title: Assessment of the ability of Malassezia pachydermatis to stimulate proliferation of canine keratinocytes in vitro.
abstract: OBJECTIVE
  To investigate the direct interaction between canine keratinocytes and live Malassezia pachydermatis and thereby determine the role of these organisms in the pathogenesis of epidermal hyperplasia associated with Malassezia dermatitis in dogs.
  
  
  METHODS
  Primary canine keratinocyte cultures established from skin samples obtained from clinically normal dogs.
  
  
  METHODS
  The proliferative response of keratinocytes co-cultured with Malassezia organisms for 1, 2, or 3 days was assessed by use of direct manual counting (to determine the number of keratinocytes in both the monolayer and the medium) and immunohistochemical staining techniques involving antibodies against proliferating cell nuclear antigen (PCNA) and another cellular proliferation marker, Ki-67. The potential cytotoxic effect of Malassezia organisms was investigated by use of an apoptosis detection kit to label keratinocytes co-cultured with M. pachydermatis that underwent apoptosis.
  
  
  RESULTS
  No stimulatory effect of Malassezia organisms on canine keratinocyte proliferation was detected via cell counting and immunohistochemical techniques. However, there was a significant increase in dead keratinocytes in the medium with increasing numbers of Malassezia organisms in the co-culture. More apoptotic cells were observed in keratinocyte monolayers co-cultured with high numbers of M. pachydermatis than there were in monolayers cultured without Malassezia organisms, and the number increased after prolonged incubation.
  
  
  CONCLUSIONS
  M. pachydermatis did not stimulate canine keratinocyte proliferation in vitro. The results suggested that the epidermal hyperplasia observed in dogs with Malassezia dermatitis is unlikely to be caused by a direct effect of the organism on the keratinocyte cell cycle, but is likely to involve other mechanisms.
authors.affiliation: Division of Veterinary Clinical Studies, The University of Edinburgh, Roslin, Midlothian, EH25 9RG, UK.
authors.forename: Tai-An
authors.initials: TA
authors.lastname: Chen
authors.forename: Richard E W
authors.initials: RE
authors.lastname: Halliwell
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Shaw
authors.forename: Peter B
authors.initials: PB
authors.lastname: Hill
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002452:Cell Count; D002478:Cells, Cultured; D003881:Dermatomycoses / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D004283:Dog Diseases / Q000382:microbiology* / Q000473:pathology; D004285:Dogs; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000382:microbiology* / Q000473:pathology; D019394:Ki-67 Antigen; D008290:Malassezia / Q000472:pathogenicity*; D018809:Proliferating Cell Nuclear Antigen; D013997:Time Factors
chemical_list: D019394:Ki-67 Antigen; D018809:Proliferating Cell Nuclear Antigen