title: Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.
abstract: BACKGROUND
  Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function.
  
  
  OBJECTIVE
  To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems.
  
  
  METHODS
  We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99.
  
  
  RESULTS
  The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis.
  
  
  CONCLUSIONS
  Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.
authors.affiliation: Shiseido Research Center, 2-12-1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kamata
authors.forename: Toshii
authors.initials: T
authors.lastname: Iida
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Fukushima
authors.forename: Junko
authors.initials: J
authors.lastname: Nomura
authors.forename: Masuyoshi
authors.initials: M
authors.lastname: Saito
authors.forename: Mami
authors.initials: M
authors.lastname: Tajima
authors.forename: Yukari
authors.initials: Y
authors.lastname: Okubo
authors.forename: Takashi
authors.initials: T
authors.lastname: Momoi
authors.forename: Ryoji
authors.initials: R
authors.lastname: Tsuboi
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Hibino
mesh_terms: D000328:Adult; D000367:Age Factors; D000596:Amino Acids / Q000378:metabolism; D000911:Antibodies, Monoclonal; D053456:Caspase 14 / Q000032:analysis / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D003876:Dermatitis, Atopic / Q000201:enzymology*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D000091344:Filaggrin Proteins; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012737:Sex Factors; D012867:Skin / Q000737:chemistry / Q000201:enzymology*; D055815:Young Adult
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D053456:Caspase 14