Peter Drevets1, Ana Chucair-Elliott1, Priyadarsini Shrestha1, Jeremy Jinkins1, Dimitrios Karamichos1,2, Daniel J J Carr3,4. 1. Department of Ophthalmology, Dean A. McGee Eye Institute, Acers Pavilion, 415A, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd., Oklahoma City, OK, 73104, USA. 2. Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. 3. Department of Ophthalmology, Dean A. McGee Eye Institute, Acers Pavilion, 415A, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd., Oklahoma City, OK, 73104, USA. dan-carr@ouhsc.edu. 4. Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. dan-carr@ouhsc.edu.
Abstract
PURPOSE: To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. METHODS: Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. RESULTS: Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. CONCLUSION: Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.
PURPOSE: To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. METHODS:Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. RESULTS: Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. CONCLUSION: Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.
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