title: Fusarium culmorum causes strong degradation of pollen allergens in extract mixtures.
abstract: BACKGROUND
  Therapeutic allergen extracts are frequently stored as mixtures to provide preparations used for specific immunotherapy. Substantial protease activity, found in certain mold extracts, has been suspected to cause a loss of allergenic activity as a result of self-degradation, as well as by means of degradation of allergens from pollen and other allergenic sources.
  
  
  OBJECTIVE
  This study was performed to address possible deterioration of individual pollen allergens during storage of extract mixtures, with a mold extract as the source of proteolytic activity.
  
  
  METHODS
  Aqueous birch and timothy pollen extracts were stored for 60 days at 6 degrees C with and without addition of an extract derived from the mold Fusarium culmorum. The stability of the pollen allergens Bet v 1, Bet v 6, Phl p 1, and Phl p 5, as well as 2 to-date-undefined F culmorum allergens was examined by using immunoblotting analysis with sera from allergic patients and allergen-specific mAbs. Furthermore, the residual allergenic activity of the pollen extracts was monitored by using the rat basophilic leukemia cell-mediator release assay. Proteolytic activity of extracts was determined by using a commercial protease assay and gelatinase zymography.
  
  
  RESULTS
  Pollen extracts were very stable, corresponding to the low proteolytic activity of these extracts. In contrast, high proteolytic activity was found for the F culmorum extract, resulting in self-degradation of mold proteins and deactivation of allergens. Similarly, the mixtures showed a strong decrease of allergenic potency in the mediator release assay. Bet v 1 and Phl p 1 were relatively stable, whereas Bet v 6 and Phl p 5 were almost entirely degraded within 1 day.
  
  
  CONCLUSIONS
  Proteases of the mold F culmorum clearly affected the overall allergenic activity of pollen extracts within a short time period. Apart from general objections against the use of mixtures of non-cross-reacting allergens, mixing of pollen extracts with extracts derived from molds for immunotherapy is not recommended unless they are applied directly after preparation of the mixture.
authors.affiliation: Paul-Ehrlich-Institut, Department of Allergology, Langen.
authors.forename: Michael
authors.initials: M
authors.lastname: Hoff
authors.forename: Marion
authors.initials: M
authors.lastname: Krail
authors.forename: Marion
authors.initials: M
authors.lastname: Kästner
authors.forename: Dieter
authors.initials: D
authors.lastname: Haustein
authors.forename: Stefan
authors.initials: S
authors.lastname: Vieths
mesh_terms: D000485:Allergens / Q000378:metabolism*; D000818:Animals; D010450:Endopeptidases / Q000494:pharmacology*; D005656:Fungal Proteins / Q000494:pharmacology*; D005670:Fusarium / Q000378:metabolism*; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011058:Pollen / Q000276:immunology*; D051381:Rats
chemical_list: D000485:Allergens; D005656:Fungal Proteins; D010450:Endopeptidases