title: Recombinant human beta 2-defensin fusion proteins as a tool to investigate defensin structure and function in small human intestinal tissue samples.
abstract: OBJECTIVE
  Effects of immune cells on the beta 2 (β2)-defensin (HBD2) expression and its antibacterial activity in the intestinal mucosa of patients with inflammatory bowel diseases remains unclear. The small size of these proteins presents a major challenge in localizing antibacterial activities in human intestinal tissue. In this study, we evaluated the detection limits at mRNA and protein level by approaching HBD2 from small tissue samples.
  
  
  METHODS
  HT-29 colonic epithelial cells were incubated with proinflammatory cytokines before HBD2 mRNA was investigated by quantitative polymerase chain reaction. The HBD2 protein was assessed by Western blot analysis using HBD2 fused with enhanced green fluorescent protein (HBD2-EGFP). Purified HBD2 fused with the glutathione-S-transferase (GST-HBD2) was used to detect antibacterial activity in a densitometric assay.
  
  
  RESULTS
  Interleukin (IL)-1β induced HBD2 mRNA in HT-29 cells; however, tumor necrosis factor-α, IL-6 and IL-17 did not. The Western blot had a sensitivity of 1.5 pmol to detect recombinant HBD2, but did not detect HBD2 in either human intestinal or IL-1β-treated HT-29 cells. HBD2-EGFP was detected by HBD2-specific Western blot within cell lysates and culture supernants of transfected HT-29 and primary cells. In nanomolar ranges, GST-HBD2 reduced bacterial growth. The HBD2 bioactivity depended on solution conditions, but not on the size of the fusion partner.
  
  
  CONCLUSIONS
  The established fusion proteins provide excellent tools to evaluate expression patterns and antibacterial effects of HBD2 in human intestinal tissue samples.
authors.affiliation: Department of General, Vascular and Thoracic Surgery, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany.
authors.forename: Annekatrin
authors.initials: A
authors.lastname: Coordes
authors.forename: Andreas
authors.initials: A
authors.lastname: Andreou
authors.forename: Ulrike
authors.initials: U
authors.lastname: Erben
authors.forename: Thorsten
authors.initials: T
authors.lastname: Stroh
authors.forename: Katja
authors.initials: K
authors.lastname: Blunert
authors.forename: Nadia
authors.initials: N
authors.lastname: Slavova
authors.forename: Britta
authors.initials: B
authors.lastname: Siegmund
authors.forename: Heinz-Johannes
authors.initials: HJ
authors.lastname: Buhr
authors.forename: Anton J
authors.initials: AJ
authors.lastname: Kroesen
mesh_terms: D017667:Adipocytes; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism*; D015169:Colony Count, Microbial; D016207:Cytokines / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000502:physiology; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D007082:Ileum / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D016207:Cytokines; C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase