title: Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins.
abstract: BACKGROUND
  Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.
  
  
  RESULTS
  A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.
  
  
  CONCLUSIONS
  This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wan
authors.affiliation: Genomics and Computational Biology Group, Centro de Pesquisas René Rachou, National Institute of Science and Technology in Tropical Diseases, Fundação Oswaldo Cruz - FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ludolf
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
authors.forename: Daniel G W
authors.initials: DG
authors.lastname: Alanine
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Owen
authors.initials: O
authors.lastname: Stretton
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Eman
authors.initials: E
authors.lastname: Ali Ali
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Nafal
authors.initials: N
authors.lastname: Al-Barwary
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Doenhoff
authors.affiliation: Center for Molecular Allergology, IDI-IRCCS, Rome, Italy; Associated Centres for Molecular Allergology, Rome, Italy.
authors.forename: Adriano
authors.initials: A
authors.lastname: Mari
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Colin M
authors.initials: CM
authors.lastname: Fitzsimmons
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David W
authors.initials: DW
authors.lastname: Dunne
authors.affiliation: Division of Medicinal Safety Science, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakamura
authors.affiliation: Genomics and Computational Biology Group, Centro de Pesquisas René Rachou, National Institute of Science and Technology in Tropical Diseases, Fundação Oswaldo Cruz - FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Guilherme C
authors.initials: GC
authors.lastname: Oliveira
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
authors.forename: Marcos J C
authors.initials: MJ
authors.lastname: Alcocer
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Franco H
authors.initials: FH
authors.lastname: Falcone
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology*; D000818:Animals; D000947:Antigens, Helminth / Q000276:immunology*; D045744:Cell Line, Tumor; D002648:Child; D015801:Helminth Proteins / Q000276:immunology*; D006801:Humans; D008297:Male; D016052:Protozoan Vaccines / Q000276:immunology*; D051381:Rats; D012550:Schistosoma mansoni / Q000276:immunology*; D055815:Young Adult
chemical_list: D000485:Allergens; D000947:Antigens, Helminth; D015801:Helminth Proteins; D016052:Protozoan Vaccines