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Abstract
Purpose
The objective of this study is to determine the expression and localization of lymphotoxin alpha (LTA) in human retinas and the functionality of one of its polymorphisms rs2229094 (C13R) (T>C), previously associated with proliferative vitreoretinopathy (PVR) development.
Materials and methods
Total RNA from three healthy human retinas were extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTA cDNA. In addition, three human eyes with retinal detachment (RD) and three healthy control eyes were subjected to immunohistochemistry (IHC) with a specific antibody against LTA. The functionality of T and C alleles was assessed by using pCEFL-Flag expression vector and transient transfection assays in COS-1 cell line. In addition, expression analysis by RT-PCR, Western blot and subcellular localization of both alleles and by immunofluorescence assay was performed.
Results
RT-PCR analysis revealed no significant levels of messenger RNA (mRNA) LTA in healthy human retinas. Sequential IHC staining showed differences between healthy human and RD retinas. No differences in mRNA and protein expression levels and in subcellular localization between both alleles were found. Both alleles were located in the cytoplasm of COS-1 cells.
Conclusion
Although results suggest lack of functionality, the differences found in IHC study and its strong association with PVR and its relationship with tumor necrosis factor locus, warrant further studies and could justify the use of this polymorphism as a valid biomarker to identify high-risk patients to develop PVR after RD.
Acknowledgments
The authors would like to thank Nieves Fernández (IOBA-University of Valladolid, Spain) for her assistance with the pictures that illustrate this work and for performing the laboratory experiments. Also, we would like to thank Fundación Centro de Investigación en Enfermedades Neurológicas (CIEN) tissue bank (Madrid, Spain) and the Hospital Clínico Universitario (Valladolid, Spain) for providing normal adult human brain sections and human eyes from donors. This article was presented in ARVO Annual Meeting, Denver, CO, May 2014, as a poster and EVER Annual meeting, Nice, France, October 2014, as a Rapid Fire. RGS is supported by SAF 2007-66394, FIS PI13/01741 and Group of Excellence Grant (GR15) from Junta de Castilla y León. The funding organization had no role in the design or conduct of this research.
Disclosure
The authors report no conflicts of interest in this work.