title: Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens.
abstract: BACKGROUND
  To assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery.
  
  
  METHODS
  Human fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST-ANOVA coupled analysis was used to compare the molecular and biochemical data.
  
  
  RESULTS
  The fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis.
  
  
  CONCLUSIONS
  AM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.
authors: IRCCS-G.B. Bietti Foundation, Rome, Italy, amicera@gmail.com.
authors: Alessandra
authors: A
authors: Micera
authors: Katerina
authors: K
authors: Jirsova
authors: Eduardo Maria
authors: EM
authors: Normando
authors: Barbara
authors: B
authors: Stampachiacchiere
authors: Graziana
authors: G
authors: Esposito
authors: Alessandro
authors: A
authors: Lambiase
authors: Stefano
authors: S
authors: Bonini
mesh_terms: D000650:Amnion / Q000378:metabolism*; D015153:Blotting, Western; D015925:Cryopreservation; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D051193:Toll-Like Receptors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D051193:Toll-Like Receptors