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Magnifying: The Truth behind Fungal Spore Counts
Abstracts AB231
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
747
Magnifying: The Truth behind Fungal Spore Counts Josh D. McLoud, Estelle Levetin, PhD, FAAAAI; University of Tulsa, Tulsa, OK. RATIONALE: National Allergy Bureau recommends that fungal spore analysis of air samples be performed at 1000x magnification. However, recent publications have calculated concentrations using a magnification of 400x. The aim of this study was to compare the concentration of airborne fungal spores using 400x and 1000x magnifications to determine the accuracy of the lower magnification. METHODS: Air samples were collected using a Burkard volumetric 7day spore trap. Sampler drums were changed weekly and slides prepared using standard methods. Slides were analyzed for fungal spores at both 400x and 1000x magnification at chosen traverses. Spore counts were converted to concentrations; resulting data were log transformed and statistically analyzed by t test to compare the concentrations of airborne fungal spores observed at each magnification. RESULTS: The 1000x magnification produced significantly higher concentrations of total spores as well as 9 individual spore types from July 2013. The mean monthly concentration of total spores counted at 400x was 8,591 spores/m3 and 13,247 spores/m3 at 1000x, t(30)529.55, p<0.0001. Among individual spore types, the greatest differences in monthly mean concentration were for Cladosporium (t(30)519.05, p<0.0001), basidiospores (t(30)512.47, p<0.0001), and ascospores (t(30)511.56, p<0.0001). The single day with the greatest difference in concentration of total spores was 27 July with levels of 22,827 spores/m3 and 34,852 spores/m3 at 400x and 1000x, respectively. CONCLUSIONS: Fungal spore counts analyzed at magnifications of 400x and 1000x demonstrate significant differences in calculated atmospheric concentrations, proving the need for an international standard of analysis at 1000x.
748
A Systematic Analysis of Pollen Transcriptomes from Plant Allergens Reveals Conserved Targets of Immune Responses Luise Sternberg1, Jason Greenbaum2, Veronique M. Schulten2, Victoria Tripple2, Denise Baker2, April Frazier2, Heidi Hofer3, Michael Wallner3, Alessandro Sette, Biol. Sci.2, Bjoern Peters2; 1La Jolla Institute for Allergy & Immunology, La Jolla, CA, 2La Jolla Institute for Allergy and Immunology, La Jolla, CA, 3University of Salzburg, Salzburg, Austria. RATIONALE: We have recently identified a set of novel antigens in Timothy grass (TG) pollen using an unbiased, integrated transcriptomic and proteomic analysis, and have shown that these antigens are prominent targets of T-cell responses. Here we determined the degree of conservation of these epitopes across multiple plant species that could be targets of cross-reactive T-cells. This would make them potential candidates for panpollen immunotherapy approaches. METHODS: RNA was extracted and sequenced from nine plant allergens (4-grass, 2-weed, and 3-tree pollens). Peptides from TG were examined for conservation across these pollens using sequence alignments, and conservation was correlated with immunogenicity. T-cell epitope cross-reactivity was determined by generating short term mono-specific T-cell lines and assessing their response across the panel of pollen extracts in IL-5 ELISPOT assays. RESULTS: We find that conservation of a peptide across pollens based on transcriptomic analysis correlates with the likelihood that it will elicit Th2 responses in allergic donors. Furthermore, If TG extract elicited high responses after culture with peptide, so did those extracts in which the peptide is conserved. Substitutions of 3 or more residues in the peptide greatly reduced the response. Thus, conserved peptides (with 2 or less substitutions) are capable of inducing cross-reactive T-cell immune responses. CONCLUSIONS: We have identified peptides that are significantly conserved across multiple plant allergen species and stimulate crossreactive T-cells. It is possible that inducing tolerance against these peptides may modulate the allergic immune response against a broad range of grass and pollen allergens.
749
Molecular Characterization & Epitope Mapping of Recombinant Rice Chitinase Naveen Arora, PhD, Ankita Mishra, Swati Sharma; CSIR - Institute of Genomics and Integrative Biology, Delhi, India. RATIONALE: The prevalence of IgE-mediated rice allergy is about 0.8% in subjects with asthma and rhinitis in India. In the present study, recombinant rice chitinase was expressed, purified and subjected to epitope mapping using in silico tools. METHODS: Rice chitinase was subcloned in pET28a+ vector, expressed and affinity purified by Ni-NTA rasin. It was analyzed for digestibility, heat stability, and IgE binding using atopic patients’ sera. Rice chitinase 3D structure was generated by homology modelling and B cell epitopes were predicted by a combination of sequence and structure based tools. RESULTS: Purified protein appeared as a single band at 24 kDa on SDSPAGE and showed significant IgE binding with 7 of 110 patients’ sera positive to different food allergens by ELISA. Rice chitinase remained stable for 60 mins on incubation with pepsin and was resistant to heat treatment at 908C for 1 h. Homology modeled 3D structure was used for epitope prediction and three B cell epitopes A1 (12–25 aa), A2 (31–45 aa) and A3 (140–166 aa) were identified for chitinase. Property Distance (PD) values were calculated for the predicted epitopes using Structural Database of Allergenic Proteins (SDAP) and showed similarity with allergens from H. brasiliensis, Persea americana (avocado) and Castanea sativa(chest nut). CONCLUSIONS: Chitinase was identified as a potential allergen and may share cross reactive epitopes with reported food allergens. However, more information needs to be gathered from the epitopic regions in predicting cross reactivity with food allergens.
MONDAY
Detection of Airborne Juniperus Pollen By Conventional and Real-Time PCR from Burkard Air Samples Rashmi Prava Mohanty, MS1, Mark A. Buchheim, PhD2, James Anderson3, Estelle Levetin, PhD, FAAAAI1; 1University of Tulsa, Tulsa, OK, 2 University of Tulsa, 3oshtech inc. RATIONALE: Microscopic detection of pollen requires considerable expertise, and identification is limited to genus or family level. Cupressaceae pollen is detected in the Tulsa atmosphere for seven months each year; however, local members of this family only pollinate from February through April. Here, we developed molecular methods to identify multiple Juniperus species from air samples. METHODS: Air samples from January to March were collected with a Burkard 7-day sampler located in Tulsa, OK. One-day segments of exposed Melenex tape were cut into smaller pieces and DNA extracted. Species-specific matK primers for J.ashei and J.virginiana were developed for conventional PCR and leafy primers/probe pairs were developed for real-time PCR for Juniperus. An air sample collected on 15 January in London, Ontario(CANADA) using a Buck Bioslide sampler was also analyzed. RESULTS: Serial dilution of J.asheii pollen demonstrated that specific matK primers were able to detect the presence of 2 to 3 pollen grains indicating this method would be useful for Burkard samples. Using matK primers, J.ashei pollen was confirmed from air samples collected in Tulsa in January as well as from Canada. Primers for matK in J.virginiana confirmed the presence of that pollen in Tulsa air sampled in February and March. Primer/probe pairs for leafy were successful in real-time PCR and can be used to quantify Juniperus pollen from Burkard samples. CONCLUSIONS: This study provides an effective alternative to microscopy for identification of pollen from Burkard air samplers and Bioslide samplers. These data also confirm the long distance transport of J.ashei pollen up to 2400 km.
746
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
747
Magnifying: The Truth behind Fungal Spore Counts Josh D. McLoud, Estelle Levetin, PhD, FAAAAI; University of Tulsa, Tulsa, OK. RATIONALE: National Allergy Bureau recommends that fungal spore analysis of air samples be performed at 1000x magnification. However, recent publications have calculated concentrations using a magnification of 400x. The aim of this study was to compare the concentration of airborne fungal spores using 400x and 1000x magnifications to determine the accuracy of the lower magnification. METHODS: Air samples were collected using a Burkard volumetric 7day spore trap. Sampler drums were changed weekly and slides prepared using standard methods. Slides were analyzed for fungal spores at both 400x and 1000x magnification at chosen traverses. Spore counts were converted to concentrations; resulting data were log transformed and statistically analyzed by t test to compare the concentrations of airborne fungal spores observed at each magnification. RESULTS: The 1000x magnification produced significantly higher concentrations of total spores as well as 9 individual spore types from July 2013. The mean monthly concentration of total spores counted at 400x was 8,591 spores/m3 and 13,247 spores/m3 at 1000x, t(30)529.55, p<0.0001. Among individual spore types, the greatest differences in monthly mean concentration were for Cladosporium (t(30)519.05, p<0.0001), basidiospores (t(30)512.47, p<0.0001), and ascospores (t(30)511.56, p<0.0001). The single day with the greatest difference in concentration of total spores was 27 July with levels of 22,827 spores/m3 and 34,852 spores/m3 at 400x and 1000x, respectively. CONCLUSIONS: Fungal spore counts analyzed at magnifications of 400x and 1000x demonstrate significant differences in calculated atmospheric concentrations, proving the need for an international standard of analysis at 1000x.
