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Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens

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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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Acknowledgments

The work was partially supported by the Italian “5×1000” tax contribution to IRCCS G.B. Bietti Foundation (2009; Rome). KJ was supported by PRVOUK-P24/LF1/3 program of the Charles University in Prague and by research project LM 2010004 BBMRI_CZ.

Financial Support

In part supported by the Italian “5×1000” tax contribution to IRCCS G.B. Bietti Foundation (Rome).

Conflict of Interest

None.

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None.

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Authors and Affiliations

  1. IRCCS-G.B. Bietti Foundation, Rome, Italy

    Alessandra Micera, Barbara Stampachiacchiere & Graziana Esposito

  2. Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic

    Katerina Jirsova

  3. CIR, Department of Ophthalmology, University Campus Bio-Medico, Rome, Italy

    Eduardo Maria Normando, Alessandro Lambiase & Stefano Bonini

  4. Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, 11–43 Bath Street, London, EC1V 9EL, UK

    Eduardo Maria Normando

  5. The Western Eye Hospital, Imperial College Healthcare Trust, Marylebone Road, London, NW1 5QH, UK

    Eduardo Maria Normando

  6. Laboratory of Ophtalmology, IRCCS-G.B. Bietti Foundation, Via Alvaro del Portillo 21, 00128, Rome, Italy

    Alessandra Micera

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  1. Alessandra Micera
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Correspondence to Alessandra Micera.

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Micera, A., Jirsova, K., Normando, E.M. et al. Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens. Graefes Arch Clin Exp Ophthalmol 252, 267–274 (2014). https://doi.org/10.1007/s00417-013-2540-z

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  • DOI: https://doi.org/10.1007/s00417-013-2540-z

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