 responses implying their antiviral antibody responses may be impaired or underdeveloped. The VP1-specific IgE could play a direct role in the asthmatic response but requires further investigation.
– [Withdrawn]
LB11
Elevation Of Anti-inflammatory Mediator Sphingosine-1phosphate Abrogate Inflammation In Allergic Rhinitis A. KleinJan, M. van Nimwegen, K. Leman, H. C. Hoogsteden; Erasmus MC, Rotterdam, NETHERLANDS. RATIONALE: Sphingosine-1-phosphate (S1P) is an anti-inflammatory mediator and an important molecule for the egress of lymphocytes from lymphoid tissue into the circulation. S1P breakdown occurs rapidly due to sphingosine-lyase(SL) in tissue’s, which can be inhibited by 2-acetyl-4tetrahydroxybutyl(THI). The anti-inflammatory role of a S1P-analogFTY720 and THI was studied in an ovalbumin(OVA) driven model of allergic rhinitis(AR). METHODS: OVA-alum sensitized animals were intranasal challenged with OVA during a 3 week period of 3 consecutive days per week. Animals were treated with (optima)l doses of 6ug FTY720 or 7.5ug THI or combined FTY720-THI or diluent. Nasal mucosal inflammation and cytokine production in OVA restimulated cervical lymph node cell suspensions (CLNs) were evaluated. RESULTS: FTY720 treatment showed significant less eosinophils and mast cells compared to diluent in the nasa[l mucosa of AR animals. IL-4, IL-5, IL-10, IL-13 and OVA specific IgE reduced significantly in FTY720 treated animals. Topical treatment with THI showed a strong reduction in mast cell numbers. Combined treatment of FTY720 and THI reduced eosinophil numbers and mast cell numbers in the nasal mucosa and reduced Th2 cytokines in CLNs in AR. Moreover, FTY720 showed to be potent enough to suppress the inflammation in a model of persistent AR. CONCLUSIONS: Elevation of the anti-inflammatory mediator S1P by adding a synthetic analog or by inhibiting the breakdown of natural occurred S1P has significant advantage to abrogate inflammation in AR. Our results may be translatable into new locally acting drugs as a powerful strategy for the treatment of AR in humans as an alternative for topical steroids treatment.
LB12