Molecular and biochemical expression of TLRs in human amniotic membrane

A comparative study of fresh and cryopreserved specimens

Alessandra Micera, Katerina Jirsova, Eduardo Maria Normando, Barbara Stampachiacchiere, Graziana Esposito, Alessandro Lambiase, Stefano Bonini

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)267-274
Number of pages8
JournalGraefe's Archive for Clinical and Experimental Ophthalmology
Volume252
Issue number2
DOIs
Publication statusPublished - Feb 2014

Fingerprint

Amnion
Toll-Like Receptors
Toll-Like Receptor 10
Membranes
Gene Expression
Innate Immunity
Wound Healing
Cicatrix
Real-Time Polymerase Chain Reaction
Polyacrylamide Gel Electrophoresis
Analysis of Variance
Anti-Inflammatory Agents
Western Blotting

Keywords

  • Amniotic membrane
  • Cytokines
  • Growth factors
  • Ocular surgery
  • Toll-like receptors

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Molecular and biochemical expression of TLRs in human amniotic membrane : A comparative study of fresh and cryopreserved specimens. / Micera, Alessandra; Jirsova, Katerina; Normando, Eduardo Maria; Stampachiacchiere, Barbara; Esposito, Graziana; Lambiase, Alessandro; Bonini, Stefano.

In: Graefe's Archive for Clinical and Experimental Ophthalmology, Vol. 252, No. 2, 02.2014, p. 267-274.

Research output: Contribution to journalArticle

Micera, Alessandra ; Jirsova, Katerina ; Normando, Eduardo Maria ; Stampachiacchiere, Barbara ; Esposito, Graziana ; Lambiase, Alessandro ; Bonini, Stefano. / Molecular and biochemical expression of TLRs in human amniotic membrane : A comparative study of fresh and cryopreserved specimens. In: Graefe's Archive for Clinical and Experimental Ophthalmology. 2014 ; Vol. 252, No. 2. pp. 267-274.
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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.",
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AU - Micera, Alessandra

AU - Jirsova, Katerina

AU - Normando, Eduardo Maria

AU - Stampachiacchiere, Barbara

AU - Esposito, Graziana

AU - Lambiase, Alessandro

AU - Bonini, Stefano

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N2 - 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.

AB - 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.

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