748
A Systematic Analysis of Pollen Transcriptomes from Plant Allergens Reveals Conserved Targets of Immune Responses Luise Sternberg1, Jason Greenbaum2, Veronique M. Schulten2, Victoria Tripple2, Denise Baker2, April Frazier2, Heidi Hofer3, Michael Wallner3, Alessandro Sette, Biol. Sci.2, Bjoern Peters2; 1La Jolla Institute for Allergy & Immunology, La Jolla, CA, 2La Jolla Institute for Allergy and Immunology, La Jolla, CA, 3University of Salzburg, Salzburg, Austria. RATIONALE: We have recently identified a set of novel antigens in Timothy grass (TG) pollen using an unbiased, integrated transcriptomic and proteomic analysis, and have shown that these antigens are prominent targets of T-cell responses. Here we determined the degree of conservation of these epitopes across multiple plant species that could be targets of cross-reactive T-cells. This would make them potential candidates for panpollen immunotherapy approaches. METHODS: RNA was extracted and sequenced from nine plant allergens (4-grass, 2-weed, and 3-tree pollens). Peptides from TG were examined for conservation across these pollens using sequence alignments, and conservation was correlated with immunogenicity. T-cell epitope cross-reactivity was determined by generating short term mono-specific T-cell lines and assessing their response across the panel of pollen extracts in IL-5 ELISPOT assays. RESULTS: We find that conservation of a peptide across pollens based on transcriptomic analysis correlates with the likelihood that it will elicit Th2 responses in allergic donors. Furthermore, If TG extract elicited high responses after culture with peptide, so did those extracts in which the peptide is conserved. Substitutions of 3 or more residues in the peptide greatly reduced the response. Thus, conserved peptides (with 2 or less substitutions) are capable of inducing cross-reactive T-cell immune responses. CONCLUSIONS: We have identified peptides that are significantly conserved across multiple plant allergen species and stimulate crossreactive T-cells. It is possible that inducing tolerance against these peptides may modulate the allergic immune response against a broad range of grass and pollen allergens.
749
Molecular Characterization & Epitope Mapping of Recombinant Rice Chitinase Naveen Arora, PhD, Ankita Mishra, Swati Sharma; CSIR - Institute of Genomics and Integrative Biology, Delhi, India. RATIONALE: The prevalence of IgE-mediated rice allergy is about 0.8% in subjects with asthma and rhinitis in India. In the present study, recombinant rice chitinase was expressed, purified and subjected to epitope mapping using in silico tools. METHODS: Rice chitinase was subcloned in pET28a+ vector, expressed and affinity purified by Ni-NTA rasin. It was analyzed for digestibility, heat stability, and IgE binding using atopic patients’ sera. Rice chitinase 3D structure was generated by homology modelling and B cell epitopes were predicted by a combination of sequence and structure based tools. RESULTS: Purified protein appeared as a single band at 24 kDa on SDSPAGE and showed significant IgE binding with 7 of 110 patients’ sera positive to different food allergens by ELISA. Rice chitinase remained stable for 60 mins on incubation with pepsin and was resistant to heat treatment at 908C for 1 h. Homology modeled 3D structure was used for epitope prediction and three B cell epitopes A1 (12–25 aa), A2 (31–45 aa) and A3 (140–166 aa) were identified for chitinase. Property Distance (PD) values were calculated for the predicted epitopes using Structural Database of Allergenic Proteins (SDAP) and showed similarity with allergens from H. brasiliensis, Persea americana (avocado) and Castanea sativa(chest nut). CONCLUSIONS: Chitinase was identified as a potential allergen and may share cross reactive epitopes with reported food allergens. However, more information needs to be gathered from the epitopic regions in predicting cross reactivity with food allergens.
MONDAY
Detection of Airborne Juniperus Pollen By Conventional and Real-Time PCR from Burkard Air Samples Rashmi Prava Mohanty, MS1, Mark A. Buchheim, PhD2, James Anderson3, Estelle Levetin, PhD, FAAAAI1; 1University of Tulsa, Tulsa, OK, 2 University of Tulsa, 3oshtech inc. RATIONALE: Microscopic detection of pollen requires considerable expertise, and identification is limited to genus or family level. Cupressaceae pollen is detected in the Tulsa atmosphere for seven months each year; however, local members of this family only pollinate from February through April. Here, we developed molecular methods to identify multiple Juniperus species from air samples. METHODS: Air samples from January to March were collected with a Burkard 7-day sampler located in Tulsa, OK. One-day segments of exposed Melenex tape were cut into smaller pieces and DNA extracted. Species-specific matK primers for J.ashei and J.virginiana were developed for conventional PCR and leafy primers/probe pairs were developed for real-time PCR for Juniperus. An air sample collected on 15 January in London, Ontario(CANADA) using a Buck Bioslide sampler was also analyzed. RESULTS: Serial dilution of J.asheii pollen demonstrated that specific matK primers were able to detect the presence of 2 to 3 pollen grains indicating this method would be useful for Burkard samples. Using matK primers, J.ashei pollen was confirmed from air samples collected in Tulsa in January as well as from Canada. Primers for matK in J.virginiana confirmed the presence of that pollen in Tulsa air sampled in February and March. Primer/probe pairs for leafy were successful in real-time PCR and can be used to quantify Juniperus pollen from Burkard samples. CONCLUSIONS: This study provides an effective alternative to microscopy for identification of pollen from Burkard air samplers and Bioslide samplers. These data also confirm the long distance transport of J.ashei pollen up to 2400 km.
746